Dissertationen zum Thema „Liver cell models“

Over the last decade a huge amount of experimental data on biological systems has been generated by modern high-throughput methods. Aided by bioinformatics, the '-omics' (genomics, transcriptomics, proteomics, metabolomics and interactomics) have listed, quantif ed and analyzed molecular components and interactions on all levels of cellular regulation. However, a comprehensive framework, that does not only list, but links all those components, is still largely missing. The biology-based but highly interdisciplinary field of systems biology aims at such a holistic understanding of complex biological systems covering the length scales from molecules to whole organisms. Spanning the length scales, it has to integrate the data from very different fields and to bring together scientists from those fields. For linking experiments and theory, hypothesis-driven research is an indispensable concept, formulating a cycle of experiment, modeling, model predictions for new experiments and, fi nally, their experimental validation as the start of the new iteration. On the hierarchy of length scales certain unique entities can be identi fied. At the nanometer scale such functional entities are molecules and at the micrometer level these are the cells. Cells can be studied in vitro as independent individuals isolated from an organism, but their interplay and communication in vivo is crucial for tissue function. Control over such regulation mechanisms is therefore a main goal of medical research. The requirements for understanding cellular interplay also illustrate the interdisciplinarity of systems biology, because chemical, physical and biological knowledge is needed simultaneously. Following the notion of cells as the basic units of life, the focus of this thesis are mathematical multi-scale models of multi-cellular systems employing the concept of individual (or agent) based modeling (IBM). This concept accounts for the entity cell and their individuality in function and space. Motivated by experimental observations, cells are represented as elastic and adhesive spheres. Their interaction is given by a model for elastic homogeneous spheres, which has been established for analysis of the elastic response of cells, plus an adhesion term. Cell movement is modeled by an equation of motion for each cell which is based on the balance of interaction, friction and active forces on the respective cell. As a fi rst step the model was carefully examined with regard to the model assumptions, namely, spherical shape, homogeneous isotropic elastic body and apriori undirected movement. The model examination included simulations of cell sorting and compression of multicellular spheroids. Cell sorting could not be achieved with only short range adhesion. However, it sorting completed with long range interactions for small cell numbers, but failed for larger aggregates. Compression dynamics of multi-cellular spheroids was apparently reproduced qualitatively by the model. But in a more detailed survey neither the time scales nor the rounding after compression could be reproduced. Based on these results, the applications consistent with the assumed simpli cations are discussed. One already established application is colony growth in two-dimensional cell cultures. In order to model cell growth and division, a two-phase model of the cell cycle was established. In a growth phase the cell doubles its volume by stochastic increments, and in a mitotic phase it divides into two daughter cells of equal volume. Additionally, control of the cell cycle by contact inhibition is included in the model. After examination of its applicability, the presented model is used for simulations of in vitro growth of mesenchymal stem cells (MSC) and subsequent cartilage formation in multi-cellular spheroids. A main factor for both processes is the oxygen concentration. Experimental results have shown, that i) MSC grow much better in vitro at low than at high oxygen concentrations and ii) the MSC progeny harvested from low oxygen culture produce higher amounts of the cartilage components aggrecan and collagen II in multicellular spheroids than the ones from high oxygen culture. In order to model these processes, IBM was extended by a stochastic model for cellular differentiation. In this model cellular differentiation is captured phenomenologically by two additional individual properties, the degree of differentiation and the lineage or cell type, which are subject to fl uctuations, that are state and environment dependent. After fitting the model parameters to the experimental results on MSC growth in monoclonal expansion cultures at low and high oxygen concentrations, the resulting simulated cell populations were used for initialization of the simulations of cartilage formation in multi-cellular spheroids. The model nicely reproduced the experimental results on growth dynamics and the observed number of functional cells in the spheroids and suggests the following explanation for the difference between the two expansion cultures: due to the stronger pre-differentiation found after expansion in high oxygen, the plasticity of these cells is smaller and less cell adopt the chondrogenic phenotype and start to produce cartilage. Moreover, the model predicts an optimal oxygen concentration for cartilage formation independent of expansion culture and a de-differentiating effect of low oxygen culture within 24h. Because all simulations comply with the concept of hypothesis-driven research and follow closely the experimental protocols, they can easily be tested and are currently used for optimization of a bioreactor for cartilage production. Cell populations are composed of individual cells and regulation of population properties is performed by individual cell, but knowledge about individual cell fates is largely missing due to the problem of single cell tracking. The IBM modeling approach used for modeling MSC growth and differentiation generically includes information of each individual cell and is therefore perfectly suited for tackling this question. Based on the validated parameter set, the model was used to generate predictions on plasticity of single cells and related population dynamics. Single cell plasticity was quantifi ed by calculating transition times into stem cell and differentiated cell states at high and low oxygen concentrations. At low oxygen the results predict a frequent exchange between all subpopulations, while at high oxygen a quasi-deterministic differentiation is found. After quantifying the plasticity of single cells at low and high oxygen concentration, the plasticity of a cell population is addressed in a simulation closely following a regeneration experiment of populations of hematopoietic progenitor cells. In the simulation the regeneration of the distribution of differentiation states in the population is monitored after selection of subpopulations of stem cells and differentiated cells. Simulated regeneration occurs on the time scales estimated from the single cell transition times except the unexpectedly fast regeneration from differentiated cells in the high oxygen environment, which favors differentiation. The latter case emphasizes the importance of single outlier cells in such system, which in this case repopulate less differentiated states with their progeny. In general, cell proliferation and regeneration behavior are in uenced by biomechanical and geometrical properties of the environment e.g. matrix stiffness or cell density. Because in the model cells are represented as physical objects, a variation of friction is linked to cell motility. The cultures of less motile cells become denser at the same size and the effects of contact inhibition of growth more pronounced. This variation of friction coe fficients allows the comparison of cultures with varying degrees of contact inhibition regarding their differentiation structure and the results suggest, that stalled proliferation is su fficient to explain the well-known differentiation effects in confl uent colonies. In addition, the composition of the simulated stem cell pool was analyzed regarding differentiation. In contrast to the established pedigree models, where stem cell can only be produced by asymmetric division, this model predicts that most of the cells in stem cell states descend from progenitor cells of intermediate differentiation states. A more detailed analysis of single cell derived clones revealed properties that could not be described by the model so far. First, a differentiation gradient was observed in larger colonies, that was the opposite of the one predicted by the model. Second, the proliferative activity turned out to depend not only on oxygen, but also to be a property of individual clones persisting over many generations. Because the relation slow growth/pre-differentiation also holds for single cell derived clones, the general model of differentiation is extended by another heritable individual property. Motivated by the decline of proliferation and differentiation in culture and the high metabolic and epigenetic activity during cell division, each division event is assumed to de-stabilize stem cell states. Consequently, in the model the cells age in terms of cell divisions determines the fl uctuations in stem cell states and the environment the mean fl uctuation strength. Including this novel concept, that links aging to growth and differentiation dynamics, into the model reproduces the experimental results regarding differentiation gradient and persistent clonal heterogeneity. The spatial differentiation pattern can largely be explained by the spatio-temporal growth pattern of the mono-clonal cell assembly: cells close to the border of the cell assembly have undergone more cell divisions than those in the interior and therefore their stem cell states are less stable. Heterogeneity of single-cell derived clones depends on the age of the first cell in the clone. When the stem cell fluctuations equal the mean fl uctuations strength, the proliferative activity passes a maximum at a certain age due to the destabilization of stem cell states. Thereafter the proliferative activity decreases, because more time is spent in non-proliferative differentiated states. Considering the number of divisions the cells have already undergone in vivo and after the initial expansion in vitro, it can be assumed that all cells have already passed this maximum. Interestingly, the model also predicts an optimal age for directed differentiation, when cells stably differentiate, but have not lost the required plasticity. According to the model, this clonal heterogeneity may be caused purely in vitro, but hypothetical simulation of in vivo aging yielded results consistent with experiments on MSC from rats of varying age. Finally, the detailed molecular regulation mechanisms in a multi-scale tissue model of liver zonation was studied, in which the key molecular components were explicitly modeled. Hence, this model resolved the intracellular regulation in higher resolution than the above considered differentiation models which had summarized the intracellular control and differentiation mechanisms by a few phenomenological, dynamical variables. The metabolic zonation of the liver is essential for many of the complex liver functions. One of the vitally important enzymes, glutamine synthetase, (GS) is only synthesized in a strictly defi ned pattern. Experimental evidence has shown that a particular pathway, the canonical wnt pathway, controls expression of the gene for GS. A model for transport, receptor dynamics and intracellular regulation mechanism has been set up for modeling the spatio-temporal formation of this pattern. It includes membrane-bound transport of the morphogen and an enzyme kinetics approach to fibeta-catenin-regulation in the interior of the cell. As an IBM this model reproduces the results of co-culture experiments in which two-dimensional arrangements of liver cells and an epithelial liver cell line give rise to different patterns of GS synthesis. The two main predictions of the model are: First, GS-synthesis requires a certain local cell number of wnt releasing cells. And second, a simple inversion of geometry explains the difference between the specifi c GS pattern found in the liver and in the co-culture experiments. Summarizing the results presented in this thesis, it can be concluded that properties such as the occurrence of memory effects and single cells pursuing fates far off the population average could be essential for biological function. Considering the role of single cells in many tissues, the use of individual based methods, that are able to take such effects into account, can be expected to be a very valuable tool for the problems of systems biology.

