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)

Kaiso is a POZ-ZF transcription factor initially identified as an interaction partner for the cell adhesion co-factor p120^ctn. Kaiso-DNA binding is inhibited by p120^ctn, implicating p120^ctn in the regulation of Kaiso transcriptional activity. In this study, Kaiso repressed transcription of a luciferase reporter carrying four copies of the sequence-specific Kaiso-binding site (4xKBS) in artificial promoter assays. Mutation of the 4xKBS which is known to disrupt Kaiso-DNA binding also abrogated Kaiso-mediated transcriptional repression. Moreover, p120^ctn inhibited Kaiso-mediated transcriptional repression via the 4xKBS, yet neither the p120^ctn deletion mutant ΔR3-ll (lacking the Kaiso binding site) or p120^ctn NLS mutant (which cannot enter the nucleus) inhibited transcriptional repression. Furthermore, in NIH 3T3 cells (which do not demonstrate a Kaiso-pl20ctn interaction), pl20ctn failed to inhibit transcriptional repression. Many POZZF transcriptional repressors recruit an HDAC complex via their POZ domain to repress transcription. To investigate the mechanism of Kaiso-mediated transcriptional repression, the POZ domain of Kaiso was deleted, which abrogated transcriptional repression. Kaiso immunoprecipitates contained HDAC activity, and the HDAC co-repressor Sin3A co-immunoprecipitated with Kaiso, implying that Kaiso recruits Sin3A to repress transcription in an HDAC-dependent manner. Lastly, Kaiso repressed transcription via a human 𝑚𝑎𝑡𝑟𝑖𝑙𝑦𝑠𝑖𝑛 promoter fragment. This suggests that the KBS element is functionally relevant and implicates 𝑚𝑎𝑡𝑟𝑖𝑙𝑦𝑠𝑖𝑛 as a Kaiso target-gene. Collectively, these data establish Kaiso as a sequence-specific, HDAC-dependent transcriptional repressor that is regulated by the adhesion co-factor p120^ctn. / Thesis / Master of Science (MSc)

The Wnt/β-catenin pathway is a fundamental regulator of embryonic development and adult tissue homeostasis. The key effector, β-catenin, is a multifunctional protein that occupies dual roles in signalling and intercellular adherens junctions. β-catenin primarily signals though the TCF/LEF transcription factors; however, many transcription factors, in addition to TCF/LEFs, interact with β-catenin, and the function of these interactions is poorly understood. To investigate novel β-catenin regulated signalling mechanisms with certainty, we developed TCF/LEF quadruple knockout (QKO) mESCs. In vitro differentiation of QKO cells reveals a neural differentiation bias, which is attenuated by overexpression of stabilized β-catenin. Our data indicate the presence of a TCF-independent β-catenin regulated neural differentiation blockade in mESCs. In addition to directly challenging the central dogma of canonical Wnt signalling, this finding has the potential to unveil new therapeutic targets for the treatment of many β-catenin-associated diseases, including forms of brain cancer that may arise from the oncogenic stimulation of neural stem cells. Furthermore, we describe an attempt to identify genome-wide TCF-independent β-catenin binding sites in QKO cells by ChIP-seq. Optimization trials provide proof of concept that the fold enrichment method of interpreting ChIP-qPCR results can be highly misleading when compared to the more comprehensive % input method of analysis. This conclusion has important implications for all fields of scientific research in which ChIP-seq methodology is employed. / Thesis / Master of Science (MSc)

By Western blotting and immunohistochemical analyses, we have demonstrated that Pin1 was overexpressed in 71.4% of hepatocellular carcinoma (HCC) and its levels correlated with the clinical survival rate. This conclusion was supported by the results from examining Pin1 protein in HCC cancer cell lines. RT-PCR was performed to examine the Pin1 transcription level in tumor part and was compared with that in non-tumor part. Our results indicated that pin1 overexpression was due to the upregulation of Pin1 transcription. Interestingly, most of the cases with upregulation of Pin1 have been shown to correlate with £]-catenin and Cyclin D1 accumulation in HCC specimens. These results were consistent with the previous studies that Pin1 caused £]-catenin and Cyclin D1 elevation in breast cancer. The concordance between hepatitis virus chronic infection and Pin1 overexpression of HCC patients was also analysis. Taken together, these data indicated that Pin1 overexpression leading to £]-catenin and Cyclin D1 accumulation might play a critical role in hepatocellular carcinogenesis and tumor progression. Pin1 levels therefore can be used as a prognostic marker for HCC, and our results suggested that Pin1 is a potential target for therapeutic intervention in hepatocellular carcinoma.

