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My PhD dissertation reports my research investigations performed on bone loss projects. 2 projects are described in this thesis. One project dealing with the effect of adipogenic DKK1 on bone loss under normal and under a high-fat-diet (HFD). The 2nd projects aimed to investigate on the potential of miRNAs to be used as potential biomarkers to predict bone fragility in T1D.

La Ténascine-C (TNC) est un composé majeur de la matrice extracellulaire tumorale et sa forte expression est directement corrélée à l’angiogenèse tumorale et au processus métastatique. Lors de ma thèse j’ai pu démontrer que la TNC dérégulait DKK1, un inhibiteur de la voie de signalisation Wnt et par ce biais augmentait l’activité de cette voie impliquée dans la cancérogenèse. La diminution de la formation des fibres de stress en présence de TNC est l’un des mécanismes majeurs qui contribue à la diminution de DKK1. L’activité de MKL1, facteur co-transcriptionnel de SRF et régulable par l’actine, s’avère diminuée en présence de TNC. Mes données indiquent que la fonction de MKL1 n’est peut-être pas le mécanisme majeur de la régulation de DKK1 par la statu de l’actine. D’autres facteurs, probablement liés aux fibres de stress d’actine pourraient être impliqués. L’augmentation de l’activité de la voie de signalisation Wnt, dépendante de DKK1, est probablement le mécanisme majeur par lequel la TNC active la progression tumorale. Cette étude a permis de mettre en évidence un nouveau mécanisme de régulation de DKK1 faisant intervenir l’intégrité du cytosquelette d’actine. / Tenascin-C (TNC) is a major component of the tumor specific extracellular matrix and its expression has been linked to tumor angiogenesis and metastasis. I demonstrated that TNC downregulates the expression of the Wnt signalling inhibitor DKK1 and by that enhances Wnt/-catenin signalling. Reduced stress fibre formation in the presence of TNC was identified as a major mechanism contributing to DKK1 downregulation. The activity of the actin-regulated SRF co-transcription factor MKL1 was found to be reduced in the presence of TNC. My results indicate that TNC-regulated MKL1 function maybe one, but not the major mechanism of DKK1 regulation by the actin status and that other factors, presumably regulated by actin stress fibres, are involved. Enhanced Wnt signalling activity downstream of TNC-induced DKK1 downregulation might be a major mechanism by which TNC promotes tumor progression. Furthermore, this study discovered a novel mechanism of regulating the Wnt inhibitor DKK1 by the integrity of the actin cytoskeleton.

Abstract This thesis focuses on mammalian kidney development and in particular on the question of how two Wnt signalling pathway genes, an antagonistic Dkk1 and an agonistic ligand Wnt5a, regulate the process. Wnts are secreted ligands that are involved in many developmental processes, including gonadal differentiation and kidney development, but also in various diseases and malformations. Wnts form a large signalling family containing 19 different glycoprotein ligands in mammals. Wnt signalling occurs via two different intracellular pathways. A canonical pathway proceeds via beta-catenin, and a non-canonical pathway utilizes other signalling molecules. Dkk1 is an antagonist of the canonical pathway and Wnt5a is considered a ligand that activates the non-canonical signalling pathway. As part of the thesis, I have studied the role of Dkk1 in kidney morphogenesis using a conditional mouse model, in which the gene is deleted in a cell specific manner from the collecting ducts. Dkk1 deficiency increased renal papilla growth and the risk of hydronephrosis. Research pointed out that the lack of Dkk1 in the collecting ducts increased cell proliferation and disturbed the balance of canonical Wnt signalling, which led to an overgrowth of renal papilla. This led to functional phenotypes including increased water reabsorption and changes in ion secretion/absorption. These changes are most likely due to altered Wnt7b signalling. The second part of the thesis examines the role of the non-canonical Wnt5a gene in kidney development with a conventional knock out mouse model. At the time work began on the thesis, no corresponding kidney phenotype had been published. The primary finding in kidneys lacking Wnt5a was an altered basement membrane organization of the collecting ducts and glomeruli. The phenotype is most likely the reason behind morphological phenotypes which vary from bilateral kidney agenesis to duplex collecting system. Notably, during the course of this study we found a mutation in the human WNT5A gene of a CAKUT patient. This is the first time Wnts have been shown to organize kidney development via basement membrane formation. / Tiivistelmä Tämän väitöskirjan tarkoituksena on ollut tutkia munuaisen kehitystä ja kuinka kaksi Wnt-signalointireitin geeniä, signalointia estävä Dkk1 ja signalointia edistävä Wnt5a säätelevät sitä. Wnt ligandit ovat eritettäviä signaalimolekyylejä, jotka ovat osallisina monissa kehitysbiologissa prosesseissa kuten sukupuolen määräytymisessä ja munuaisen kehityksessä. Myös monissa taudeissa on havaittu muuntuneita Wnt geenien tuottotasoja. Wnt-geenit muodostava suuren signalointimolekyyliperheen, johon lukeutuu 19 jäsentä nisäkkäillä ja Wnt-signointi on jaettu perinteisesti kahteen signalointiryhmään. Dkk1 on kanonisen Wnt-signaloinnin estäjä ja Wnt5a:ta pidetään pääsaantiöisesti ei-kanonisena Wnt-ligandina. Väitöskirjassani olen tutkinut Dkk1 geenin toimintaa kohdennetussa Dkk1-poistogeenisessä hiiressä, jossa geenin toiminta on poistettu spesifisesti munuaisen kokoojaputkista. Dkk1:n puutos johtaa munuaisen papillan kasvuun ja lisää riskiä hydronefroksen muodostumiseen. Tutkimukset osoittivat että Dkk1:n puutos aiheuttaa lisääntynyttä solujakautumista kokoojaputkissa, jolloin Wnt-signaloinnin muutos aiheuttaa papillan ylikasvua. Ylikasvusta seuraa lisääntynyttä veden takaisin imeytymistä ja muutoksia ionien erittämisessä ja takaisin imeytymisessä. Todennäköisimmin muutokset johtuvat muuntuneesta Wnt7b signaloinnista, jota Dkk1 normaalisti säätelee. Väitöskirjan toisessa osassa tutkittiin ei-kanonisen reitin Wnt5a ligandin roolia munuaisen kehityksessä käyttäen poistogeenistä hiirimallia, jossa Wnt5a:n roolia munuaisenkehityksessä ei ollut julkaistu työn aloituksen aikaan. Wnt5a:n puutoksen havaittiin vaikuttavan tyvikalvon järjestymiseen kokoojaputkissa ja munuaiskeräsessä. Tyvikalvon häiriö on todennäköisin syy morfologisiin muutoksiin, jotka vaihtelevat molempien munuaisen puuttumisesta kaksois-kokoojatiehyen muodostumiseen. Työssä osoitetaan ensimmäistä kertaa kuinka Wnt-signalointireitin proteiinit säätelevät munuaisen kehitystä tyvikalvon muodostuksen kautta.

