Renal dysplasia is a disease characterized by developmental abnormalities of the kidney that affect 1 in 250 live births. Depending on the severity of the renal abnormalities, this disorder can lead to childhood kidney failure, adult onset chronic kidney disease, and hypertension. Currently, the best treatment options for patients with renal dysplasia are renal dialysis and kidney transplant. Our limited understanding of the pathogenesis of renal dysplasia has prevented the development of better treatment strategies for those patients. A hallmark of renal dysplasia is an expansion of loosely packed fibroblast cells, termed renal stroma. Markedly elevated levels of β-catenin have been reported in the expanded stromal population in patients with dysplastic kidneys. Yet, the contribution of stromal β-catenin to the pathogenesis of renal dysplasia is not known. Additionally, the role of stromal β-catenin in the developing kidney is not clear. The overall hypothesis of this PhD thesis is that β-catenin in stromal cells controls key signalling molecules that regulate proper kidney development. Furthermore, we hypothesize that elevated levels of β-catenin contribute to the pathogenesis of renal dysplasia. To mimic the human condition, we generated a mouse model that overexpresses β-catenin specifically in the stroma (termed β-catGOF-S). In addition, to gain a better understanding of its role in kidney development, we generated a second mouse model deficient for β-catenin exclusively in stromal cells (termed β-catS-/-). The goal of this study is to utilize these models to understand the role of stromal β-catenin in kidney formation and investigate its contribution to renal dysplasia. The first objective defines the contribution of stromal β-catenin to the genesis of renal dysplasia. We provide evidence for a mechanism whereby the overexpression of stromal β-catenin disrupts proper differentiation of stromal progenitors and leads to an expansion of stroma-like fibroblast cells and vascular morphogenesis defects. In the second objective, we establish a mechanism where stromal β-catenin modulates Wnt9b signaling in epithelial cells to control proliferation of the nephron progenitors. In the third objective, we define a role for stromal β-catenin in proper formation and survival of the medullary stroma. Finally, in a technical report, we outline a protocol to isolate stromal cells in the developing kidney and provide potential downstream applications to further our understanding of stromal β-catenin in the developing kidney. Taken together, our findings establish a crucial role for stromal β-catenin in the genesis of renal dysplasia and demonstrate, using two mouse models, that stromal β- catenin must be tightly regulated for proper formation of the stroma lineages and development of the kidney. / Thesis / Doctor of Philosophy (PhD)

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b-catenin plays a dual role as a structural component of adherens junctions and as a transcriptional coactivator through its interactions with E-cadherin and TCF/LEF transcription factors, respectively. Normally, free b-catenin in cytoplasma is regulated by proteosome-dependent degradation system. In malignant tumor cell , deregulation of b-catenin degradation results in its aberrant accumulation, and leading to cancer. The goals of this study were to explore the reason of aberrant b-catenin accumulation in cervical carcinoma and evaluate the correlation between b-catenin¡BE-cadherin and p53 in different FIGO stage. Seventy paraffin embedded specimen with different FIGO stage were included in this study. Immunohistochemical staining was performed using anti-b-catenin polyclonal antibody and anti-p53 polyclonal antibody respectively and direct sequencing methods to analyze the mutation of CTNNB1 exon 13. The results showed 58 cases (82.8%) displayed cytoplasmic/nuclear b-catenin and no mutations in exon 13 of b-catenin gene, whereas no significant correlation between b-catenin expression level and tumor metastasis. However, b-catenin expression intensity had significant correlation with tumor size (p=0.008) and inversely correlated with E-cadherin (p=0.027) in different FIGO stage. The other way, the p53 staining intensity was significant correlated with b-catenin expression intensity (p=0.013) . Therefore, we suggest that mutations of CTNNB1 exon 13 may not be a reason for aberrant b-catenin accumulation in cervical carcinoma and aberrant p53 may play an important factor in accumulation of b-catenin.

No description available.

No description available.

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.

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.

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.

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.