Orientador: Antonio Gonçalves de Oliveira Filho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T04:54:32Z (GMT). No. of bitstreams: 1 Patricio_FlaviaRezendePereira_M.pdf: 2174141 bytes, checksum: 0011ea137b3f271896d984e54bb4f4d6 (MD5) Previous issue date: 2009 / Resumo: Tumores do córtex da supra-renal (TCSR) em crianças são raros e correspondem a 0,2% dos tumores da infância. Estes tumores são endocrinologicamente ativos, causando na maioria das vezes, virilização do paciente associada, ou não, a um aumento de cortisol. O tratamento dos TCSR é principalmente cirúrgico, sendo a cirurgia com o procedimento de ressecção completa do tumor e sem ruptura a principal modalidade terapêutica. No entanto, a distinção entre tumores benignos e malignos, baseada exclusivamente na histologia, pode ser difícil de ser realizada. Os fatores prognósticos são baseados quase exclusivamente no estadiamento da doença, o qual leva em conta o peso e volume tumoral e a disseminação metastática do tumor. Estudos clínicos e experimentais sugerem que a propagação metastática em alguns tumores está relacionada aos níveis de E-caderina e ?-catenina, que são moléculas presentes no tecido epitelial normal, estando envolvidas diretamente na adesão intercelular. A análise da expressão destas moléculas pode fornecer dados que permite identificar grupos de pacientes mais propensos à evolução tumoral desfavorável, proporcionando, assim, um tratamento mais adequado e individualizado a estes pacientes. Com o objetivo de analisar a expressão da E-caderina e ?-catenina em crianças com TCSR e sua correlação com a evolução da doença, foi realizada uma revisão retrospectiva dos prontuários de 33 crianças com diagnóstico de TCSR tratadas no Centro Infantil Boldrini (Janeiro de 1998 a Janeiro de 2005). Foram coletados e analisados dados referentes ao sexo, idade, manifestações clínicas, estadiamento, tratamento e evolução dos pacientes. Para a análise imunoistoquímica, foi empregada a técnica de multitissue array com anticorpos específicos para E-caderina e ?-catenina em 30 tumores de crianças com TCSR e uma adrenal normal. Houve predominância do sexo feminino na amostra e a apresentação clínica mais freqüente foi a virilização. Nesta série, observou-se que crianças com idade inferior a dois anos apresentaram melhor prognóstico e, também que a ruptura e recidiva tumoral apresentaram influência negativa na sobrevida dos pacientes. A análise imunoistoquímica mostrou expressão da E-caderina em 73,3% e ?-catenina em 83,3% das crianças que apresentavam TCSR. Além disso, não foi verificada sua expressão na glândula adrenal normal. Quando avaliada a relação da expressão da E-caderina e ?-catenina com os estádios evolutivos da doença, não foi verificada associação significativa entre as variáveis. A positividade da E-caderina e ?-catenina na membrana celular, citoplasma ou núcleo, verificou-se que a expressão na membrana celular mostrou associação significativa com mau prognóstico / Abstract: Adrenal cortical tumor (ACT) in children are rare and they correspond to 0,2% of the tumors of the childhood. They are usually active, causing mainly virilization of patient associate or not with increased levels of corticoids. The treatment of the TCSR is mainly surgical, being the surgery with complete resection of the tumor without spillage the therapeutic mainstay. The distinction between benign and malignant tumors based exclusively on the histology can be difficult and very often uncertain. The prognostic factors are based almost exclusively on the staging of the disease, who takes into account the weight and volume tumoral and the metastatic dissemination. Clinical and experimental studies suggest that metastatic dissemination in some tumors is related with the levels of E-cadherin and ?-catenin. These molecules are found in the normal epithelial tissues and are strongly related with intercellular adhesion. The analysis of the expression of these molecules maybe can allow identifying groups of patients with higher risk of presenting unfavorable outcomes and ensuing appropriate and individualized treatment. With the objective of analyzing the expression of E-cadherin and ?-catenin in children with ACT and its correlation with the evolution of the disease, a retrospective chart review of 33 children with diagnosis of ACT treated at Centro Infantil Boldrini (January of 1998 through January of 2005). Data regarding sex, age, clinical presentation, staging, treatment and outcome were collected and analyzed. Multitissue array technique using specific antibodies for E-cadherin and ?-catenin was done in 30 tumors from children with ACT and 1 normal adrenal tissue. There was predominance of the feminine sex and the most frequent clinical presentation was virilization. In these series children with age bellow two years had a better outcome and tumoral spillage and relapse have had negative influence in the survival of the patients. Immunohistochemical analysis showed expression of E-cadherin in 73,3 % and ?-catenin in 83,3 % of the children who had ACT but showed no expression in the normal adrenal tissue. When the relationship between the expression of E-cadherin and ?-catenin with the stages of the disease was analyzed, no significant association was found. When analyzed the expression of E-cadherin and ?-catenin in the cellular membrane, cytoplasm or nucleus, its presence in the membrane of the cell was found as associated with poor outcome. As far we know, this is the first study to evaluate the expression of E-cadherin and ?-catenin in children with ACT and, although with small number of patients due to the rarity of the disease, it apparently shows some relationship with prognosis. When the relationship between the expression of E-cadherin and ?-catenin with the stages of the disease was analyzed, no significant association was found. When analyzed the expression of E-cadherin and ?-catenin in the cellular membrane, cytoplasm or nucleus, its presence in the membrane of the cell was found as associated with poor outcome. As far we know, this is the first study to evaluate the expression of E-cadherin and ?-catenin in children with ACT and, although with small number of patients due to the rarity of the disease, it apparently shows some relationship with prognosis / Mestrado / Cirurgia / Mestre em Cirurgia

