The adult prevalence of obesity in the United States exceeds 30% and obesity is associated with increased cancer risk and poor prognosis, including postmenopausal breast cancer. p53 is a tumor suppressor gene that responds to diverse cellular stress including DNA damage, oxidative stress and hypoxia. p53 is mutated in most human cancers, including postmenopausal breast cancer, and is involved in the regulation of lipogenic enzymes. However, the links between p53 and obesity in postmenopausal breast cancer are poorly understood. Here we test the hypothesis that the effect of obesity on mammary tumor growth is impacted by p53 status. The aim of this study was to determine how p53-deficient mammary tumor cells (relative to p53 wild-type cells) respond to obesity-driven tumor growth. To test this hypothesis, we used ovariectomized (OVX) C57BL/6 mice randomized to a control diet (n=40) or a diet-induced obesity (DIO) regimen (n=40) for 10 weeks. At the time, DIO mice were approximately 40% heavier (p<0.001) and had 45% greater adiposity (p<0.001) than control mice. Mice were then injected (in the 4th mammary fat pad) with either p53-deficient (p53+/-) or p53 wild-type (p53+/+) MMTV-Wnt-1 mammary tumor cells. Mice were monitored for tumor growth, killed when moribund, and tumors were collected at study end point. We found an interaction between diet and p53 status, with p53+/+ Wnt-1 tumors grown in DIO mice developing the more aggressive morphology compared to p53+/+ Wnt tumors in control mice while the observation was not seen in p53+/- Wnt tumors. From histopathological analysis we also discovered that the DIO regimen promotes local invasion of mammary tumor cells and alters the morphology of MMTV-Wnt-1 p53+/+ mammary tumors. Specifically, p53+/+ Wnt tumors grown in DIO mice displayed disorganized ductal structures characteristic of p53+/- tumors grown in control mice, and DIO exacerbated this aberrant morphology in p53+/- Wnt tumors. Moreover, immunohistological analyses showed that DIO reduces p53 protein expression while elevating Ki-67 expression only in the p53+/+ Wnt mammary tumors. These results suggest that p53 and DIO have interactive effects on mammary tumor growth, as p53+/+ Wnt tumors growing in DIO mice resulted in higher tumor grade similar to p53+/- Wnt tumors. / text

Epithelial to mesenchymal transition (EMT) is an essential process during embryogenesis for the development of organ systems, including the heart and its vasculature. The development of both coronary vessels and heart valves depends on EMT. In this dissertation, we first present data demonstrating that increasedoligosaccharide hyaluronan (o-HA) levels after EMT induction within atrioventricular (AV) valves leads to a decrease in EMT due to the induction of VEGF expression. Regulated EMT inhibition prevents the formation of hyperplastic valves. Next, we show that the proepicardium, which provides the precursor cells required for epicardial and coronary vessel development, migrates to the developing heart via direct contact of multicellular proepicardial villi to the developing myocardium. This shifts the paradigm from a migration consisting of floating cysts to one of direct contact and differential adhesion forces to form the initial epicardium. A subset of epicardial cells undergoes EMT, migrates into the developing heart, and differentiates into cardiac fibroblast, vascular endothelial, and smooth muscle cells. In order to more effectively study epicardial EMT in vitro, we developed several new methods for the in vitro study of coronary vessel development. We developed an improved protocol for isolating embryonic myocyte cells, for use in co-cultures with epicardial cells. This co-culture system allows investigation into the effects of myocyte derived soluble factors uponepicardial EMT and mesenchymal cell differentiation. We also present a protocol for isolating epicardial clonal colonies from an epicardial cell line derived from the ImmortoMouse. These clones provided direct evidence that the epicardium is a heterogeneous population of cells. These unique clones allow for to study into specific epicardial cell lineages and phenotypes. Finally, we provide data defining the expression of Wnts within the developing heart and the role may play during epicardial EMT. We conclude that canonical Wnts are both necessary and sufficient to inhibit epicardial EMT. These results provide the first direct evidence for a role of Wnt proteins during coronary vessel development. Collectively our results provide significant advancements in our understanding of EMT regulation during cardiac development.