Background and objectives: Nonalcoholic steatohepatitis (NASH), which is characterized by concurrent existence of hepatic steatosis and predominantly lobular necroinflammation, represents the more advanced stage in the spectrum of nonalcoholic fatty liver disease (NAFLD). NASH exhibits dramatically increased risk of progression to end-stage liver diseases than simple steatosis. Therefore, the progression of hepatic steatosis to steatohepatitis is the crucial step in the development of obesity-related NASH. Toll like receptor 4 (TLR4), a master regulator of innate immunity, is the principal receptor for endotoxin, which is a central mediator of liver inflammation associated with both alcoholic and nonalcoholic liver disease. However, due to a lack of suitable animal models which fully recapitulate the natural history of obesity-induced NASH, the precise pathophysiological function of TLR4 signaling in the development of this disease remains poorly understood. The objective of this study is to investigate the role of TLR4 in mediating inflammatory responses in obesity-induced NASH using both in vivo and ex vivo approaches, and to unveil cellular and molecular mechanisms responsible for TLR4 actions. Key findings: 1. To address the role of TLR4 in the pathogenesis of NASH, we crossed ApoEdeficient mice (ApoE-/-) with TLR4 mutant mice (TLR4-/-) to generate ApoE-/- /TLR4 wild type mice (ApoE-/-/TLR4-WT) and ApoE-/-/TLR4-/- mice. Noticeably, when fed with high fat high cholesterol (HFHC) diet, ApoE-/-/TLR4-WT mice developed the typical pathology of NASH (hepatic steatosis, lobular inflammation, and hepatocyte ballooning) in the context of obesity and metabolic syndrome, suggesting HFHC-fed ApoE-/- mice as a suitable animal model for NASH. 2. TLR4 inactivation protected ApoE-/- mice against HFHC diet-induced liver injury, as indicated by a significant improvement in liver histology, a a marked reduction in serum ALT activity, a dramatic repression of inflammatory infiltrates, as well as an obvious decrease in hepatic production of pro-inflammatory cytokines. 3. In ApoE-/-/TLR4-WT mice, TLR4 expression was selectively elevated in Kupffer cells in response to HFHC diet feeding. 4. The activation of XBP1, a transcription factor involved in endoplasmic reticulum stress, was markedly elevated in liver of ApoE-/-/TLR4-WT mice fed with HFHC diet, whereas this change was abrogated in HFHC diet-fed ApoE-/-/TLR4-/- mice. 5. In rat primary Kupffer cells, treatment with anti-oxidants blocked endotoxininduced activation of XBP1 and NF-κB, leading to decreased cytokine production. In addition, siRNA-mediated knockdown of XBP1 inhibited NF-κB activation and cytokine production resulted from the treatment with the TLR4 agonist LPS. 6. In ApoE-/-/TLR4-WT mice, adenovirus-mediated expression of dominant negative XBP1 had no obvious effect on HFHC diet-induced hepatic steatosis and ROS production, but markedly decreased lobular inflammation, NF-κB activation, cytokine production in the liver and significantly reduced serum levels of ALT. Conclusions: These findings support the role of TLR4 in Kupffer cells as a key player in mediating the progression of simple steatosis to NASH, by inducing ROS-dependent activation of XBP1. In light of the obligatory role of XBP1 in TLR4-induced liver inflammation and injury, therapeutic interventions that inhibit TLR4/XBP1 activation may represent a promising strategy for treatment of NASH.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

Each year liver cancer kills more than half a million people, making it the third leading cause of cancer-related death worldwide. Annual incidence continues to rise steadily, both domestically and globally, increasing the burden of this disease. Advancements in the ability to obtain detailed molecular profiles of tumors have led to the successful development of targeted therapies for a number of different cancers. Unfortunately, however, the molecular pathogenesis of liver cancer is poorly understood relative to many other types of malignancies. Thus, the identification of factors contributing to the development and progression of liver tumors is a major goal of current research. In pursuit of this goal, I have utilized the Sleeping Beauty (SB) transposon system as a tool for forward genetic mutagenesis screening in mice. The SB system recapitulates the kinetics of spontaneous tumor development in humans by providing a stepwise accumulation of mutations. Micro-evolutionary processes within a developing tumor lead to the selective expansion of cells harboring mutations that confer some kind of selective advantage. By identifying the most prevalent mutation events within a specific tumor type across a large number of independent samples, a list of genes implicated as being involved in tumorigenesis can be generated. Using this approach, the Dlk1-Dio3 imprinted domain was identified as a site of frequent mutation in SB-induced hepatocellular carcinomas (HCCs). I discovered that the mechanistic basis for recurrent selection of transposon insertion within this domain in liver tumors involved activated expression of Retrotransposon-like 1 (Rtl1). I also found that RTL1 activation is a common event in human HCC, suggesting that it could potentially be beneficial as a therapeutic target in a subset of patients. Etiological factors related to liver cancer development are varied, but are linked by the fact that each provides a chronic liver injury stimulus that promotes the development of hepatic fibrosis. In fact, ˜ 90% of human HCC occurs in this context, and yet the majority of mouse liver cancer models fail to account for this important environmental component of the disease. I have conducted a screen for genetic drivers of liver cancer in the presence or absence of hepatic fibrosis. Comparison of mutation profiles between fibrotic and non-fibrotic tumors revealed largely non-overlapping sets of candidate genes, indicative of a differential selective pressure for mutations depending on the fibrotic context of the liver. Driver mutations identified preferentially in the presence of liver fibrosis have a high likelihood of relevance to human disease, given the similarities in environmental context and kinetics of mutation acquisition. Consistent with this idea, multiple genes with well-established roles in human HCC were found to be preferentially mutated in SB-induced tumors developed in a fibrotic liver. Before a candidate cancer gene identified in an animal model system can have an impact on human disease, its proposed role in tumorigenesis must be validated. Existing techniques for validation of putative liver cancer genes suffer from significant limitations including high cost, low throughput, and a level of complexity that prohibits widespread utilization. I have contributed to the generation of a novel tool for in vivo validation of candidate genes that is not subject to these limitations. By combining elements of recombinant adenoviral vectors and the piggyBac transposition system, we have generated a highly flexible gene delivery system with significant advantages over existing techniques. The Ad-PB system has broad accessibility and applicability, making it a valuable tool for advancing efforts to improve cancer therapies.

L’un des enjeux de l’industrie pharmaceutique aujourd’hui est de développer des modèles de foie in vitro fidèles pour améliorer la prédictivité des études précliniques, notamment l’étude de la toxicité et de l’efficacité des médicaments candidats. Ces dernières années, l’ingénierie tissulaire, approche multidisciplinaire pour développer des tissus, a mené au développement de nouvelles méthodes de culture cellulaire. Parmi elles, les cultures de cellules en 3D ou en perfusion ont permis d’obtenir des activités hépatiques similaires à celles observées in vivo. L’objectif de cette thèse est de combiner ces deux méthodes de culture cellulaire pour créer un modèle de foie in vitro encore plus fidèle. Pour cela, nous cherchons à développer un cryogel d’alginate intégré en micropuce avec des propriétés mécaniques adaptables à celles du foie en fonction de l’état physiologique à reproduire (foie sain ou pathologique). Dans la première partie, nous développons et caractérisons le cryogel d’alginate au niveau microscopique et macroscopique, à l’extérieur (échantillons cylindriques) puis à l’intérieur de la biopuce. Trois paramètres sont étudiés ici : la température de cryopolymérisation, la concentration d’alginate ainsi que la quantité d’agents réticulants. Les propriétés mécaniques, la porosité, l’absorption, l’interconnectivité des pores et la résistance au flux sont analysés.La deuxième partie vise à cultiver des cellules hépatiques au sein de ce nouveau dispositif. Pour cette étude de faisabilité la lignée cellulaire HepG2/C3A est utilisée. Les résultats montrent des cellules viables et fonctionnelles (production d’albumine, transformation d’APAP). De plus, nous observons une structure tissulaire 3D, qui se maintient après retrait du cryogel d’alginate. La dernière partie a pour but de complexifier le modèle hépatique, notamment par des co-cultures. Pour se rapprocher de la structure du sinusoïde, des cellules hépatiques sont cultivées avec des cellules endothéliales (HUVEC) selon deux approches. De plus, la possibilité de suivre des cellules tumorales circulantes (MDA-MB-231) dans le système est étudiée
Today, one of the challenges for the pharmaceutical industry is to develop accurate in vitro liver models to improve the predictability of preclinical studies, in particular the study of the toxicity and efficacy of drug candidates. In recent years, tissue engineering, a multidisciplinary approach to develop tissues, has led to the development of new cell culture methods. Among them, cell cultures in 3D or in perfusion allowed to obtain hepatic activities similar to those observed in vivo. The objective of this thesis is to combine these two cell culture methods to create an even more accurate in vitro liver model. To do so, we are seeking to develop an alginate cryogel integrated into a microchip with mechanical properties adaptable to those of the liver depending on the physiological state to be reproduced (healthy or pathological liver).In the first part, we develop and characterize the alginate cryogel at the microscopic and macroscopic level, outside (cylindrical samples) and then inside the biochip. Three parameters are studied here: the cryopolymerization temperature, the alginate concentration and the quantity of cross-linking agents. Mechanical properties, porosity, absorption, pore interconnectivity and flow resistance are analyzed. The second part aims to culture liver cells within this new device. For this feasibility study the HepG2/C3A cell line is used. The results show viable and functional cells (albumin production, APAP transformation). In addition, we observe a 3D tissue structure, which is maintained after removal of the alginate cryogel. The last part aims to complexify the hepatic model, in particular by co-cultures. To get closer to the sinusoid structure, liver cells are cultured with endothelial cells (HUVEC) according to two approaches. In addition, the possibility to follow circulating tumor cells (MDA-MB-231) in the system is studied

