The high-mobility group A protein 2 (HMGA2) is a non-histone chromatin factor highly expressed in fetal tissue and malignant tumors but rarely detected within normal adult tissues. The clinical implications and biological functions of HMGA2 in endometrial carcinoma are largely unknown. Here we report that HMGA2 expression was barely detected in benign endometrium samples (2 of 28 samples). However, HMGA2 expression increased significantly from precancerous lesion endometrial glandular dysplasia (7 of 17, 41.2%), to serous endometrial intraepithelial carcinoma (5 of 8, 62.5%) and to full blown endometrial serous carcinoma (39 of 59, 66.1%). Functional characterization of HMGA2 revealed that the gene has both tumor growth promotion and metastasis. In addition, HMGA2 induced epithelial-mesenchymal transition (EMT) through modulation vimentin and β-catenin. Furthermore, HMGA2 overexpression started from endometrial serous precancers, non-invasive cancers, as well as in full blown carcinomas in a p53 knockout mouse model we recently established in our laboratory. Our findings suggest that HMGA2 may serve as a useful diagnostic marker in the assessment of endometrial serous cancer and its precursor lesions.

Epithelial to Mesenchymal Transition (EMT) is a cellular transformation process that is employed repeatedly and ubiquitously during vertebrate morphogenesis to build complex tissues and organs. Cellular transformations that occur during cancer cell invasion are phenotypically similar to developmental EMT, and involve the same molecular signalling pathways. EMT processes are diverse, but are characterised by: a loss of cell-cell adhesion; a gain in cell-matrix adhesion; an increase in cell motility; the secretion of proteases that degrade basement membrane proteins; an increased resistance to apoptosis; a loss of polarisation; increased production of extracellular matrix components; a change from a rounded to a fibroblastic morphology; and an invasive phenotype. This thesis focuses explicitly on endocardial EMT, which is the EMT that occurs during vertebrate embryonic heart development. The embryonic heart initially forms as a tube, with myocardium externally, endocardium internally, with these tissue layers separated by a thick extracellular matrix termed the cardiac jelly. Some of the endocardial cells in specific regions of the embryonic heart tube undergo EMT and invade the cardiac jelly. This causes cellularised swellings inside the embryonic heart tube termed the endocardial cushions. The emergence of the four chambered double pump heart of mammals involves a complex remodelling that the endocardial cushions play an active role in. Even while heart remodelling is taking place, the heart tube is operating as a single-circulation pump, and the endocardial cushions are performing a valve-like function that is critical to the survival of the embryo (Nomura-Kitabayashi et al. 2009). As the endocardial cushions grow and remodel, they become the valve leaflets of the foetal heart. The endocardial cushions also contribute tissue to the septa (walls) of the heart. Their correct formation is thus essential to the development of a fully functional, fully divided, double-pump system. It has been shown that genetic mutations that cause impaired endocardial EMT lead to the development of a range of congenital heart defects (Fischer et al. 2007). An extensive review is conducted of existing experimental investigations into endocardial EMT. The information extracted from this review is used to develop a multiscale conceptual model of endocardial EMT, including the major protein signalling pathways involved, and the cellular phenotypes that they induce or inhibit. After considering the requirements for computational simulations of EMT, and reviewing the various techniques and simulation packages available for multi-cell modelling, cellular Potts modelling is selected as having the most appropriate combination of features. The open source simulation platform Compucell3D is selected for model development, due to the flexibility, range of features provided and an existing implementation of multiscale models; that include subcellular models of reaction pathways. Based on the conceptual model of endocardial EMT, abstract computational simulations of key aspects are developed, in order to investigate qualitative behaviour under different simulated conditions. The abstract simulations include a 2D multiscale model of Notch signalling lateral induction, which is the mechanism by which the embryonic heart tube is patterned into cushion and non-cushion forming regions. Additionally, a 3D simulation is used to investigate the possible role of contact-inhibited mitosis, upregulated by the VEGF protein, in maintaining an epithelial phenotype. One particular in vitro investigation of endocardial EMT (Luna-Zurita et al. 2010) is used to develop quantitative simulations. The quantitative data used for fitting the simulations consist of cell shape metrics that are derived from simple processing of the imaging results. Single cell simulations are used to investigate the relationship between cell motility and cell shape in the cellular Potts model. The findings are then implemented in multi-cell models, in order to investigate the relationship between cell-cell adhesion, cell-matrix adhesion, cell motility and cell shape during EMT.

