No description available.

No description available.

M.Sc. Thesis Dissertation, August 2019, McMaster University / Renal dysplasia, defined as the abnormal development of kidney tissue, is the leading cause of kidney disease in children. While there are numerous causes of renal dysplasia (i.e. genetic, environmental and epigenetic factors), there is no cure to this abnormal defect. Kidney development occurs by two main processes: branching morphogenesis, which forms the collecting duct system, and nephrogenesis, which generates the nephrons, the functional units of the kidney. Our previous studies have demonstrated that β-catenin, a dual-function protein involved in cell adhesion and gene transcription, regulates branching morphogenesis and nephrogenesis. Furthermore, we discovered that nuclear β-catenin levels are increased in kidneys from patients with renal dysplasia, suggesting β-catenin can be a potential therapeutic target to modulate kidney development and renal dysplasia. Quercetin is a flavonoid that reduces β-catenin levels and inhibits its transcriptional activity, leading to improved outcomes in cancer and in kidney fibrosis. The role of quercetin in kidney development and in abnormal defects that arise during kidney development is yet to be examined. Using embryonic mouse kidney organ culture, I found that quercetin treatment resulted in a dose-dependent disruption in branching morphogenesis and nephrogenesis. In addition, quantitative reverse-transcriptase PCR revealed a decreased expression of β-catenin target genes essential for kidney development (i.e. Pax2, Six2 and GDNF). Immunohistochemistry for β-catenin demonstrated that quercetin reduced nuclear β-catenin expression and increased cytoplasmic and membrane-bound expression in a dose-dependent manner. These results were confirmed by Western blot analysis. These novel findings demonstrate that quercetin treatment resulted in decreased levels of nuclear β-catenin, resulting in a decrease in its transcriptional activity which manifested in alterations in kidney developmental processes, suggesting quercetin is effective at reducing nuclear β-catenin in wild-type embryonic kidneys. Next, to determine whether quercetin has any effects on renal dysplasia, I utilized transgenic mice models that overexpress β-catenin in select cells of the embryonic kidney. These models recapitulate the defects observed in human renal dysplasia, including disorganized branching morphogenesis and disrupted nephrogenesis. Quercetin treatment of embryonic dysplastic kidneys resulted in a partial rescue of renal dysplasia which was evident in marked improvements in branching morphogenesis and nephrogenesis, as well as an increase in the number of properly-developing nephrons in the kidney tissue. Analysis of β-catenin expression in quercetin-treated dysplastic kidneys revealed a decrease in nuclear levels and an increase in cytoplasmic and membrane-bound levels, resulting in a reduced expression of target genes (Pax2, Six2, and GDNF). Finally, this partial rescue of renal dysplasia was associated with an improved and organized E-cadherin expression in quercetin-treated dysplastic kidneys, suggesting a possible molecular mechanism of quercetin action in resolving abnormal kidney development. Overall, my findings demonstrate, for the first time, that quercetin reduces β-catenin transcriptional activity in normal and dysplastic kidneys and reduces the severity of defects in renal dysplasia. / Thesis / Master of Science in Medical Sciences (MSMS)

No description available.

Die evolutionär konservierten morphogenen Signalwege Wnt/β-Catenin und Hedgehog (Hh) spielen vor allem in der Embryogenese, Zelldifferenzierung und Kanzerogenese eine große Rolle. Es ist bekannt, dass es zwischen Komponenten der beiden Signalkaskaden zu verschiedensten Wechselwirkungen und Hintergrundreaktionen in unterschiedlichsten Organismen und Geweben kommt. Ziel dieser Arbeit war die Aufklärung einzelner mole- kularer, transkriptioneller Prozesse hinter diesen Kreuzreaktionen in primären Hepatozyten und dem Hepatozellulären Karzinom. Dafür sind die beiden Signalwege durch verschiedenste Einflüsse, wie dem Einsatz von siRNAs, transgenen Mausmodellen und rekombinanten Proteinen gegen einzelne Faktoren der Hedgehog, aber auch der Wnt/β-Catenin Kaskade in ihrer Genexpression verändert und die Reaktionen der Signalwegskomponenten mittels der quantitativen Real-Time-PCR untersucht worden. Neben dem Organismus Maus haben einzelne vergleichende Experimente auch auf der humanen Ebene zum Erkenntnisgewinn beigetragen. Durch die Einflussnahme auf den Hedgehog Signalweg in murinen Hepatozyten wird deutlich, dass die Antworten auf die einzelnen Veränderungen in den Signalkaskaden sehr vielschichtig und umfangreich sind. Abhängig vom induzierten bzw. reprimierten Gen, aber auch vom gelenkten Signalweg variieren die Genexpressionen auf unterschiedlichste, und zum Teil gegenläufige Weise. Ferner wird deutlich, dass es zu Unterschieden in der Genantwort bezüglich der verschiedenen Organismen Maus und Mensch, aber auch zu Variationen der Interaktionen in den diversen Gewebe bzw. Zelltypen kommt.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 94-105).