La mort cellulaire joue un rôle central dans le développement et la progression des maladies du foie. Quel qu’en soit l’agent étiologique, il en résulte une destruction des hépatocytes, conduisant à une inflammation et à une prolifération compensatoire. En outre, la disparition continue de cellules peut aboutir au développement d’une fibrose, d’une cirrhose, voire d’un carcinome hépatocellulaire, la 3ème cause de décès par cancer. L'expression ou la sécrétion de ligands de mort, tels que le TNF-α, le FAS L et le TRAIL, par les cellules inflammatoires constitue le principal facteur de la progression des maladies du foie. En aval des récepteurs de ces ligands de mort ou de motifs moléculaires associés aux pathogènes (PAMP), la protéine kinase 1 interagissant avec le récepteur (RIPK1) influence le destin de la cellule, que ce soit pour survivre ou pour mourir par apoptose dépendante de caspases ou par nécroptose dépendante de RIPK3 / MLKL. Elle pourrait donc constituer une cible thérapeutique potentielle pour réguler le destin des hépatocytes. RIPK1 peut jouer un rôle distinct sur la mort ou la survie, grâce, respectivement, à ses fonctions kinase ou d’échafaudage. Dans cette optique, nous avons déjà montré le rôle protecteur de RIPK1 dans des modèles animaux d'hépatite aiguë induite par la ConA et le LPS. Au cours de mon travail de thèse, l'objectif était d'évaluer le rôle de RIPK1 dans des modèles murins d’hépatites aiguës (hépatite virale fulminante, dommages au foie induits par le CCl4 ou l'acétaminophène [APAP]) et d'hépatites chroniques (régime à haute teneur en gras et en cholestérol [HFHCD]). Nos résultats ont démontré que RIPK1 protège les hépatocytes du TNF-α sécrété par les macrophages au cours de l'hépatite fulminante induite par le virus MHV3. Ces données révèlent les risques potentiels d'aggravation d'une infection par le VHB chez les personnes atteintes de polymorphisme ou de mutations amorphes homozygotes déjà décrites pour le gène RIPK1. En outre, nous avons établi que RIPK1 dans les cellules parenchymateuses du foie n’influence pas les lésions hépatiques induites par l’APAP chez la souris. L'inhibition supplémentaire de l'activité kinase de RIPK1 chez les souris Ripk1LPC-KO n'a pas limitée les dommages hépatiques, révélant que l'activité kinase de la RIPK1 dans les cellules hépatiques non-parenchymateuses ne contribue pas aux lésions hépatiques induites par l’APAP. Sinon, nous avons démontré que RIPK1 dans les cellules parenchymateuses du foie préserve partiellement le foie des lésions induites par le CC14, lésions ne dépendant pas du TNF-α. Enfin, nous avons montré que RIPK1 dans les cellules parenchymateuses hépatiques avait tendance à limiter le développement de la fibrose induite par HFHCD dans la NASH murine et qu'une intervention alimentaire pouvait améliorer la fibrose hépatique chez la souris atteinte de NASH. Quant au rôle de l'activité kinase de la RIPK1 dans la NASH, elle reste à être explorée pour évaluer son intérêt thérapeutique
Cell death plays central role in the development and progression of liver diseases. Irrespective of the etiological agents, it results in hepatocyte destruction, leading to inflammation and compensatory proliferation. In addition, the persistent cell demise can lead into fibrosis and ultimately hepatocellular carcinoma, the 3rd leading cause of cancer related death. Expression or release of death ligands, such as TNF-α, FAS L and TRAIL, by inflammatory cells remains the key players in the progression of liver diseases. Downstream of death ligand receptors or PAMPs, receptor interacting protein kinase 1 (RIPK1) influences the fate of cell, whether to survive or to die by caspase-dependent apoptosis or by RIPK3/MLKL-dependent necroptosis and could therefore be potential targets in regulating cell death. RIPK1 can have distinct pro-death or pro-survival role, regulated by its kinase or scaffolding functions, respectively. In line with this, we have already shown the protective role of RIPK1 in animal models of acute hepatitis induced by ConA, LPS. In my PhD work, the objective was to assess the role of RIPK1 in animal models of acute (fulminant viral hepatitis, CCl4 and acetaminophen [APAP] induced liver damage) and chronic hepatitis (High Fat High Cholesterol diet [HFHCD]-induced NASH). Our results demonstrated that RIPK1 protects hepatocytes from TNF-α secreted from macrophages during viral induced fulminant hepatitis. These data emphasize the potential worsening risks of an HBV infection in people with polymorphism or homozygous amorphic mutations already described for the RIPK1 gene. Besides, we established that RIPK1 in liver parenchymal cells does not influence APAP-induced liver injury in mice. Additional inhibition of RIPK1 kinase activity in Ripk1LPC-KO mice did not improve hepatic damage, revealing that RIPK1 kinase activity in liver non-parenchymal cells does not contribute to APAP-induced liver injury. Otherwise, we demonstrated that RIPK1 of liver parenchymal cells partly preserves the liver from CCl4-induced damage, lesions that do not depend on TNF-α . Finally, we showed that RIPK1 in liver parenchymal cells has a tendency to protect from HFHCD-induced fibrosis in murine NASH and that dietary intervention can improve liver fibrosis in mice with NASH. As for the role of RIPK1-kinase activity in NASH, it remains to be explored to evaluate its therapeutic interest

Die Leber ist das am häufigsten von Metastasen betroffene Organ und kann daher als Modellorgan für metastatische Invasion dienen. Aus diesem Grund war es das Ziel dieser Dissertation Genexpressionsprofile zu verstehen und metastasierungs- sowie invasionsassoziierte Gene zu identifizieren. Differentielle Genexpression wurde in drei Systemen überprüft: Einem syngenen Mausmodell, einem Xenograftmodell sowie in fünf Gewebeproben von Patienten. Genexpressionprofile des syngenen Mausmodells und der Patientenproben zeigten, dass man die Invasionsfront als Ganzes betrachten, um möglichst viele über-lappende Gene zu finden. Globale Genexpressionstudien, die auf den Wirtsteil der Invasionsfront zeigten bemerkenswerte Überrepräsentation z. B. der „GO-terms“ „extrazelluläre Matrix“, Zellkommunikation“, „Antwort auf biotischen Stimulus“, Strukturmolekülaktivität“ und „Zellwachstum“. Marker der Aktivierung hepatischer Sternzellen überrepräsentiert in der invasionsfront, was die Durchführbarkeit einer Analyse differentieller Genexpression im genomweiten Rahmen anzeigt. Globale Genexpressionsstudien, auf den Tumorzellen in der in vitro Situation, in vivo und in der Invasionsfront zeigten insgesamt einen Anstieg zellulärer Spezialisierung von der in vitro zur Invasionsfront. Sezernierte proangiogenetische Chemokine zeigten eine Hochregulation in der Invasionsfront. Das beta catenin Gen war in der Invasionsfront 9.6 fach erhöht im Vergleich zur in vitro Situation. Die Überprüfung der transkriptionellen Aktivierung von beta catenin über die Prüfung der Promotoraktivität zeigte einen 18.4 fachen Anstieg in den Tumorzellen der Invasionsfront. Weiterhin war die Promotoraktivität (an Hand der Aktivität der mRNA des Alkalischen Phosphatase Reportergens) im Tumorinneren 3.5 fach höher als in der Zellkultur, was für einen transkriptionellen Mechanismus der beta catenin Regulation zusätzlich zu den posttranslationalen Mechanismen spricht.
Liver is most frequently populated by metastases and may therefore serve as a model organ for metastatic invasion. So the aim of this thesis is to understand the gene expression profiles and identify metastasis and invasion related genes. Differential gene expression was examined in three systems: A syngeneic mouse model, a xenograft model and five clinical specimens. Gene expression profiles of a syngenic mouse model and human clinical specimen revealed that the invasion front should be considered as a whole to find more overlapping potential target genes. Global gene expression studies on the host part of the invasion front, revealed a pronounced overrepresentation of GO-terms (e.g. “extracellular matrix”, “cell communication”, “response to biotic stimulus”, “structural molecule activity” and “cell growth”). Hepatic stellate cell activation markers were over-represented in the invasion front demonstrating the feasibility of a differential gene expression approach on a genome wide scale. Global gene expression studies of the tumor cells in vitro, in vivo and tumor part of the invasion front revealed an overall increase of cellular specialization from in vitro to the invasion front. Secreted angiogenic cytokines were found to be up regulated in the invasion front. Beta catenin gene of “cell adhesion” GO term was elevated 9.6 fold in invasion front compared to in vitro. Evaluation of transcriptional up-regulation of beta catenin by promoter activity showed an 18.4 fold increase in the tumor cells of the invasion front as compared to those from the faraway tumor. Promoter activity assessed by soluble human placental alkaline phosphatase reporter gene mRNA was 3.5 fold higher in the inner parts of the tumor than in vitro cells indicating a transcriptional mechanism of beta catenin regulation in addition to the posttranslational regulatory mechanisms.

The difference between the number of patients needing transplantation for chronic liver disease and the number of organ donors is growing, drawing attention to the urgent requirement for novel therapies. Chronic liver injury is commonly caused by viral hepatitis, alcohol consumption, obesity and metabolic disorders. Prolonged liver injury leads to fibrosis, hepatic scarring and eventually cirrhosis. This project is based on previous studies demonstrating the therapeutic effects of bone marrow-derived macrophages (BMDM) in a murine model of liver fibrosis. BMDM facilitated fibrosis regression and improved liver regeneration. Pro-resolution macrophages exhibited increased expression of MMPs, growth factors and phagocytosis-related genes. However, macrophages derived from bone marrow are inherently heterogeneous and difficult to genetically manipulate. To overcome this limitation, our laboratory has established a protocol whereby pure populations of macrophages can be produced in significant numbers from murine embryonic stem cells (ESC) in vitro, providing an essentially limitless source of macrophages. The first goal of this project was to compare macrophages derived from ESCs (ESDM) with classical BMDM. ESDM displayed characteristic macrophage morphology, could be activated and responded to different cytokines in vitro, and were functionally phagocytic. However, they displayed some differences in their gene expression profile, and were found to be less phagocytic than BMDM. We then assessed whether ESDM could be used in the treatment of a murine model of hepatic injury induced by carbon tetrachloride administration. ESDM therapy helped in the regression of liver fibrosis, down-regulated the number of fibrogenic myofibroblasts, and activated liver progenitor cells. However, a higher number of ESDM compared to BMDMs were required to exert that effect. To assess whether ESDM may be similar to yolk sac derived tissue-resident macrophages, rather than monocyte-derived, we compared their behaviour in a Kupffer cell repopulation assay. Macrophages were depleted using liposomal clodronate treatment then animals were transplanted with either ESDM or BMDM. We demonstrated that ESDM were more efficient than BMDM at repopulating the Kupffer cell compartment and reversing the effects of liposomal clodronate treatment in mice. It is well known that macrophages are very difficult to genetically modify. So our strategy was to genetically modify ESC and then differentiate them to macrophages that carry the modification. By genetically modifying ESCs, we attempted to produce pro-fibrolytic ESDM that over-express MMP12 which is a member of the matrix metalloproteinase family of genes that mainly degrades elastin, an extracellular matrix component. We initially employed a Tet-On 3G expression system to create an ESC line where MMP12 could be expressed in an inducible manner in differentiated macrophages. However, although this inducible strategy functioned in undifferentiated ESCs we could not induce the expression of MMP12 in differentiated macrophages. In an attempt to overcome possible gene-silencing issues, we designed and constructed an expression strategy such that Mmp12 was expressed specifically in macrophages. The ESC line was built such that Mmp12 expression would be driven by the promoter of macrophage colony stimulating factor-1 receptor gene (Csf-1r or c-fms). Using the CRISPR/Cas9 strategy, we successfully targeted the Mmp12 cDNA to the Csf-1r locus but ESDM that were differentiated from targeted ESC lines did not express Mmp12. Thus, despite having adopted two independent strategies, we have failed to generate genetically modified macrophages. As a first step to translate the therapeutic effects of macrophages into the clinical setting, we optimized a feeder- and serum-free protocol to efficiently generate macrophages from human induced pluripotent stem cells.