Glioblastoma (GBM) is a very aggressive and invasive tumour that relapses within nine months of diagnosis and remains incurable despite advances in multimodal therapy including surgical resection, chemotherapy and radiation. Poor patient outcome has been correlated to specific markers of brain tumour initiating cells (BTIC) and intratumoural heterogeneity (ITH), which have also been associated with treatment resistance and tumour recurrence. ITH can be explained at the cellular level by the existence of multiple populations of cancer cells, including some which have acquired stemness properties like self-renewal, proliferation, and multilineage differentiation, also known as cancer stem cells (CSCs). In brain tumours, CSCs or BTICs, have been shown to be resistant to both chemotherapy and radiation treatment, allowing them to escape therapy and consequently generate for tumour recurrence. As a result, therapies that focus on targeting the BTIC compartment within the bulk GBM tumour would provide better treatment and prognosis for patients. To profile GBM BTICs we conducted two transcriptomic screens. The first compared GBM BTICs to neural stem cells (NSCs), their healthy counterparts, and for the second we developed a pipeline utilizing a dynamic BTIC patient-derived xenograft (PDX) model of human GBM recurrence allowing for the profiling of GBM BTICs at engraftment, after chemoradiotherapy delivery in a phase we have termed "minimal residual disease" (MRD), and at tumour recurrence. In this study, Dickkopf-1 (DKK1) was identified as a potential therapeutic target for GBM from each transcriptomic screen and was studied using short hairpin knockdowns, blockade with monoclonal antibodies, and subsequent functional stem cell assays. / Thesis / Master of Science (MSc) / Glioblastoma (GBM) is a very aggressive tumour that relapses within nine months of diagnosis and remains incurable despite chemotherapy, radiation, and surgery. Relapse is believed to be caused by the presence of a wide variety of cell types, including cancer stem cells (CSCs), which have been shown to be resistant to both chemotherapy and radiation in GBM. As a result, therapies that focus on targeting the CSCs within the bulk GBM tumour would provide better treatment for patients. In this study, we analyzed this cell population by conducting two screens. The first compared the level at which genes are expressed in GBM CSCs in comparison to how they are expressed in their healthy counterparts, neural stem cells, whereas the second compared the primary patient GBM tumour to its relapsed form in a mouse model of the disease. In this study, the protein Dickkopf-1 (DKK1) was identified and validated as a potential therapeutic target of GBM using well established molecular and stem cell functional assays.