La glande surrénale est un organe endocrinien d’une importance vitale de par son rôle dans le maintien de l’homéostasie corporelle. Pour assurer cette fonction, le cortex surrénalien est divisé en différentes zones qui produisent des hormones spécifiques. Les mécanismes de la mise en place de cette zonation au niveau embryonnaire ainsi que de son maintien tout au long de vie sont encore inconnus de nos jours. Les gènes Rspo1 et 3 sont exprimés de manière très spécifique au niveau de la capsule mais ils codent pour des protéines secrétées qui agissent sur les cellules adjacentes de la zone glomérulée afin d’induire l’activation de la voie canonique Wnt/β-caténine. La délétion du gène Rspo3 pendant le développement embryonnaire entraine un défaut d’activation des voies Shh et Wnt/β-caténine et donc en conséquence un défaut de la mise en place de la zonation. Sa fonction reste également essentielle au cours de la vie adulte puisqu’elle assure à la fois le maintien de l’homéostasie tissulaire et de la zone glomérulée. L’absence du gène Rspo1, n’affecte pas le développement ni la zonation ou l’homéostasie de la glande surrénale. Par contre, si on l’exprime de façon ectopique dans tous le cortex surrénalien, entrainant une activation anormale de la voie Wnt/β-caténine dans cette zone, on peut observer une hyperplasie des glandes surrénales. A partir de 1 an d’âge, cette hyperplasie surrénalienne entraine une formation de tumeurs. Ce travail démontre donc que la capsule par le biais du gène Rspondin 3 agit comme un centre de signalisation capable de contrôler à la fois l’homéostasie par le remplacement des cellules endommagés et le maintien de la zonation de la glande surrénale / The adrenal gland is an endocrine organ of vital importance because of its role in the maintenance of body homeostasis. To ensure this function, the adrenal cortex is divided into different areas that produce specific hormones. The mechanisms of the establishment of this zonation at the embryonic level as well as its maintenance throughout life are still unknown today. The Rspo1 and 3 genes are expressed very specifically at the capsule level but they encode secreted proteins that act on the adjacent cells of the zona glomerulosa in order to induce the activation of the Wnt / β-catenin canonical pathway. The deletion of the Rspo3 gene during embryonic development leads to a lack of activation of the Shh and Wnt / β-catenin pathways and hence a lack of zonation. Its function is also essential during adult life since it ensures both the maintenance of tissue homeostasis and the glomerular zone. The absence of the Rspo1 gene does not affect development, zonation or homeostasis of the adrenal gland. On the other hand, its ectopical expression in all the adrenal cortex leads to an abnormal activation of the Wnt / β-catenin pathway in this area and thus to an hyperplasia of the adrenal glands. From 1 year of age, this adrenal hyperplasia leads to the formation of tumors. This work demonstrates that the capsule through the Rspondin 3 gene acts as a signaling center capable of controlling both homeostasis by replacing damaged cells and maintaining the zonation of the adrenal gland

The liver is an essential organ for cholesterol homeostasis. If this process becomes dysregulated, cardiovascular disease (CVD) develops. Zinc-fingers and homeoboxes 2 (Zhx2) as an important hepatic gene regulator and contributes to CVD. BALB/cJ mice, with mutant Zhx2 allele, have fewer atherosclerotic plaques compared to other strains on a high fat diet. In my dissertation, I focused on the liver phenotype in BALB/cJ mice on a high-fat diet and found increased liver damage compared to wild-type Zhx2 mice. These data indicates that reduced Zhx2 in the liver leads to CVD resistance, but increases liver damage. Therefore, Zhx2 has an important role in lipid metabolism and liver function. Hepatic alpha-fetoprotein (AFP) is expressed abundantly in the fetal liver and repressed after birth regulated through three enhancers (E1, E2, and E3). E3 activity is restricted to a single layer of hepatocytes surrounding central veins (pericentral region) along with glutamine synthetase (GS). In my dissertation, I explore pericentral gene regulation in the adult liver. A GS enhancer (GSe) also exhibits pericentral activity which, along with E3, is regulated by the β-catenin signaling pathway. Orphan receptors, Rev-erbα, Rev-erbβ, and RORα, contribute to E3 activity elucidating a potential mechanism for zonation.