The major objective of this work was the development of an in vitro model for liver homeostasis which would allow the future study of early events in cell proliferation and cell death. The model which was set up involves growing T51B rat liver epithelial cells with a single dose of 1nM epidermal growth factor (EGF). This results in a period of hyperplasia where the cells reach double the control cell numbers two days after EGF addition. This is then followed by a decrease in cell numbers and the cell density returns to around the confluent control level five days after EGF addition. The model was investigated to ascertain whether the decrease in cell numbers three to five days after EGF addition was due to an increase in apoptosis. The results from light and electron microscopy studies, from the electrophoresis of T51B cell DNA and from the quantification of nuclear fragmentation indicated that the cells do die via an increase in apoptosis. The electron microscopy studies also show that healthy T51B cells can phagocytose apoptotic bodies. This suggests that the model is more physiological than other in vitro models of apoptosis. Cell growth studies and EGF binding studies were carried out in order to try to determine which events, if any, are EGF specific. The results from these studies suggest that occupancy of the low affinity binding site of the EGF receptor is responsible for the hyperproliferation seen when the T51B cells are grown with high doses of EGF. These studies also suggest that the apoptosis could be triggered by the down-regulation of the receptor, in a manner analogous to the removal of a trophic hormone in other systems. Thus this work describes the development and characterization of an in vitro model of liver homeostasis which closely parallels in vivo systems where animals are given mitogenic stimuli, and it also provides a good system for studying the role of EGF in cell proliferation and apoptosis.

Abstract The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancers stemming from epithelial cells and are the most common cancer type. The aim of this study was to understand the differentiation and malignant transformation of epithelial Madin-Darby canine kidney (MDCK) cells and to analyse the electrophysiological parameters which regulate their transport capacity. Emphasis was placed on comparing different culture environments, both in 2D and 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst and lumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cells were capable of both water secretion and reabsorption. The cells were able to perform these functions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluid was not required. Taken together, these results validate MDCK cells as a good basic model for studying kidney function. Next, the aim was to analyse the effect of 2D and 3D culture environments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysis revealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switching the untransformed cells from 2D environment to 3D. This downregulation of survivin occurs in adult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2D cells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherin expression. The last part was to study further the factors controlling survivin expression and its significance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cells remained in contact with the extracellular matrix. If MDCK cells were denied of ECM contacts they were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if cells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though they had proper cell-matrix contacts. Taken together, these results highlighted the importance of cellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid apoptosis
Tiivistelmä Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmisten yleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksena oli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistumista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimintaa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologisten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solujen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tulokset osoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyperpolarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutosta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksen perusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjen vaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujen MDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muutoksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen, kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin väheneminen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetut solut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeeni sai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuksen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen ja surviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-solut eivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jos solut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentävät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivat apoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tulokset korostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilaistumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta

Introduction: Hepatic stellate cells (HSC) and myofibroblasts may be relevant stromal drivers of human hepatocellular carcinoma (HCC). It was hypothesised that targeted inhibition of αv integrin-mediated TGF-β activation, by HSC or hepatocytes, may result in reduced peri-tumoural and intra-tumoural extracellular matrix formation, and reduced hepatic carcinogenesis. The role of HSC in acute liver injury is less well characterised. It was anticipated that integrin signalling on HSC and hepatocytes might also be relevant in the acute setting. The emerging technique of intravital microscopy (IVM) allows detailed, real-time investigation of the cellular processes involved in hepatocyte injury, cell death and repair. It was hypothesised that this could be coupled with mouse models of HCC and acute liver injury, to perform sequential imaging under anaesthesia. Aims: (i) To determine the effect of targeted inhibition of αv integrins on HSC and hepatocytes, during hepatocarcinogenesis, in a mouse model of HCC. (ii) To investigate the effect of targeted inhibition of αv and other integrins on HSC, hepatocytes, and liver sinusoidal endothelial cells (LSEC), during acute liver injury, in the mouse model of paracetamol-induced liver injury. (iii) To develop IVM of the liver, via an abdominal imaging window, with optimisation of surgical and imaging techniques, to allow sequential imaging of the same animal. Methods: The diethylnitrosamine (DEN)-induced mouse model of hepatocarcinogenesis was used, and PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were employed to deplete αv integrins on HSC and hepatocytes respectively. Tumours were harvested at 40 weeks post-DEN. Tumour size and number was evaluated in all animals. PDGFRβ-Cre;αvfl/fl and Alb-Cre;αvfl/fl mice were used in the paracetamol model, to investigate the role of αv integrins in acute liver injury. PDGFRβ-Cre;β8fl/fl and Alb-Cre;β 8fl/fl animals were also tested in this model. The role of integrins in liver sinusoidal endothelial cells (LSEC) during paracetamol-induced liver injury was evaluated using Cdh5-Cre mice. IVM of the liver was performed by surgical implantation of an abdominal imaging window, consisting of a titanium ring and coverslip, secured in place with a purse string suture. Fluorescent reporter mice were used to identify hepatic and vascular architecture, and other label-free microscope technologies were utilised to image collagen, lipid distribution, necrotic areas and blood flow within tissues. Results: In large cohorts of PDGFRβ-Cre;αvfl/fl, Alb-Cre;αvfl/fl, and control animals, there was no difference in mean tumour size or number, at 40 weeks. Targeted inhibition of α v integrins and β 8 integrin on hepatocytes, HSC or LSEC was not protective in paracetamol-induced liver injury. IVM of the liver can be performed on animals with HCC and throughout paracetamol-induced liver injury, to obtain high quality, real-time images of multiple cell lineages and the hepatic microenvironment. Conclusions: The role of TGF-β in HCC pathogenesis is complex and context-dependent. Targeted loss of αv integrin did not result in reduction in tumour burden in this non-cirrhotic model of HCC. IVM of the liver is a powerful tool to quantify inflammatory infiltrates and assessment of vascular remodelling throughout the course of acute liver injury and regeneration, providing insights into the biological processes determining recovery.

Bibliography: leaves 142-170.
Fumonisin B₁ (FB₁‚ ) is a carcinogenic mycotoxin produced by the fimgus Fusarium moniliforme in maize, and is hepatotoxic and hepatocarcinogenic in rats. The goal of this dissertation was to characterise the FB₁-fed rat as a model for liver injury and carcinogenesis, and to examine the role of oval ('progenitor') cells during these processes. Male Fischer 344 rats were fed FB₁ 250 mg/kg diet for five weeks, and this basic feeding regimen was modified in individual experiments. Short-term feeding of FB₁ caused a severe 'toxic' hepatitis, apoptosis and regeneration of hepatocytes, fibrosis, proliferation of OV-6 positive oval cells, and formation of GST pi positive hepatic foci and nodules. Oval cells were noted inside some of the hepatic nodules. There were marked increases in the expression of mRNA transcripts for mature TGF-β1 and c-myc in livers of FB₁-fed animals. The overexpression of TGF-β1 by hepatocytes may be responsible for the prominent apoptosis and fibrosis seen with liver injury due to FB₁. Increased expression of c-myc and TGF-β1 may cooperate during FB₁-induced promotion of liver tumours, possibly by providing an environment that selects for the growth of TGFβ1-resistant transformed liver cells. In rats given FB₁ in the presence of dietary iron overload, FB₁ augmented iron-induced lipid peroxidation in the liver. However, dietary iron loading appeared to protect against the cancer-promoting properties of FB₁, possibly due to a stimulatory effect on hepatocyte regeneration. Long-term feeding of FB₁ caused fibrosis and regenerative nodules, dysplastic hepatic nodules, cholangiofibrotic lesions, intraductal cholangiocarcinomas, and a hepatocellular carcinoma. 2-Acetylaminofluorene enhanced the effects of FB₁ in the liver, presumably by blocking hepatocyte regeneration in response to FB₁ toxicity. Proliferating oval cells were found inside/adjacent to GST pi positive lesions, dysplastic nodules, and cholangiofibrotic lesions, suggesting that oval cells may be involved in FBI-induced hepato- and cholangiocarcinogenesis in the liver. Furthermore, the OV-6 antigen was expressed by proliferating oval cells and bile ductules, hepatic nodules, cholangiofibrotic lesions, and cystic lesions, indicating that all of these cells may have a common ('stem') cell of origin. In conclusion, the FB₁-fed rat is a promising model for the study of liver injury, oval ('progenitor') cell proliferation, and carcinogenesis.