The transforming growth factor beta (TGFβ) is a cytokine that regulates a plethora of cellular processes such as cell proliferation, differentiation, migration and apoptosis. TGFβ signals via serine/threonine kinase receptors and activates the Smads to regulate gene expression. Enigmatically, TGFβ has a dichotomous role as a tumor suppressor and a tumor promoter in cancer. At early stages of tumorigenesis, TGFβ acts as a tumor suppressor by exerting growth inhibitory effects and inducing apoptosis. However, at advanced stages, TGFβ contributes to tumor malignancy by promoting invasion and metastasis. The pro-tumorigenic TGFβ potently triggers an embryonic program known as epithelial-mesenchymal transition (EMT). EMT is a dynamic process whereby polarized epithelial cells adapt a mesenchymal morphology, thereby facilitating migration and invasion. Downregulation of cell-cell adhesion molecules, such as E-cadherin and ZO-1, is an eminent feature of EMT. TGFβ induces EMT by upregulating a non-histone chromatin factor, high mobility group A2 (HMGA2). This thesis focuses on elucidating the molecular mechanisms by which HMGA2 elicits EMT. We found that HMGA2 regulates a network of EMT transcription factors (EMT-TFs), such as members of the Snail, ZEB and Twist families, during TGFβ-induced EMT. HMGA2 can interact with Smad complexes to synergistically induce Snail expression. HMGA2 also directly binds and activates the Twist promoter. We used mouse mammary epithelial cells overexpressing HMGA2, which are mesenchymal in morphology and highly invasive, as a constitutive EMT model. Snail and Twist have complementary roles in HMGA2-mesenchymal cells during EMT, and tight junctions were restored upon silencing of both Snail and Twist in these cells. Finally, we also demonstrate that HMGA2 can epigenetically silence the E-cadherin gene. In summary, HMGA2 modulates multiple reprogramming events to promote EMT and invasion.

This thesis investigates modeling of modular multilevel converters (MMCs) using an averaging method known as extended-frequency dynamic phasors. An MMC can be used as an inverter or a rectifier in high voltage direct current (HVDC) system. This research develops a dynamic phasor model for an MMC operated as an inverter. Extended-frequency dynamic phasors are used to model a system with only interested harmonics present. The developed model is capable of capturing both the low and high-frequency dynamic behavior of the converter depending on the requirements of the study to be performed. The selected MMC model has 5 submodules per arm (6-level converter), nearest level control, capacitor voltage balancing, direct control and phase-locked loop (PLL) synchronization. With the above features, the developed dynamic phasor model is validated with electromagnetic transient model is developed using PSCAD simulation software. The results are compared at transient and steady state with disturbances. The main computational advantage of this modeling is achieving less simulation time with inclusion of harmonics of interest. / October 2016

Platelets play a crucial role for blood hemostasis, the process that prevents bleeding. In addition, platelets have been demonstrated to promote cancer progression and cancer related complications like metastasis and thrombosis. Platelets can affect cancer related diseases either directly or by interacting with other blood cells or molecules in the circulation of individuals with cancer. The current thesis addresses the role of platelets in tumor progression and tumor-induced systemic effects of cancer, with a special focus on the effects on the vasculature. In the first paper, the role of platelets in tumor progression in histidine-rich glycoprotein (HRG)-deficient mice was addressed. We report that HRG-deficient mice show enhanced tumor growth, epithelial to mesenchymal transition (EMT) and metastasis. The enhanced platelet activity in the absence of HRG is responsible for the accelerated tumor progression. In the second paper, we demonstrate that platelet-derived PDGFB is a central player to keep the tumor vessels functional. Moreover, in a pancreatic neuroendocrine carcinoma model with PDGFB-deficient platelets, spontaneous liver metastasis was enhanced. With this finding we identify a previously unknown role of platelet derived PDGFB. In the third paper, we found that TBK1 mediates platelet-induced EMT by activation of NF-kB signaling, which suggest that TBK1 contributes to tumor invasiveness in mammary epithelial tumors. In the last paper, we report that the vascular function in organs that are neither affected by the primary tumor, nor represent metastatic sites, is impaired in mice with cancer. We show that tumor-induced formation of intravascular neutrophil extracellular traps (NETs), a fibril matrix consisting of neutrophils with externalized DNA and histones, granule proteases and platelets, are responsible for the impaired peripheral vessel function.

No description available.