Colorectal cancer is estimated to cause approximately 50,000 deaths each year in the United States. Epidemiological studies have demonstrated an inverse association between sunlight exposure, which stimulates the formation of vitamin D in the skin, and colorectal carcinoma. Laboratory studies report that metabolites of vitamin D, acting through the vitamin D receptor (VDR), regulate cellular proliferation, differentiation and apoptosis. In addition, VDR contains a polymorphic variant, FokI, which results in two different isoforms of VDR. We have demonstrated a differential suppression of β-catenin transcriptional activity by these isoforms in the presence of 1,25(OH)₂D₃ (1,25D). Epidemiological evaluation of metachronous colorectal adenoma formation indicates that VDR includes several single nucleotide polymorphisms (SNPs) which influence the odds of developing colorectal adenoma. In addition, we have found full length Adenomatous Polyposis Coli (APC), a frequently mutated tumor suppressor gene in colorectal cancer, augments both the interaction of VDR and β-catenin as well as the suppression of β-catenin transcriptional activity in the presence of 1,25D. We have also demonstrated in epidemiological studies that the presence of a T-A haplotype in APC codons 486 and 1822, respectively, reduces the odds of any metachronous adenoma by 27% [odds ratio (OR), 0.73; 95% confidence interval (95% CI), 0.59 – 0.91]. Taken together, these data support not only a protective role for vitamin D acting through the VDR, but also for an important role of heritable polymorphic variation in VDR and APC in carcinogenesis.

Medulloblastomas are tumours of cerebellar origin and are thought to arise from the malignant transformation of progenitor cells in the developing cerebellum. A number of developmental signalling pathways are required for the precise cell specification, proliferation, migration and differentiation involved in forming the mature cerebellum and it is the dysregulation of these processes that can lead to the eventual formation of a tumour. Genes encoding components of the canonical Wnt/β-catenin signaling pathway are mutated in around 15% of medulloblastomas and germline mutations that activate this pathway are known to predispose to medulloblastoma. Despite this, the contribution of Wnt/β-catenin signaling to normal cerebellum development is not yet well understood and the developmental origins of medulloblastoma arising from activation of this pathway are only beginning to be revealed. Therefore, the aims of this thesis were to characterise the spatio-temporal nature of Wnt/β-catenin signalling during cerebellum development and to investigate its function, with the broad goal of informing our understanding of how medulloblastoma arises from oncogenic activation of Wnt/β-catenin signalling. To address the first aim I utilised a LacZ expressing Wnt/β-catenin signalling reporter mouse to characterize the spatio-temporal pattern of Wnt/β-catenin pathway activation during cerebellum development. Analysis of LacZ reporter expression revealed a pattern of transient Wnt/β-catenin activity in discrete cell populations throughout cerebellum development. I found that Wnt/β-catenin activity is present during the early specification of granule cells at the cerebellar rhombic lip but not during the expansion of this cell population at later stages. During perinatal development Wnt/β-catenin activity shifts to the cerebellar ventricular zone, a known germinal centre for GABAergic interneurons and glia, and was observed in cells radiating out from this region. By early postnatal development the expression of the Wnt/β-catenin reporter became progressively restricted to the developing Bergmann glia population. To investigate the function of Wnt/β-catenin in these cell lineages and how its dysregulation could contribute to medulloblastoma, I used a combination of ex vivo organotypic culture, in utero electroporation and tissue-specific gene targeting to manipulate components of the pathway. Culturing slices of E18.5 cerebellum in the presence of small molecule activators of the Wnt/β-catenin pathway revealed a reduction in the expression of glial markers Sox9 and GFAP. In addition, interneuron lineage marker Pax2 was also reduced, supporting the conclusion that dysregulation of Wnt/β-catenin signalling affects the generation of cell lineages from the ventricular zone. To investigate this hypothesis further, I constitutively activated the Wnt/β-catenin signalling pathway in the developing cerebellum using Cre-Lox gene targeting to knock out Apc, a negative regulator of the pathway, in ventricular zone derived lineages. Cre-induced recombination of Apc resulted in nuclear accumulation of β-catenin, a sign that the pathway had become ectopically activated. Furthermore, a reduction in the expression of Sox9 and Pax2 was also observed in these mutant cells. From these data, I conclude a potential role for Wnt/β-catenin signaling in the regulation of glial/interneuron progenitors. Combined, these data support a model where Wnt/β-catenin signalling could perform multiple functions in specification of the granule lineage, regulation of glial/interneuron progenitors and in glial differentiation/maturation. Importantly, dysregulation of progenitor self-renewal and differentiation is widely acknowledged to promote tumourigenesis. Thus, the data in this thesis support a potential mechanism for the development of medulloblastoma from the dysregulation of ventricular zone progenitors.