Les travaux antérieurs ont montré que les myofibroblastes portaux (PMFs) contribuaient de manière significative à la fibrogenèse et à l'angiogénèse dans la fibrose hépatique. L'objectif principal de cette thèse était de cartographier les cellules mésenchymateuses portales, et plus particulièrement la niche des cellules souches mésenchymateuses portales. Nous avons caractérisé la variété des cellules mésenchymateuses portales du foie de souris. Résultat important, nous avons identifié une population de cellules mésenchymateuses portales ayant les caractéristiques de cellules souches mésenchymateuses, désignées cellules souches mésenchymateuses portales (PMSCs), qui ont la capacité de se transformer en PMFs in vitro. Nous avons identifié Slit2 comme un marqueur des PMSCs par scRNA-seq et bulk RNA-seq. In vivo, nous avons mis en évidence l'expansion de PMSCs dans le foie de modèles murins de fibrose hépatique et de patients ayant une maladie chronique du foie. Nous avons identifié des signatures transcriptomiques spécifiques des PMSCs d’une part et des cellules étoilées du foie (CEF), de l’autre. Les résultats obtenus par l’utilisation de ces marqueurs, renforcent nos conclusions selon lesquelles les PMSCs s’accumulent de façon corrélée avec la fibrogenèse et l'angiogenèse, tandis que la signature des CEFs ne varie pas. En conclusion, nos travaux apportent des éléments à la connaissance des populations de cellules mésenchymateuses portales du foie. Ils ont permis d’identifier et caractériser les PMSCs ainsi que les myofibroblastes qui en dérivent, ouvrant de nouvelles perspectives dans le domaine des thérapies ciblées et des biomarqueurs pour la pratique clinique
Previous work has demonstrated that portal myofibroblasts (PMFs) significantly contributed to liver fibrogenesis and modulated angiogenesis in liver fibrosis. The main aim of this thesis was to elucidate the landscape of portal mesenchymal cells, with a particular focus on a portal mesenchymal stem cell niche. We characterized the murine normal liver portal mesenchymal cell landscape. Importantly, we revealed a portal mesenchymal cell population with the features of mesenchymal stem cells (MSCs), designated portal mesenchymal stem cells (PMSCs) that possessed the ability to give rise to PMFs in vitro. Furthermore, we identified Slit2 as a new marker of PMSCs based on scRNA-seq and bulk RNA-seq analysis. In vivo, we observed PMSC expansion (measured by the expression of Slit2) in liver from both animal fibrosis models (DDC and CDAA) and patients with chronic liver disease (NASH, PSC and other liver disease). Notably, we defined the specific gene signatures for PMSCs and hepatic stellate cells (HSCs), respectively. By using these markers, we provide further evidence indicating that PMSCs expand in correlation with fibrogenesis and angiogenesis in different murine and human liver diseases, whereas the HSCs gene signatures did not vary. In conclusion, our work collectively offers insights into the components and functions of the mammalian liver portal mesenchymal cell populations, and in particular, identify and characterize PMSCs and their derived myofibroblasts, opening up the possibility for the development of novel targeted drugs or biomarkers of clinical significance with increased precision

INTRODUÇÃO: Transplantes parciais de fígado em crianças têm sido realizados com maior freqüência, enfatizando a importância do estudo da regeneração hepática, bem como dos efeitos de drogas imunossupressoras sobre a mesma. A regeneração do parênquima é o resultado do balanço entre multiplicação celular e apoptose, esta última definida como morte celular programada. Neste processo, estão envolvidas expressões de genes pró-apoptóticos (Bak e Bax) e anti-apoptóticos (Bcl-XL e Bcl-2). Dentre as proteínas relacionadas à proliferação hepatocitária, destaca-se a interleucina-6 (IL-6). Embora o modelo de ressecção de 70% da massa hepática de ratos adultos seja amplamente utilizado para estudos de regeneração, não há trabalhos com animais em crescimento. MÉTODOS: Na presente pesquisa, foi padronizado o modelo de hepatectomia parcial em ratos recém-nascidos e em recém-desmamados: realizou-se ligadura com fio de algodão do pedículo dos lobos esquerdo lateral, esquerdo medial e direito medial, seguida da ressecção do parênquima desses lobos. Os fígados remanescentes foram imediatamente pesados e comparados com os pesos dos fígados de animais controles. Para caracterização dos modelos de regeneração, 40 ratos recém-nascidos e 30 recém-desmamados foram submetidos à hepatectomia descrita e mortos nos dias subseqüentes (1, 2, 3, 4 e 7), e o fígado residual submetido a análises de peso e histologia convencional. A seguir, 36 animais (18 recém-nascidos e 18 recém-desmamados) foram divididos nos seguintes grupos: controle, cirurgia simulada e hepatectomia. Um dia após, utilizando métodos moleculares (técnica do RT-PCR), estudou-se a expressão do gene da IL-6, dos genes pró-apoptóticos e anti-apoptóticos nos fígados desses animais e, por meio de métodos imunoistoquímicos, analisou-se a presença de antígenos relacionados à proliferação celular (PCNA) e apoptose (TUNEL) em lâminas preparadas pela técnica do \"tissue microarray\". Em outros 36 ratos (18 recém-nascidos e 18 recém-desmamados), foram administradas drogas imunossupressoras (metilprednisolona, ciclosporina A ou tacrolimus, separadamente) no ato da hepatectomia, e o parênquima hepático remanescente submetido às mesmas análises moleculares e imunoistoquímicas, um dia após. RESULTADOS: A ressecção do parênquima hepático correspondeu a 70% da massa total do fígado. A mortalidade relacionada à hepatectomia nos animais recém-nascidos e recém-desmamados foi 30% e 0% respectivamente. Na análise histológica observou-se maior quantidade de mitoses em hepatócitos no terceiro dia nos recém-nascidos e no segundo dia nos recém-desmamados, com normalização da arquitetura do parênquima até o 7º dia e recuperação total do peso de ambos. No animal recém-nascido, notou-se intensa esteatose associada ao processo regenerativo. A hepatectomia provocou aumento na expressão do gene da IL-6 no fígado residual e diminuição da expressão dos genes pró-apoptóticos em ambos os modelos, além de aumento do anti-apoptótico Bcl-2 nos animais recém-desmamados. O estudo realizado sobre o efeito das drogas imunossupressoras mostrou resultados diferentes daqueles descritos em animais adultos, não havendo alteração no número de células em proliferação (PCNA positivas) ou apoptose (TUNEL positivas). As drogas não tiveram efeito sobre a expressão do gene da IL-6. Metilprednisolona e tacrolimus ocasionaram aumento da expressão do gene anti-apoptótico Bcl-2 em ambos os modelos; metilprednisolona e ciclosporina provocaram aumento na expressão do gene pró-apoptótico Bak nos ratos recém-nascidos. CONCLUSÕES: os modelos de regeneração hepática em ratos recém-nascidos e recém-desmamados foram exeqüíveis e adequados para a pesquisa; a hepatectomia promoveu estímulo da proliferação de hepatócitos com inibição da apoptose; as drogas imunossupressoras utilizadas não exerceram efeito sobre a proliferação de hepatócitos porém provocaram aumento da expressão de genes relacionados a apoptose.
INTRODUCTION: Partial liver transplantation has been performed in children with increasing frequency, and this emphasizes the importance of the studies of hepatic regeneration and the effects of immunosuppressive drugs on this phenomenon. Liver regeneration is controlled by the balance between cell proliferation and apoptosis (defined as a programmed cell death that results from the expression of pro-apoptotic genes - Bax and Bak - and anti-apoptotic genes - Bcl-2 and Bcl-XL). Among proteins related to hepatocyte proliferation, interleukin-6 is an important one. Although the adult rat model of 70% hepatectomy has been widely utilized for studies of liver regeneration, there are no studies using growing animals. METHODS: In the current paper, two experimental models were created utilizing newborn and weaning rats: using a cotton thread, the vascular hilum and the hepatic vein were ligated and the left lateral, left medial and right medial lobes were resected. The remaining liver was immediately harvested and weighted to be compared to control livers. The animals were sacrificed on days 1, 2, 3, 4, and 7 after the operation, the remnant livers were weighted and harvested for histological examinations. Then, 36 animals (18 newborn and 18 weaning animals) were divided into the following groups: control, sham and hepatectomy. One day after, the expressions of IL-6 gene, pro-apoptotic and anti-apoptotic genes were studied in the remnant livers. Immunohistochemical stainings for cell antigens related to cell proliferation (PCNA) and apoptosis (TUNEL) were also performed utilizing tissue microarray sections. In another group of 36 animals (18 newborn and 18 weaning animals), immunosuppressive drugs were administered just after the hepatectomy (methylprednisolone, cyclosporine A or tacrolimus separately), and the remnant liver submitted to the same molecular and immunohistochemical studies. RESULTS: The resected liver corresponded to 70% of the total liver weight. The mortality rates after hepatectomy were 30% and 0% for newborn and weaning rats, respectively. The histological examinations showed a great number of mitoses of hepatocytes on the third day in newborns and on the second day in weaning rats, and normalization of histological aspects by 7 days after hepatectomy and weight recuperation. In the newborn group liver regeneration was related to an intense steatosis. Hepatectomy promoted an increase in the expression of IL-6 gene of the remnant liver, a decreased expression of pro-apoptotic genes in both models, and an increased expression of anti-apoptotic Bcl-2 gene in weaning rats. The study of the effects of immunosuppressants showed different results from those described in adult animals, with no alterations in the number of cells in proliferation (PCNA positive) and apoptosis (TUNEL positive). Drugs had no effect in expression of IL-6 gene. Methylprednisolone and tacrolimus promoted an increased expression of anti-apoptotic gene Bcl-2. In addition, methylprednisolone and cyclosporine promoted an increase in the expression of the pro-apoptotic gene Bak in newborn rats. CONCLUSIONS: The experimental models were feasible and adequate for the current investigations; hepatectomy stimulated hepatocyte proliferation and inhibited hepatic cells apoptosis; the utilized immunosuppressant drugs did not affect hepatocyte proliferation although an increased expression of apoptosis-related genes was verified.