High mobility group A2 (HMGA2) is a non-histone protein highly expressed during the development but is low or absent in most adult tissues. Epithelial-mesenchymal transition (EMT) plays a critical role in prostate cancer progression and metastasis. HMGA2 has been shown to promote EMT in separate studies. Interestingly, wild-type HMGA2 and truncated (lacking the 3’UTR) HMGA2 isoforms are overexpressed in many cancers. However, there are no studies on the role of each isoform in prostate cancer progression. We hypothesized that wild-type and truncated HMGA2 promotes prostate cancer progression by different mechanisms. We analyzed the expression of HMGA2 in the prostate panel by western blot analysis and the localization in prostate tissue microarray by immunohistochemistry. We stably overexpressed wild-type and truncated HMGA2 cDNA in LNCaP cells and measured the expression and the localization of HMGA2 as well as EMT markers. We also performed the migration and cell viability assays. We analyzed phospho-ERK in cells overexpressing HMGA2 as well as inhibition with U0126 (MAPK inhibitor). To explore the role of truncated HMGA2, we measured the reactive oxygen species (ROS) concentration by DCFDA dye, as well as analyzing Jun-D as a putative downstream effector of HMGA2. Additionally, we knocked down Jun-D and performed the migration and cell viability assays. We treated ARCaP-M mesenchymal cells with camalexin, a 3-thizol-2-yl-indole (a natural product, as a candidate to target HMGA2) in vitro and in vivo in nude mice. Our results showed an increase in nuclear HMGA2 expression with prostate cancer progression as compared to normal tissue. LNCaP cells overexpressing wild-type but not truncated HMGA2 displayed nuclear localization and induced EMT via the ERK1/2 pathway, and this effect could be reversed by treating the cells with U0126. Conversely, truncated HMGA2 displayed cytoplasmic expression and increased prostate cancer migration via increasing Jun-D expression and ROS; this could be antagonized by Jun-D knockdown. Finally, treating ARCaP-M aggressive prostate cancer cells with camalexin reduce its expression in vitro and in vivo. In conclusion, both wild-type and truncated HMGA2 induce prostate cancer progression by different mechanisms which may be targeted by camalexin.

The Wilms' tumour suppressor gene Wt1 is a key regulator of embryonic development and tissue homeostasis. In humans, mutation in the gene may lead to childhood kidney cancer, severe glomerular kidney diseases, gonadal dysgenesis and, in rare cases, heart diseases. The importance of WT1 in embryonic development is related to its crucial function in the regulation of two cellular plasticity processes: the epithelial to mesenchymal transition (EMT) and the reverse process, the mesenchymal to epithelial transition (MET). WT1 expression persists during the waves of EMT and MET that generate certain mesodermal tissues. In fact, WT1 is a major regulator of both transitions and it is essential for the survival of mesenchyme progenitors. Furthermore, it has been proposed that WT1 is required for the derivation of progenitors from different mesothelia, possibly through an EMT. Progenitors expressing WT1 are believed to differentiate into different cell types, giving rise to coronary vasculature, adipocytes and hepatic stellate cells. In my PhD I aimed to investigate the instructive role of different WT1 isoforms. To address this, the first goal was to generate a single plasmid that would accommodate all necessary components of an inducible system, in order to derive cellular models for the inducible expression of WT1 single isoforms. Second, I aimed to understand the processes that the single variants were sufficient to drive. Therefore, I started with the establishment of two cellular models for the inducible expression of the main four isoforms of WT1 (with or without the exon 5 and/or the KTS, here referred as +/+, +/-, -/+ and -/-). I cloned different plasmids carrying doxycycline inducible WT1 isoforms and derived single stable clones in two epithelial kidney cell lines that do not express WT1: the MDCK and the IMCD3 cells. I then analysed the expression profiles of the clones, using either microarray or RNA sequencing, and performed cellular assays to characterize the cells after WT1 induction. Overall, WT1 induction did not affect cell growth, cell cycle, cell migration or anchorage independent growth, suggesting that the expression of WT1 in these two cell lines does not change their oncogenic potential. The expression analysis of the MDCK cells suggested that the induction of WT1 isoforms activates an inflammatory response, leading to the overexpression of several cytokines. Moreover, the -/+ isoform speciffically caused the upregulation of fibrotic markers and the rearrangement of the actin cytoskeleton. Interestingly, the expression of the mesothelial marker UPK3B increased following the induction of the -/+ isoform. Because the expression of the -/+ variant led to the most signifficant isoform-specific changes in both cell lines, I focused on this isoform for the validation of the transcriptomic data of the IMCD3 cells. I confirmed that the induction of WT1 -/+ in the IMCD3 cells leads to the upregulation of fibrotic markers, increases cell adhesion and activates the AKT and MAPK pathways. Moreover, there was a significant overexpression of different mesothelial markers and, importantly, of key regulators and markers of developmental processes, such as adipogenesis, skeletal and cartilage development, as well as angiogenesis. I then dissected the timing of expression of some specific markers and regulators, analysing the levels of the genes at different time points after WT1 -/+ induction. The preliminary results intimate that WT1 -/+ might induce epithelial cells in the direction of cartilage-skeletal tissue and fat, possibly through a mesothelial intermediate. The data also suggest that the induction of this isoform initiates an EMT, possibly followed by an MET, as the levels of expression of the differentiation markers and regulators increase. To validate the proposed instructive role of WT1, it will be of crucial importance to determine both RNA and protein levels of markers and regulators at even later time points, both in IMCD3 cells and in a model of inducible embryonic stem cells, which is currently under development. In the future, it will be important to address the relevance of these findings in vivo and to dissect the molecular mechanisms.