Master of Science - Biology / á-catenin plays a crucial role in cell adhesion. Expression levels of á-catenin have been shown to be decreased in almost all tumours studied. The levels of the epidermal growth factor receptor (EGFR) were shown to be increased in oesophageal squamous cell carcinoma (OSCC) cell lines. á-catenin therefore, may play a part in linking the EGF pathway or other signal transduction pathways, bringing about some of the changes in the OSCC cell lines. The á-catenin gene from five OSCC cell lines was sequenced. Three out of five OSCC cell lines studied were found to harbour mutations. One of the mutations resulted in a change in the amino acid sequence of á-catenin. It was concluded that this alteration may not have affected the functioning of á-catenin. á-catenin was largely expressed at the plasma membrane with some weaker cytoplasm/nuclear expression occurring in all of the OSCC cell lines. Treatment of the OSCC cells with EGF for a 12 hour period resulted in no noticeable change in the expression levels of á- catenin. The results obtained from this study indicated that á-catenin could play a role in signal transduction pathways in the OSCC cell lines.

La réponse antivirale innée constitue la première réaction d’une cellule à une infection virale. Il s’agit d’une réponse rapide, transitoire, non-spécifique, ubiquitaire qui a été conservée sous différentes formes au cours de l’évolution. La synthèse d’interféron β (IFNβ) joue un rôle essentiel dans l’établissement de la réponse antivirale innée chez les vertébrés. L’expression du gène Ifnb1 codant pour l’IFNβ est finement régulée au niveau transcriptionnel, ce qui permet de passer de la répression de son expression en absence d’infection à son activation après infection par un virus. Bien qu’une forte et rapide expression d’IFNβ soit bénéfique lors d’une infection virale, une expression anormale d’IFNβ peut entrainer des troubles physiologiques importants (réactions auto-immunes, dérégulations inflammatoires). En s’intéressant plus particulièrement à la séquence promotrice du gène Ifnb1, codant pour l’IFNβ, nous avons identifié deux sites de liaison potentiels pour les facteurs de la famille des TCFs (T-Cell Factor). En présence de β-caténine nucléaire, les complexes TCF/β-caténine se forment au niveau des sites de liaison aux TCFs et recrutent des complexes co-activateurs de la transcription. Dans une première partie de ce travail, nous avons étudié le rôle des complexes TCF/β-caténine dans la régulation de l’expression d’Ifnb1 aussi bien en absence que suite à une infection virale. Nous avons ensuite montré que ce mécanisme était fonctionnel car il permettait aux cellules traitées par un inhibiteur de GSK3 (qui induit une accumulation de β-caténine) de mieux se protéger contre l’effet cytopathique induit par le VSV. Lors de l’étude des fonctions biologiques ciblées par le Virus de la Fièvre de la Vallée du Rift (VFVR) auquel j’ai participé, il est apparu que cet arbovirus hautement pathogène ciblait via sa protéine non-structurale NSs, les promoteurs de gènes codant pour des nombreux acteurs de la voie Wnt/β-caténine et de l’adhésion cellulaire. Dans une deuxième partie de ce travail, j’ai analysé l’effet d’une infection par la souche pathogène (+NSs) ou non-pathogène (-NSs) du VFVR sur le taux de β-caténine et la présence de celle-ci au niveau des jonctions adhérentes. Les résultats obtenus montrent que l’infection par la souche pathogène de VFVR entraine une diminution du taux global de β-caténine ex et in vivo ainsi que la redistribution de celle-ci en dehors des jonctions adhérentes, couplée à une très forte désorganisation du cytosquelette d’actine de la cellule infectée. Cette perturbation du réseau d’actine est corrélée à la dérégulation de l’expression de certains gènes codant pour des protéines affectant la morphologie cellulaire. / No abstract available