Die Entwicklung von Dickdarmkrebs wird durch eine Reihe von Lebens- und Essgewohnheiten sowie Umweltfaktoren begünstigt. Den letzteren beiden sind Substanzen zuzurechnen, die bei der Zubereitung der Nahrung entstehen und mit ihr aufgenommen werden. Zu diesen Verbindungen gehört das 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridin (PhIP) aus der Substanzklasse der heterozyklischen aromatischen Amine. Es entsteht bei der Erhitzung zahlreicher proteinhaltiger Nahrungsmittel und die Zielorgane in Nagerstudien stimmen mit der Häufung von Krebsinzidenzen in westlichen Industrienationen überein. Dieser Zusammenhang konnte jedoch bis heute nicht endgültig bewiesen werden. Fütterungsversuche mit Ratten wurden mit Konzentrationen der Substanz durchgeführt, die weit über der menschlichen Exposition liegen. Durch das Verfüttern einer humanrelevanten Dosis PhIP sollte geklärt werden, ob auch geringe Konzentrationen dickdarmkrebstypische Mutationen, präneoplastische Läsionen oder Tumore induzierten. Die mit humanrelevanten Dosen gefütterten Tiere wiesen weniger Läsionen als die Hoch-Dosis-PhIP-Gruppe auf, in der allerdings keinerlei maligne Tumoren des Dickdarms auftraten. Hinweise auf dickdarmkrebstypische Mutationen fanden sich ebenfalls in beiden Gruppen, wobei hier keine Dosisabhängigkeit beobachtet werden konnte. Die Sequenzierung ergab ein deutlich von Literaturdaten abweichendes Spektrum. In Bezug auf das verwendete Tiermodell wurden erhebliche Abweichungen in der Empfindlichkeit der Tiere gegenüber der Substanz im Vergleich zu ähnlichen Studien festgestellt. Beide Fütterungsgruppen zeigten deutlich weniger Läsionen; als mögliche Gründe wurden Unterschiede in der Futterzusammensetzung und –zubereitung sowie in der Tierhaltung und –herkunft ausgemacht. Es konnte erstmalig ein Zusammenhang zwischen PhIP in niedrigen Dosen in der Nahrung und der Induktion von Entzündungen gezeigt werden. Diese waren sowohl makroskopisch als auch histologisch sichtbar, der genaue Mechanismus ihrer Entstehung ist jedoch unbekannt.<br><br> Die zusammenfassende Betrachtung aller Ergebnisse lässt vermuten, dass PhIP allein über lange Zeiträume aber in geringen Dosen verabreicht nicht für die hohe Zahl an Krebserkrankungen in westlichen Industrienationen ursächlich ist. / The development of colon cancer is associated with several nutritional, life style, and environmental factors. Among the environmental factors probably involved are substances formed during food processing and taken up with food. One of these substances is the heterocyclic aromatic amine (HAA) 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed during the heating of proteinaceous food such as meat and fish. In rodent studies the target organs for HAA-derived cancer development are identical with human organs showing high tumor incidences in western countries. Whether there is an association between exposure to PhIP and high tumor incidences in humans is still uncertain. The amount of PhIP administred to rodents in several studies was far above the levels of human exposure towards HAA. Thus, the aim of this study was to elucidate whether low concentrations of the substance are able to induce finger-print colon cancer gene mutations, preneoplastic lesions or tumors in rats. Animals fed with high amounts of PhIP developed fewer lesions than animals fed with a human-relevant concentration of PhIP. However none of the groups developed tumors of the colon. Both groups showed finger-print mutations for colon cancer, but not in a dose-dependent manner. Sequencing showed that the mutations were different from the known mutation spectum of PhIP. The susceptibility of the F344 rats to PhIP used in this study differed from that in previous feeding studies, with both groups showing much less lesions of the colon. Differences in composition and processing of the animal diets as well as animal maintenance and –origin may explain this discrepancy. For the first time an association between low doses of PhIP in the diet and induction of inflammation was shown. Signs of inflammation were observed macroscopically as well as in histological slices, but the mechanism of its induction remains to be clarified.<br><br> Taken together the results suggest that a chronical exposure to low doses of PhIP alone is not sufficient to explain the high incidences of colon cancer in western countries.