La tumorigenèse est influencée par des facteurs génétiques et environmentaux. La surnutrition est une cause d'obésité et d'accumulation pathologique de lipides dans le foie, la stéatose, qui peut évoluer en stéato-hépatite. Obésité et stéato-hépatite contribuent à l'augmentation de l'incidence du diabète dans le monde entier. Le diabète et l'obésité sont des facteurs de risque pour le cancer du foie (El-Serag et al., Clin Gastroenterol Hepatol, 2006). Cet projet de thèse élucide les mécanismes moléculaires liant l'activation de la voie de signalisation de l'insuline, les maladies du foie gras et le développement du cancer du foie, et il propose de nouvelles stratégies thérapeutiques. La délétion hépato-spécifique du gène codant le suppresseur de tumeurs Phosphatase and tensin homolog (PTEN) est un modèle de cancer du foie associé à la stéatose (Horie et al., J Clin Invest, 2004). Nous avons utilisé ce modèle, où la cascade de signalisation PI3K/mTOR est activée, pour démontrer que l'induction de l'expression du facteur de transcription et récepteur nucléaire Peroxysome Proliferator-Activated Receptor gamma (PPARγ) est responsable de la stéatose et de la glycolyse aérobie (effet Warburg). Son activité est spécifiquement dépendente de l'activité d'un effectuer en aval de la voie PI3K/mTOR, la protéine sérine/thréonine kinase, AKT2 (Panasyuk et al., Nat Comm, 2012). Sur la base de ces observations, nous avons construit l'hypothèse que PPARγ est un important régulateur de la croissance pathologique et du développement des adénocarcinomes hépatiques associés aux stéato-hépatites. Avec mon travail de thèse j'ai pu démontrer que l'expression et l'activité de PPARγ sont essentielles pour le développement du cancer du foie dans le modèle de souris invalidées pour PTEN dans les hépatocytes. Par ailleurs, la délétion de PPARγ dans le foie dépourvu de PTEN joue un rôle protecteur de la tumorigenèse, confirmant que PPARγ est effectivement placé en aval de AKT2. De plus, nous avons découvert que PPARγ est induit dans des échantillons de carcinome hépatocellulaire humains. Ces échantillons sont caractérisés par leur agressivité (haut taux prolifératif et bas niveau de différentiation) et aussi par l'activation de la voie PI3K/AKT. L'analyse des échantillons humains au stade pré-carcinome (adénomes) nous a permis de démontrer que l'ARN de PPARγ est le plus exprimé dans les adénomes caractérisés par un haut degré de stéatose. Ils sont caractérisés par la perte de fonction du facteur de transcription Hepatocyte Nuclear Factor 1α (HNF1α). Nous avons identifié HNF1α comment un nouveau régulateur négatif de la transcription de PPARγ. Nous avons aussi découvert que l'expression et l'activité de HNF1α sont inhibées par AKT2, induisant l'expression de PPARγ et son activité pro-tumorigénique. Finalement, la sensitibilité de PPARγ aux ligands, naturels et exogènes, nous a encouragé à tester des traitements pharmacologiques pour moduler son activation. La stimulation de l'activité de PPARγ avec l'agoniste synthétique Pioglitazone a conduit à une aggravation des symptomes dans le foie. Par contre, son inhibition par un antagoniste sélectif, SR2595, était thérapeutique, resultant en une réduction de signes pré-tumoraux et tumoraux dans le foie des souris invalidé par PTEN. En résumé, nos études chez l'humain et la souris, révèlent une nouvelle signature d'interaction entre les facteurs de transcription HNF1α et PPARγ et la voie de signalisation de l'insuline, suggérant de nouvelles stratégies thérapeutiques possibles pour le traitement d'une sous-classe spécifique de cancer du foie lié aux stéato-hépatites
Tumorigenesis is influenced by genetic and environmental factors. Overnutrition leads to obesity and fatty liver disease, contributing to increase diabetes incidence worldwide. Diabetes and obesity are independent risk factors for liver cancer development (El-Serag et al., Clin Gastroenterol Hepatol, 2006). This PhD project elucidates the molecular mechanisms linking activated insulin signalling pathway, fatty liver disease and liver cancer development and proposes novel therapeutic strategies. The hepatocytes-specific deletion of tumour suppressor Phosphatase and tensin homolog (PTEN) is a model of steatosis-associated liver cancer (Horie et al., J Clin Invest, 2004). Using this model of activated PI3K/mTOR signalling, our laboratory discovered that the nuclear receptor transcription factor Proliferator-Activated Receptor gamma (PPARγ) is induced in PTEN-null liver. My group demonstrated that in the liver PPARγ contributes to steatosis and aerobic glycolysis. Its activity specifically requires a downstream effector in the PI3K/mTOR pathway, the serine/threonine-specific protein kinase AKT2 (Panasyuk et al., Nat Comm, 2012). Based on these observations, we hypothesized that PPARγ might be an important regulator of pathological growth and development of steatohepatitis-associated liver adenocarcinomas. In my PhD work, I demonstrated that PPARγ expression and activity is essential for liver cancer in PTEN mutants. Moreover, PPARγ is induced in human samples of Hepatocellular Carcinoma (HCC) characterized by poor differentiation accompanied by the activation of PI3K/AKT pathway. We could attribute to PPARγ a specific role in tumour formation as it is required for abnormal liver growth and steatosis in mice at pre-tumoral age. In addition, deletion of PPARγ in PTEN mutants protected animals form liver tumorigenesis placing PPARγ downstream of activated AKT2. Analysing human samples of pre-carcinoma lesions characterized by high steatotic rate, we demonstrated that PPARγ transcript levels are increased in a specific subgroup of adenomas characterized by loss-of-function mutations in the Hepatocyte Nuclear Factor 1α (HNF1α). We identified HNF1α as a novel direct negative regulator of PPARγ transcription. We also revealed HNF1α expression and activity inhibited by AKT2 and thereby inducing PPARγ pro-tumorigenic action. Finally, the sensitivity of PPARγ to natural and exogenous ligands encouraged us to perform treatments to pharmacologically modulate PPARγ activity. Further activation of PPARγ with its synthetic ligand pioglitazione dramatically aggravates liver disease. While PPARγ inhibition by selective antagonist SR2595 allowed to reduce the pre-tumoral and tumoral signs of PTEN-null mice. In sum, our studies in men and mice reveal a novel pro-tumorigenic network of transcription factors HNF1α and PPARγ downstream of activated insulin signalling pathway, suggesting possible strategies for treatment of a subgroup of steatohepatitis-associated liver cancer

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

To examine the relationship between liver injury and the appearance of bone marrow derived hepatic cells we performed sex-mismatched bone marrow transplants in mice, with subsequent liver injury. Co-labeling for a marker of donor bone marrow origin and a marker of liver epithelial phenotype allowed us to identify rare marrow-derived hepatocytes at various time points following liver damage. The number of marrow-derived hepatocytes was low, however, and did not allow us to determine if liver-specific injury upregulated this process from baseline. We conclude that while marrow-derived hepatocytes are found, the low level of occurrence in this study makes it impossible to draw a clear temporal relationship between liver damage, recovery and the appearance of donor-derived cells. In addition, we cannot say whether liver-specific damage upregulates this phenomenon.

The incidence of Type 2 Diabetes Mellittus (T2DM) is increasing world wide. In Africa the limited access to health care and the insidious course of the disease lead to more severe illness and diabetic complications. There is a need to find alternative approaches to treatment and prevention that address the problems and needs of Africa. Sutherlandia frutescens (S.frutescens) is a traditional herbal plant with known anti-diabetic properties, the precise mechanism of action of S.frutescens is not known. In order to develop new approaches for treatment and prevention of T2DM the pathophysiology of T2DM must be understood. T2DM is the final outcome of a multi-organ disease characterized by early defects in muscle, adipocytes, hepatocytes and pancreatic β-cells. In this study the role of the liver was investigated because of its central role in glucose and lipid metabolism. It is hard to differentiate between all the influences in an in vivo model, so the aim of this study was to develop an in vitro model of T2DM in Chang liver cells and to determine if S.frutescens can reverse the state of insulin resistance in this model. Different culture media conditions were screened to identify a method that can be used as the T2DM model in Chang liver cells. Serum free medium (MCBD-201) supplemented with human diabetic serum, (2.5%-10%), high insulin concentrations (0.1μM-1μM), high fructose concentrations (1-10mM). and a combination of high insulin and high fructose was used for this screening. Chang liver cells cultured in MCBD-201 medium supplemented with 1mM fructose and 0.1μM insulin showed reduced glucose uptake and increased lipid accumulation. The effect of two S.frutescens extracts, two anti-diabetic drugs, metformin and ciglitazone, and a hypolipidemic drug ciprofibrate were determined and shown to increase glucose uptake and reduce lipid accumulation. It was postulated that exposing the cells to excess nutrients in the form of high fructose would stimulate the cells to become adipogenic and accumulate lipids, which would interfere with the glucose uptake and induce insulin resistance. Gene expression of PPARγ, PPARα, and SREBP-1 transcription factors regulating lipid metabolism was determined in Chang liver cells cultured in insulin resistance inducing medium over a 48 hour time course. The expression of PPARγ, known to stimulate adipogenesis was increased after 6, 24 and 48 hours of exposure (P(H1)<0.0001). The expression of PPARα, known to stimulate β-oxidation expression, was significantly decreased after 24 hours of exposure (P(H1)<0.0001). The presence of the plant extracts in the insulin resistance inducing media protect against this increase in adipogenesis and decrease in β-oxidation after 48 hours of exposure by increasing PPARα expression and decreasing PPARγ expression. A PCR Array was performed which identified 32 more potential molecular targets of S.frutescens. Five of the 32 targets identified with the PCR Array were validated using qRT-PCR. These genes play a role in lipid and glucose metabolism and protection against oxidative stress and inflammation. In summary a cellular model of insulin resistace in hepatocytes has been established and the capacity of S.frutescens to reverse this process has been demonstrated by acting as a dual PPARγ/α agonist. New genes have been identified in the development of insulin resistance and as targets of S.frutescens.

End stage liver disease represents a common end point for a number of disease processes, the only current treatment for which is liver transplantation, but the demand for donor organs exceeds the supply. This has led to the use of more marginal donors with an increase in the rates of complications. Mesenchymal stem/stromal cells (MSC) are a multipotent cell capable of modulating the immune system through a number of different processes and represent a potential therapy in post transplantation liver injury. In this study I describe the prospective isolation and culture expansion of murine MSC. In in vitro assays MSC suppressed T lymphocytes and following stimulus with inflammatory cytokines MSC secreted a number of cytokines including Il-10. In the MDR2-/- model intravenous MSC therapy led to a reduction in liver injury with an increase in restorative macrophages. Subcutaneous administration of MSC showed no beneficial effect. MSC were also tested in a hepatic ischaemia reperfusion injury model where no effect was seen. In summary MSC were able to suppress lymphocyte proliferation and secrete anti-inflammatory cytokines in vitro, and in vivo they were able to reduce liver injury in the MDR2-/- model but not the hepatic ischaemia reperfusion injury mode.