Au cours de la progression des cancers dorigine épithéliale, on observe une disparition des jonctions intercellulaires et une réorganisation de leurs composants. Par ailleurs, cette progression tumorale saccompagne également dune surexpression de certaines chimiokines par les cellules tumorales. Dans ce travail, nous nous sommes attachés à étudier la régulation potentielle de ces chimiokines par certaines molécules dadhérence. Nous avons plus particulièrement examiné linfluence de la caténine beta et de ZO-1 sur lexpression des chimiokines étant donné leur particularité de pouvoir effectuer la navette entre la membrane et le noyau et leur implication dans des voies de signalisation. Dans un premier chapitre de résultats (chapitre III.1.1), nous rapportons notre étude concernant la régulation de MCP-1/CCL2 par la voie de signalisation caténine beta Publication 1 Nos travaux détaillant la régulation de lIL-8/CXCL8 par ZO-1 font lobjet dun second chapitre de résultats (chapitre III.1.2) Publication 2 Parallèlement à notre axe principal de recherche centré sur la régulation de lexpression des chimiokines, nous avons également participé à des travaux montrant linfluence de la voie de signalisation caténine beta sur la régulation de la vimentine lors de la transition épithélio-mésenchymateuse (TEM) associée à la progression tumorale. Un troisième chapitre de résultats est consacré à lexposé de ces travaux (chapitre III.2). Publications 3, 4, 5

Epithelial mesenchymal/-myofibroblast transition (EMT/EMyT) has emerged as one of the central mechanisms in wound healing and tissue fibrosis. The main feature of EMyT is the activation of a myogenic program, leading to the induction of the α-smooth-muscle actin (SMA) gene in the transitioning epithelium. Recent research suggests that intercellular contacts are not merely passive targets, but are active contributors to EMT/EMyT. Indeed, our group showed previously that contact uncoupling or injury is necessary for TGFβ to induce EMyT (two-hit paradigm). Further, our previous work also revealed that Smad3, the main TGFβ-regulated transcription factor, binds to the Myocardin Related Transcription Factor (MRTF), the prime driver of SMA promoter, and inhibits MRTF’s transcriptional activity. During EMyT, Smad3 eventually degrades, which liberates the MRTF-driven myogenic program. However the mechanisms whereby cell contacts regulate the fate of Smad3 and MRTF during EMyT are poorly understood. Accordingly, the central aim of my studies was to explore the role of intercellular contacts, in particular that of Adherens Junction (AJs) in the induction of the myogenic reprogramming of the injured epithelium. This thesis describes two novel molecular mechanisms through which AJs impact EMyT. In the first part, we show β-catenin, an AJs component and transcriptional co-activator counteracts the inhibitory action of Smad3 on MRTF. Moreover we reveal that β-catenin is necessary to maintain MRTF stability via protecting MRTF from proteasomal degradation. Thus, β-catenin is an indispensable permissive factor for SMA expression. In the second part, we demonstrate that contact injury and TGFβ suppress the expression of the phosphatase PTEN. EMyT-related reduction or absence of PTEN potentiates Smad3 degradation. EMyT is associated with enhanced phosphorylation of the T179 residue in Smad3 linker region, and this event is necessary for Smad3 degradation. PTEN silencing increases the stimulatory effect of contact uncoupling and TGFβ on SMA promoter activity and SMA protein expression. Thus, the integrity of intercellular contacts regulates the level of PTEN, which in turn controls Smad3 stability through impacting on T179 phosphorylation. This new knowledge holds promises for targeted therapies and more effective prevention of the currently incurable fibroproliferative and fibrocontractile diseases.