No description available.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Satellite cells are muscle stem cells that are responsible for the growth and repair of skeletal muscle tissue. Satellite cells typically exist in a quiescent state in their niche between the sarcolemma and basal lamina. In response to muscle tissue injury, activated satellite cells, otherwise known as myoblasts, migrate to the site of injury where they proliferate and subsequently differentiate and fuse to repair damaged myofibers. The success of muscle growth and repair is highly dependent on the speed and degree to which these myoblasts migrate, proliferate and differentiate. This overall process, referred to as myogenesis, is largely controlled by the myogenic regulatory factors, a group of basic helixloop- helix transcription factors including MyoD, Myf5, myogenin and Mrf4. It has recently been found that the Wnt family of secreted signalling proteins are highly involved in the regulation of developmental processes such as myogenesis. Wnt proteins are a family of 21 highly-conserved, secreted, cysteine-rich signalling molecules which are found in all multi-cellular organisms. Wnt signalling is highly versatile and is initiated by the binding of extracellular Wnt to cell-surface Frizzled receptors (Fz). It is highly dependent on both the Wnt isoform and Fz type and may initiate one of three known signalling pathways. Wnt3A and Wnt7A are of particular interest as they have previously been linked with myogenesis. C2C12 myoblasts over-expressing Wnt3A have been seen to have reduced levels of motility and terminal differentiation. Wnt7A is suspected to maintain a healthy satellite cell pool by regulating self-renewal; injection of recombinant Wnt7A into mouse leg muscle resulted in increased satellite cell numbers. In vitro Wnt studies have typically involved the treatment of mouse cells with conditioned medium containing Wnt, often at unknown concentrations. In our study we wished to test the effects of known concentrations of recombinant Wnt3A and Wnt7A on mouse C2C12 and donor-derived human skeletal muscle myoblasts (HSkM) in vitro. Wnt3A and Wnt7A were seen to increase the rate of C2C12 migration in a dose dependent manner. HSkM cells treated with 10 ng/ml Wnt3A also displayed increased motility. Neither Wnt3A nor Wnt7A were seen to have any significant effects on the proliferation of C2C12 or HSkM cells. Wnt3A (10ng/ml and 100 ng/ml) but not Wnt7A was seen to decrease C2C12 terminal differentiation as measured by expression of myosin heavy chain (MyHC). Subsequent confocal microscopy revealed that Wnt3A significantly reduced the percentage of MyoD+ C2C12 nuclei during differentiation. A reduction in nuclear MyoD would support the observed impaired commitment to differentiation. However, donor-derived human skeletal muscle myoblasts treated with 10 ng/ml Wnt3A were not seen to have significantly reduced nuclear MyoD levels or terminal differentiation; the reason for this is unclear but may relate to a number of factors including the concentration of Wnt, Fz and co-receptor profiles and the presence of specific extracellular matrix and serum factors. These studies provide new insight into the role of Wnts in myogenesis and lay the foundation for future work on Wnt3A and Wnt7A. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2014.

No description available.

WNT ligands are secreted proteins that act as signals between cells. WNTs activate several interconnected signaling pathways that are required for embryonic development as well as tissue homeostasis in adults. The X-chromosomal Porcn gene encodes a membrane-bound O-acyl transferase that is required for the acylation of all 19 WNT ligands encoded in the mammalian genome. Non-acylated WNTs fail to be secreted from the producing cell and thus do not activate downstream signaling targets. In my thesis research, I have investigated the function of Porcn in mouse embryonic development. In vitro, I have shown that Porcn is required for canonical WNT signaling in ES cells and further, for their differentiation into endodermal and mesodermal derivatives. Taking advantage of a mouse line carrying a conditional (floxed) Porcn allele that I have generated, I have focused my studies on the early embryonic roles of Porcn using Cre recombinase-mediated and X chromosome inactivation-based ablation of Porcn function in vivo. I have found that the earliest requirement for Porcn in mouse development is the induction of gastrulation. In contrast to findings from in vitro studies, I have provided evidence that Porcn is not required for pre-implantation development in vivo. Dissecting embryonic and extra- embryonic roles of Porcn, I have been able to show that Porcn is required in the extra-embryonic chorion in order to mediate chorio-allantoic fusion, whereas ablation in the extra-embryonic visceral endoderm had no apparent effects. The extra-embryonic requirement for Porcn results in a parent-of-origin effect in Porcn heterozygous females due to X chromosome inactivation. In contrast to the placentation defect causing embryonic lethality of maternal allele mutants, deletion of the paternal allele caused variable fetal defects resulting in perinatal lethality with only rare survivors to adulthood. Both fetuses and adults represent a mouse model for Focal Dermal Hypoplasia (FDH), the syndrome caused by mutations in the human PORCN gene. My studies highlight the importance of PORCN-mediated WNT signaling for gastrulation, placentation, and fetal development, but suggest that endogenous WNT secretion does not play an essential role in either implantation or blastocyst lineage specification.