The unfolded protein response (UPR) is activated by protein misfolding stress in the endoplasmic reticulum (ER). The UPR is a transcriptional program that aims to maintain ER folding capacity, where imbalances between protein load and processing ability is termed ER stress. Signal transduction of the UPR begins with 3 ER-resident transmembrane sensors: PERK, IRE1 and ATF6. All sensors initiate downstream signaling cascades which culminate in improved protein folding, transcriptional upregulation of genes encoding ER chaperones, and mechanisms to reduce translational and transcriptional ER load, therefore re-establishing ER homeostasis. The signaling cascades of each sensor are distinct but cooperative, and involve a significant amount of crosstalk, feedback and overlap. Indeed, there are many pathological and physiological conditions have an effect on ER protein burden, and therefore on activation of the UPR. Increases in protein load in professional secretory cells, hypoxic conditions in a tumor mass, obesity all induce cause changes in the ER folding environment. Although we understand how the UPR contributes to relieve ER stress under acute conditions (e.g. pharmacological treatment) much less is understood about the contributions to physiological processes and chronic stress conditions. Our overall goal was to understand how the UPR is activated during physiological settings, the mechanisms it uses to maintain folding capacity under these setting and the specific components responsible for adapting the response to various stresses. We first decided to understand a chronic stress from a transgenic approach. By creating a knockout mouse, the genetic deletion functions as a stress and we can understand its physiological role. By compounding two genetic deletions in UPR components (ATF6α and p58IPK) we provide evidence for the developmental role these components play. Homozygous deletion ATF6α bears no gross histological phenotype yet causes synthetic lethality when combined with p58IPK deletion. This also reveals that the UPR is able to adapt to genetic impairment of protein folding in vivo. Next, to better understand these chronic states, we established an experimentally tractable chronic stress treatment in vivo. Our treatment suppressed ATF6α dependent chaperone expression through an mRNA degradative mechanism, which led to long term changes in UPR expression. We determined that chronic conditions can change the sensitivity of the UPR to ER stress, potentially as an adaptive consequence. We also showed that sensitivity to ER stress can be changed during chronic stress. Finally we simulated the UPR in a computational ordinary differential equation (ODE) model in order to determine how various stresses and component interactions determine the output of the UPR. We built a series of equations to describe the UPR signaling network, entrained it on experimental data and refined it through the use of transgenic knockout cells. Our model was robust enough to recreate experimental measurements of UPR components when tested in parallel with knockout cells. We found that stress sensitivity is dependent on the crosstalk and negative feedback connections of the UPR. This study has enhanced our understanding of activation of the UPR under non-acute settings. It demonstrates that the UPR is a signaling hub with a broad output range that is capable of handling a variable degree of insults because of the intrinsic properties of the signaling network. This provides a better understanding for the contributions of the UPR to physiological stresses and certain chronic diseases.

A state of chronic metabolic inflammation and activation of the cell stress response in organs such as liver and adipose tissue are important pathogenic adaptations with the onset of obesity and the metabolic syndrome. The extent to which these processes are modulated by the early life nutritional experience is not well established, especially in large animal models. The overall aim of this thesis was to identify whether nutritional programming during prenatal and postnatal development enhances metabolic inflammation and cell stress response of obesity. A nutritional model of fetal growth restriction achieved by maternal nutrient restriction (NR) to 60% of requirements during late gestation (110 days to term at 147 days) in twin bearing sheep was used. Combination of prenatal and postnatal nutritional interventions were studied with the following three study protocols: 1. Offspring of twin bearing sheep born to mothers nutrient restricted or fed to appetite were separated after weaning at 3 months of age and then exposed to either restricted physical activity leading to obesity or to unrestricted activity and remained lean. 2. Following maternal NR, both twins or only one twin were reared on their mother’s milk during suckling period in order to achieve a relatively faster growth rate in the latter. 3. Twin offspring of sheep randomised to NR or feeding to requirement during late gestation were separated after birth and randomised to either formula feeding or being fed by the mother until weaning followed by obesogenic rearing. Total body weight of sheep in the obese group was raised by ~30% and was unaffected by any intervention. Obesity led to an increased insulin response to the glucose tolerance test, together with hepatic triglyceride deposition, and adipocyte hypertrophy with macrophage infiltration in omental adipose tissue. NR exacerbated obesity associated hepatic triglyceride deposition and upregulated gene expression of hepatic autophagy and omental unfolded protein response. Formula feeding of sheep offspring following NR was associated with slower weight gain and decreased gene expression for MTOR. Sheep offspring fed by mother as singleton gained weight at faster rate during suckling period as compared to offspring fed by their mothers as twins. Neither postnatal interventions exacerbated the state of obesity associated metabolic inflammation and cell stress response. It is possible that the increased hepatic autophagic gene expression is a reflection of defective autophagy and future work should include study of markers of autophagic function. Possible mechanisms of upregulated omental adipose UPR in offspring of sheep undergoing NR could include a programmed decrease in adipocyte number or selective survival of preadipocytes with effective ER stress response. Such adaptations followed by obesity would predispose the adipocytes to initiate inflammation and cell death pathways.

Este trabalho apresenta um método numérico para simular escoamentos viscoelásticos bidimensionais governados pela equação constitutiva Giesekus [Schleiniger e Weinacht 1991]. As equações governantes são resolvidas pelo método de diferenças finitas numa malha deslocada. A superfície livre do fluido é modelada por partículas marcadoras possibilitando assim a sua visualização e localização. O cálculo da velocidade é efetuado por um método implícito enquanto a pressão é calculada por um método explícito. A equação constitutiva de Giesekus é resolvida pelo método de Euler modificado explícito. O método numérico desenvolvido nesse trabalho é verificado comparando-se a solução numérica com a solução analítica para o escoamento de um fluido Giesekus em um canal. Resultados de convergência são obtidos pelo uso de refinamento de malha. Os resultados alcançados incluem um estudo da aplicação do modelo Giesekus para simular o escoamento numa contração planar 4:1 e o problema de um jato incidindo sobre uma placa rígida, em que o fenômeno jet buckling é simulado.
This work presents a numerical method to simulate two-dimensional viscoelastic flows governed by the Giesekus constitutive equation [Schleiniger e Weinacht 1991]. The governing equations are solved by the finite difference method on a staggered grid. The free surface of the fluid is modeled by tracer particles thus enabling its visualization and location. The calculation of the velocity is performed by an implicit method while pressure is calculated by an explicit method. The Giesekus constitutive equation is resolved by the explicit modified Euler method. The numerical method developed in this work is verified by comparing the numerical solution with the analytical solution for the flow of a Giesekus fluid in a channel. Convergence results are obtained by the use of mesh refinement. Results obtained include a study of the application of the Giesekus model to simulate the flow through a 4:1 contraction and the problem of a jet flowing onto a rigid plate where the phenomenon of jet buckling is simulated.

Thesis (Ph.D.)--University of Florida, 2005.
Typescript. Title from title page of source document. Document formatted into pages; contains 162 pages. Includes Vita. Includes bibliographical references.

In vitro disease models are powerful platforms for the development of drugs and novel therapies. Stem-cell based approaches have emerged as cutting-edge tools in disease modelling, allowing for deeper insights into previously unknown disease mechanisms. Hence the significant role of these disease-in-a-dish methods in therapeutics and translational medicine. Impedance sensing is a non-invasive, quantitative technique that can monitor changes in cellular behaviour and morphology in real-time. Bioimpedance measurements can be used to characterize and evaluate the establishment of a valid disease model, without the need for invasive end-point biochemical assays. In this work, two stem cell-based disease models-on-a-chip are proposed for acute liver failure (ALF) and age-related macular degeneration (AMD). The ALF disease model-on-a-chip integrates impedance sensing with the highly-differentiated HepaRG cell line to monitor in real-time quantitative and dynamic response to various hepatotoxins. Bioimpedance analysis and modelling has revealed an unknown mechanism of paracetamol hepatotoxicity; a temporal, dose-dependent disruption of tight junctions (TJs) and cell-substrate adhesion. This disruption has been validated using ultrastructural imaging and immunostaining of the TJ-associated protein ZO-1. Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world with a need for disease models for its currently incurable forms. Human induced pluripotent stem cells (hiPSCs) technology offers a novel approach for disease modelling, with the potential to impact translational retinal research and therapy. Recent developments enable the generation of Retinal Pigment Epithelial cells from patients (hiPSC-RPE), thus allowing for human retinal disease in vitro studies with great clinical and physiological relevance. In the current study, the development of a tissue-on- a-chip AMD disease model has been established using RPE generated from a patient with an inherited macular degeneration (case cell line) and from a healthy sibling (control cell line). A reproducible Electric Cell-substrate Impedance Sensing (ECIS) electrical wounding assay was conducted to mimic RPE damage in AMD. First, a robust and reproducible real-time quantitative monitoring over a 25-day period demonstrated the establishment and maturation of RPE layers on microelectrodes. A spatially-controlled RPE layer damage that mimicked cell loss in AMD was then initiated. Post recovery, significant differences in migration rates were found between case and control cell lines. Data analysis and modelling suggested this was due to the lower cell-substrate adhesion of the control cell line. These findings were confirmed using cell adhesion biochemical assays. Moreover, different-sized, individually-addressed square microelectrode arrays with high spatial resolution were designed and fabricated in-house. ECIS wounding assays were performed on these chips to study immortalized RPE migration. Migration rates comparable to those obtained with ECIS circular microelectrodes were determined. The two proposed disease-models-on-a-chip were then used to explore the therapeutic potential of the antioxidant N-Acetyl-Cysteine (NAC) on hiPSC-RPE and HepaRG cell recovery. Addition of 10 mM NAC at the end of a 24h paracetamol challenge caused a slight increase in the measured impedance, suggesting partial cell recovery. On the other hand, no effect on case hiPSC-RPE migration has been observed. More experiments are needed to examine the effect of different NAC concentrations and incubation periods. The therapeutic potential of electrical stimulation has also been explored. A preliminary study to evaluate the effect of electrical stimulation on RPE migration has been conducted. An externally applied direct current electric field (DC EF) of 300 mV/mm was found to direct the migration of the immortalized RPE cell line (hTERT-RPE1) perpendicular to the EF. The cells were also observed to elongate and to realign their long axes perpendicular to the applied EF. The proposed tissue-on-a-chip disease models are powerful platforms for translational studies. The potential of such platforms has been demonstrated through revealing unknown effects of acetaminophen on the liver as well as providing deeper insights into the underlying mechanisms of macular degeneration. Combining stem cell technology with impedance sensing provides a high throughput platform for studying patient-specific diseases and evaluating potential therapies.