L’ostéoporose est une maladie caractérisée par une faible masse osseuse et une détérioration du tissu osseux. Cette condition entraîne une plus grande fragilité osseuse et des risques de fractures. Plusieurs études ont associé l’ostéoporose à la faible densité osseuse des mandibules, à la perte d’attache parodontale, à l’augmentation de la hauteur de la crête alvéolaire et à la chute des dents. Cette étude vise à comprendre les mécanismes sous-jacents cette perte osseuse. En effet, au cours du développement des souris, PITX1 joue un rôle clé dans l'identité des membres postérieurs et dans le bon développement des mandibules et des dents. Son inactivation complète chez la souris mène à un phénotype squelettique sévère. Tandis que, son inactivation partielle provoque des symptômes apparentés à l'arthrose avec une augmentation de la masse osseuse au niveau de l’os cortical et de l’os trabéculaire. Inversement, une étude antérieure chez des jumelles monozygotiques discordantes pour l’ostéoporose, montrent une augmentation d’environ 8.6 fois du niveau d’expression du gène Pitx1 chez la jumelle ostéoporotique. Collectivement, ces données nous ont poussés à investiguer sur le rôle du facteur de transcription PITX1 dans le métabolisme osseux normal et pathologique. Dans ce contexte, des souris transgéniques Col1α1-Pitx1 sur-exprimant Pitx1 spécifiquement dans le tissu osseux sous le promoteur du collagène de type-I (fragment 2.1kpb) ont été générées et phénotypiquement caractérisées. Ces résultats ont révelé que les souris transgéniques Col1α1-Pitx1 présentaient un phénotype similaire à celui des patients ostéoporotiques accompagné d'une perte de dents et des problèmes dentaires et parodontaux. De plus, cette étude a révélé que la surexpression de Pitx1 induit une altération de l’homéostasie osseuse via l’inactivation de la voie de signalisation Wnt/β-caténine canonique. Cette hypothèse a été appuyée par le fait que le traitement des souris transgéniques Col1α1-Pitx1 avec du chlorure de lithium, un activateur de la voie Wnt canonique, prévient le phénotype ostéoporotique chez ces souris. Finalement, cette étude établit un rôle crucial de PITX1 dans la régulation de la masse osseuse et une implication possible dans l’ostéoporose et les maladies parodontales via l’inactivation de la voie de signalisation Wnt/β-caténine canonique. / Osteoporosis is a progressive bone disease that is characterized by a decrease in bone mass and density leading to an increased risk of fracture. Several studies have linked osteoporosis to the low bone density of the mandibles, the periodontal attachment loss, increasing of the alveolar crest heigh and tooth loss. This study aims to understand the mechanisms underlying bone loss. During mouse development, PITX1 plays a key role in the identity of the hindlimb and the proper development of the jaws and teeth. Interestingly, PITX1-null mice displayed severe skeletal phenotype. Whereas, ageing PITX1+/- progressively developed OA-like lesions in cartilage associated with a drastic increase in cortical and trabecular bone formation. Conversely, expression analysis of osteoblasts derived from monozygotic twins discordant for osteoporosis reported an 8.6-fold up-regulation of Pitx1 expression in osteoblasts from osteoporotic twins when compared with healthy ones. Collectively, these data prompted us to investigate the role of PITX1 in normal and pathological bone metabolism. In this context, transgenic Col1α1-Pitx1 mice over expressing Pitx1 specifically in bone tissue under the type-I collagen promoter (2.3kb fragment) were generated and phenotypically characterized. These data suggested that Pitx1 overexpression induces an osteoporosis like phenotype accompanied with oral bone loss, edentulousness, dental and periodontal problems. These results suggested that Pitx1 overexpression induces alteration of bone homeostasis via the inactivating of the the Wnt/β-catenin canonical pathway. This hypothesis was supported by the fact that treatment with lithium chloride, a Wnt canonical activator, rescued the phenotype. Finally, this study establishes a crucial role of PITX1 in the regulation of bone mass and a possible involvement in the developpement of osteoporosis and periodontal disease via the inactivation of the Wnt/β-catenin canonical pathway.

<p>Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the <i>MEN1</i> and <i>VHL</i> tumour suppressor genes, is still elusive. The protein product of the <i>MEN1</i> gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.</p><p>Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous <i>Men1</i> mice. For comparison, normal and <i>MEN1</i> non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between <i>Men1</i> expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in <i>Men1</i> mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and <i>Men1</i> mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of <i>Men1</i> and wt mice were observed.</p><p>In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.</p><p>Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.</p>

Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the MEN1 and VHL tumour suppressor genes, is still elusive. The protein product of the MEN1 gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling. Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous Men1 mice. For comparison, normal and MEN1 non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between Men1 expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in Men1 mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and Men1 mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of Men1 and wt mice were observed. In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression. Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.