WNT ligands are secreted proteins that act as signals between cells. WNTs activate several interconnected signaling pathways that are required for embryonic development as well as tissue homeostasis in adults. The X-chromosomal Porcn gene encodes a membrane-bound O-acyl transferase that is required for the acylation of all 19 WNT ligands encoded in the mammalian genome. Non-acylated WNTs fail to be secreted from the producing cell and thus do not activate downstream signaling targets. In my thesis research, I have investigated the function of Porcn in mouse embryonic development. In vitro, I have shown that Porcn is required for canonical WNT signaling in ES cells and further, for their differentiation into endodermal and mesodermal derivatives. Taking advantage of a mouse line carrying a conditional (floxed) Porcn allele that I have generated, I have focused my studies on the early embryonic roles of Porcn using Cre recombinase-mediated and X chromosome inactivation-based ablation of Porcn function in vivo. I have found that the earliest requirement for Porcn in mouse development is the induction of gastrulation. In contrast to findings from in vitro studies, I have provided evidence that Porcn is not required for pre-implantation development in vivo. Dissecting embryonic and extra- embryonic roles of Porcn, I have been able to show that Porcn is required in the extra-embryonic chorion in order to mediate chorio-allantoic fusion, whereas ablation in the extra-embryonic visceral endoderm had no apparent effects. The extra-embryonic requirement for Porcn results in a parent-of-origin effect in Porcn heterozygous females due to X chromosome inactivation. In contrast to the placentation defect causing embryonic lethality of maternal allele mutants, deletion of the paternal allele caused variable fetal defects resulting in perinatal lethality with only rare survivors to adulthood. Both fetuses and adults represent a mouse model for Focal Dermal Hypoplasia (FDH), the syndrome caused by mutations in the human PORCN gene. My studies highlight the importance of PORCN-mediated WNT signaling for gastrulation, placentation, and fetal development, but suggest that endogenous WNT secretion does not play an essential role in either implantation or blastocyst lineage specification.

Molecular Mechanisms of Hematopoietic Stem Cell Development- The Role of Retinoic Acid Signaling Bhaskar Chanda For the Doctor of Philosophy Medical Biophysics University of Toronto 2013 Abstract During mouse embryonic development, the formation of blood or hematopoiesis occurs in multiple phases. The first phase or primitive hematopoiesis generates a restricted subset of blood cell lineages but is devoid of lymphoid and hematopoietic stem cell (HSC) potential. The next phase of hematopoiesis, also known as definitive hematopoiesis, is characterized by its ability to generate multilineage hematopoietic progenitors and HSCs from a specialized population of endothelial cells known as hemogenic endothelium (HE). Such endothelial to hematopoietic transitions (EHT) have been recently observed at a clonal level, however, molecular mechanisms that underlie EHT leading to the specification of HSCs have remained poorly understood. Here we show that retinoic acid (RA) signaling plays a pivotal role in embryonic hematopoiesis and HSC development. RA signaling inhibits primitive hematopoiesis, and promotes definitive hematopoiesis. This inductive effect of RA signaling extends to the specification of HSCs. Activation of the RA signaling pathway ex vivo in AGM-derived HE dramatically enhanced the repopulating potential, whereas its conditional inhibition in vivo abrogated HSC development. These repressive and inductive effects of RA signaling were mediated primarily via retinoic acid receptor (RAR)- &alpha;. We further analyzed the mechanistic basis of RA signaling with a combined use of cellular, molecular and biochemical assays, and show that &beta;-catenin dependent Wnt signaling is the downstream mediator of RA signaling. Collectively, this thesis provides new insight into molecular mechanisms that control embryonic hematopoiesis and identify the RA pathway as a key regulator of definitive hematopoiesis and HSC specification.

The USP4 is a deubiquitinating enzyme found elevated in certain human lung and adrenal tumours. USP4 has a very close relative, USP15, which has caused great difficulty in studying only one or the other. We have had generated two antibodies specific to USP4 and USP15, and have confirmed that the two do not cross react. Although there have been previous findings of interacting partners, possible substrates and pathways in which it is involved, the biological role of USP4 is mostly unknown. We have used these antibodies to determine that USP4 and USP15 expression differs across tissue and cell types, and that expression changes as the organism ages. We have shown that USP4 plays a role in canonical Wnt signaling, perhaps by stabilizing Beta-catenin, and identified GRK2 as a kinase, phosphorylating USP4. These data have provided enough information to form a hypothesis, implicating USP4 with the destruction complex in the Wnt signaling pathway.