Este trabalho teve como objetivo desenvolver um protocolo para a diferenciação in vitro de células-tronco mesenquimais (CTM) em hepatócitos e a padronização de um modelo animal de fibrose hepática induzida por dimetilnitrosamina (DMN) para ensaios pré-clínicos de transplante de CTM. CTM isoladas de fontes variadas apresentaram morfologia fibroblastóide e aderência ao plástico e o padrão de marcadores de superfície celular esperado na análise por citometria de fluxo. A capacidade de diferenciação osteogênica e adipogênica dessas células foi comprovada pelas colorações de vermelho de alizarina, oil red e azul de toluidina, respectivamente, confirmando, que as células isoladas para este estudo se comportaram como CTM conforme proposto pela Sociedade Internacional de Pesquisa em Células-tronco. A diferenciação hepática foi avaliada quanto à morfologia e capacidade das células diferenciadas de estocar glicogênio confirmada por PAS (ácido periódico-Schiff), de sintetizar albumina confirmada por imunofluorescência, além da capacidade de expressar genes hepato-específicos verificada por ensaios de PCR em tempo real. Com base na literatura para diferenciação hepática, diferentes protocolos de um, dois e três passos foram testados. CTM humanas mostraram capacidade de produzir e estocar glicogênio e de sintetizar albumina, apenas quando diferenciadas com protocolos de três etapas, porém sem uma expressão aumentada dos genes hepato-específicos albumina, α-fetoproteína e c-Met. Uma etapa de diferenciação endodérmica, previamente aplicada à diferenciação hepática, aumentou a capacidade de produzir e estocar glicogênio das CTM diferenciadas. Para a padronização do modelo de fibrose hepática induzida por DMN, foram realizados experimentos de dose-resposta e foi verificado o efeito da hepatectomia em modelos mistos DMN/hepatectomia. A injúria hepática e o efeito do transplante de CTM foram avaliados por análise macroscópica dos fígados, histologia das biópsias de fígados corados com HE e tricromo de Masson e parâmetros bioquímicos séricos. Alterações macroscópicas, histológicas e nos níveis séricos de fosfatase alcalina indicam a indução da fibrose hepática nos ratos Wistar tratados com DMN na dose de 10 µg/g de peso animal por três dias consecutivos durante quatro semanas, mas não observamos nenhum efeito induzido pela hepatectomia. Porém, este modelo com DMN se mostra semelhante a estágios iniciais de uma fibrose hepática. O transplante de 1 x 107 CTM de veia de cordão umbilical humano (VCUH) no modelo de injúria hepática induzida por DMN não resultou em melhora da fibrose, diminuição dos níveis séricos de fosfatase alcalina e nem em ganho de peso dos animais quando comparados aos animais tratados com PBSA após a injúria hepática (grupo placebo). Em conjunto, esses resultados sugerem que CTM humanas se diferenciam após tratamentos mais complexos, onde os indutores hepatogênicos são sequencialmente adicionados ao meio de modo a mimetizar a sinalização durante o desenvolvimento embrionário. O transplante de CTM de VCUH parece não ter efeito positivo em um modelo pré-clínico de injúria hepática similar a estágios iniciais de fibrose. Financiado por CNPq (573578/2008-7) e FAPESP (2007/54260-2).
This study aimed to develop an in vitro differentiation protocol of mesenchymal (MSC) stem cells to hepatocytes and to standardize an animal model for hepatic fibrosis induced by dimethylnitrosamine (DMN) for preclinical transplant assays of MSC. MSC isolated from various sources presented fibroblastoid morphology, plastic adherence, and the expected pattern of cell surface markers by flow cytometry analysis. The capacity of osteogenic, adipogenic and chondrogenic differentiation of these cells was confirmed by alizarin red, oil red and toluidine blue staining, respectively, confirming that the cells isolated for this study behave as MSC, as proposed by the International Society for Stem Cell Research. Hepatogenic differentiation was evaluated by analysis of cell morphology, capacity to store glycogen confirmed by PAS (periodic acid-Schiff), albumin synthesis confirmed by immunofluorescence, as well as hepatic-specific gene expression verified by real time PCR assays. Based on the published literature on hepatic differentiation, several protocols of one, two, and three steps were tested. Human MSC differentiated solely when treated in a three step-protocol, showing the ability to produce and store glycogen and synthesize albumin; however the expression of hepatic-specific genes such as albumin, α-fetoprotein and c-Met was not increased. An endoderm differentiation stage, added to the hepatic differentiation protocol, increased the capacity to produce and store glycogen of differentiated MSC. In order to standardize the model of liver fibrosis induced by DMN, dose-response experiments were performed and the effect of hepatectomy in mixed models DMN/hepatectomy was observed. Severity of liver injury and the effect of cell transplantation were evaluated by macroscopic analysis of the livers, histology of liver biopsies stained with HE and Masson\'s trichrome, and evaluation of serum biochemical parameters. The macroscopic and histological observations, and altered alkaline phosphatase serum levels indicated the success in inducing liver fibrosis in DMN-treated rats at a dose of 10 µg/g of animal weight for three consecutive days, during four weeks, without any additional effect upon hepatectomy. Transplanting 1 x 107 umbilical cord MSC in the model of liver injury induced by DMN did not result in improvement of the fibrosis, decrease of alkaline phosphatase serum levels, or in weight gain of the treated animals compared to animals treated with PBSA after liver injury (placebo group). Together, these results suggest that human MSC are capable of differentiating to hepatocyte-like cells after more complex protocols, where hepatogenic inducers are sequentially added to the medium in order to mimic signaling that occurs during fetal development. Transplantation of undifferentiated umbilical cord MSC did not have any positive effect in a preclinical liver injury model characterized by an early stage of fibrosis. Supported by CNPq (573578/2008-7) and FAPESP (2007/54260-2).

Background & Aims: Despite advances in surgical care and chemotherapeutic regimens, the five-year survival rate for Stage IV Hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. YAP1 and β-Catenin co-activation occurs in 80% of children’s HB; however, a lack of conditional genetic models precludes exploration of tumor maintenance and therapeutic targets. Thus, the clinical need for a targeted therapy remains unmet. Given the predominance of YAP1 and β-catenin activation in children’s tumors, I sought to evaluate YAP1 as a therapeutic target in HB. Approach & Results: Herein, I engineered the first conditional murine model of HB using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A, constitutive β-CateninDelN90, and a luciferase reporter to murine liver. Tumor regression was evaluated using in vivo bioluminescent imaging, and tumor landscape characterized using RNA sequencing, ATAC sequencing and DNA foot-printing. Here I show that YAP1 withdrawal in mice mediates >90% tumor regression with survival for 230+ days. Mechanistically, YAP1 withdrawal promotes apoptosis in a subset of tumor cells and in remaining cells induces a cell fate switch driving therapeutic differentiation of HB tumors into Ki-67 negative “hbHep cells.” hbHep cells have hepatocyte-like morphology and partially restored mature hepatocyte gene expression. YAP1 withdrawal drives formation of hbHeps by modulating liver differentiation transcription factor (TF) occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and can rescue liver damage in mice. Conclusions: YAP1 withdrawal, without modulation of oncogenic β-Catenin, significantly regresses hepatoblastoma, providing the first in vivo data to support YAP1 as a therapeutic target for HB. Modulating YAP1 expression alone is sufficient to drive long-term regression in hepatoblastoma because it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.

Le diabète mellitus, également désigné comme la maladie du siècle, est une pathologie mortelle qui affecte le système endocrinien. Les mécanismes liés à la rupture de la boucle de rétroaction, qui régule le métabolisme et induit le diabète, ne sont pas entièrement connus. La compréhension des mécanismes d'action de l'insuline est donc essentielle pour le développement de stratégies thérapeutiques efficaces afin du lutter contre cette maladie. Par conséquent, il est impératif de trouver un modèle robuste et fiable, capable de surmonter les limites de la culture cellulaire traditionnelle en 2D et de l'expérimentation animale, pour la recherche sur le diabète. L'objectif de cette thèse est de développer un nouveau modèle de co‐culture foie‐pancréas en utilisant des systèmes microphysiologiques avancés (MPs) afin d’aborder plus efficacement le mécanisme impliqué dans la régulation endocrinienne hépatique et pancréatique. Ce travail met en évidence la capacité des systèmes multi‐organes sur puce qui combinent la compartimentation avancée des cellules en 3D, la microfluidique et la technologie des cellules souches pluripotentes induites (iPSC), pour atteindre une complexité biologique élevée et des fonctions rarement reproduites par une seule de ces technologies d’ingénierie tissulaire
Diabetes mellitus (DM) or the so called disease of the century is a life threatening dysfunction that affects the endocrine system. The mechanisms underlying the break in the feedback loop that regulates the metabolism and the consequent diabetes induction are not fully known. Understanding the mechanisms of insulin action is therefore crucial for the further development of effective therapeutic strategies to combat DM. Accordingly, it is imperative to find a robust and reliable model for diabetes research able to overcome the limitations of traditional 2D in vitro cell culture and animal experimentation. The aim of this thesis is to develop a new liver‐pancreas co‐culture model using advanced microphysiological systems (MPs) to tackle more effectively the mechanism involving the hepatic and pancreatic endocrine regulation. This work highlights the power of multi organ‐on‐chip systems that combines the advanced 3D‐cell compartmentalization, microfluidics and induced pluripotent stem cells (iPSC) technology to achieve a high biological complexity and functions that are rarely reproduced by only one of these tissue engineering technologies

Uveal melanoma is the most common primary malignant intraocular tumour in adults and despite advances in treatment of the primary tumour, the 10-year survival rate remains unchanged. The most frequent cause of death for patients of this disease is liver metastases. Removal of the primary tumour before clinical presentation of metastases, however, has no effect on patient outcome.
In order to understand the interactions between single malignant cells or sub-clinical metastases and affected organs, we have successfully developed a novel animal model of uveal melanoma. We utilized the unique properties of green fluorescent protein, a skin-flap in vivo imaging technique, and nude mice to accomplish this goal. The precision of green fluorescent protein imaging has allowed us to observe single cells interacting with organ tissues and reveal that these malignant cells are only capable of surviving in the liver.