B-catenin is a transcriptional co-activator in the Wnt signaling pathway. Upon Wnt stimulation, cytosolic B-catenin translocates into the nucleus where it forms complexes with members of the TCF /LEF family of transcription factors to activate gene transcription. Translocation into the nucleus is followed by transcriptional activation of B-catenin's target genes, which are involved in proliferation, angiogenesis and oncogenesis, is a crucial step in the progression of a subset of cancers. The cellular expression of B-catenin is known to be regulated by phosphorylation. However, the mechanisms(s) responsible for B-catenin nuclear entry is not well understood. Recently, B-catenin was reported to be post-translationally modified by O-glycosylation in breast cancer cells. We investigated whether O-glycosylation regulates the signal transduction properties of the protein. Our results indicated that while there are higher levels of total B-catenin in the nucleus of two prostate cancer cell lines (DU-145 and LNCaP) compared to that in a normal prostate epithelial cell line (PNT1A), most of it was in the unglycosylated form. Also, the normal prostate cell line exhibited higher levels of O-glycosylated B-catenin in both the nucleus and cytosol than what was seen in the two prostate cancer cell lines. We carried out further experiments using PUGNAc, a non-cytotoxic reversible inhibitor of O-GlcNAcase, which causes a time dependent increase in cellular levels of O-glycosylated B-catenin. Treatment of prostate cancer cells with PUGNAc caused a decrease in the expression of B-catenin in the nucleus with increasing cellular O-glycosylation of the protein suggesting that O-glycosylation was hindering B-catenin nuclear translocation. Additional studies showed that O-glycosylation of B-catenin decreased that transcriptional activity of a TopFlash reporter plasmid and the protein expression of two B-catenin target genes. Our results suggest that O-glycosylation of B-catenin may represent a novel mechanism important in the regulation of the nuclear localization and transcriptional activity of B-catenin. / Thesis / Master of Science (MS)

No description available.

Student No. 9404705H MDent Research Report School of Oral Sciences / Expression of the E-cadherin/β-catenin complex in oral squamous cell carcinoma and its correlation with histomorphology and metastasis. The immunohistochemical expression of E-cadherin and β-catenin was examined in 30 primary oral squamous cell carcinomas in patients with (n=19) and without (n=11) nodal metastasis, as confirmed on histopathological examination of the resected regional lymph nodes. The corresponding primary and nodal metastases tissue samples were available for 17 patients. The 30 primary carcinomas were histologically graded according to the invasive tumour front grading system and by conventional Broders’ criteria. None of the 30 primary carcinomas showed homogenous, membranous E-cadherin and β-catenin expression when compared to the normal oral squamous epithelium. Staining was heterogeneous in 73% (22/30) and in 77% (23/30) of the primary carcinomas stained for E-cadherin and β-catenin respectively. There was a highly significant reduction (P<0,001) of both E-cadherin and β- catenin expression with progression from the well-differentiated areas to the less differentiated tumour cells at the invasive tumour front. At the invasive tumour front, however, irrespective of the nodal status and invasive tumour front grading score, 28/30 (93%) tumours showed loss of E-cadherin expression. Loss of β-catenin expression was recorded in 22/30 (73%) cases. These findings indicate that E-cadherin and β-catenin play a key role in the loss of differentiation of tumour cells in oral squamous cell carcinoma and while they may be permissive for metastasis, in isolation, E-cadherin and β-catenin are probably not predictive of metastatic potential in oral squamous cell carcinoma.