Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references (leaves 97-114). Also available in electronic version.

Thesis (doctoral)--University of Wisconsin-Madison, 2000. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, Aug. 2006. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.)--Indiana University, 2006. / Title from screen (viewed on Apr. 27, 2007) Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI) Includes vita. Includes bibliographical references (leaves 181-239)

Orientadores : Gustavo Antonio de Souza, Marcelo Alvarenga / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-07-28T18:11:59Z (GMT). No. of bitstreams: 1 CezarJunior_OlavoPedroso_D.pdf: 210070 bytes, checksum: 373d42d115f8ec8b7f306b27f88b8dbc (MD5) Previous issue date: 2001 / Doutorado

The eukaryotic cell nucleus is a highly organised organelle, with distinct specialised sub- compartments responsible for specific nuclear functions. Within the context of this functional framework, the genome is organised, allowing contact between specific genomic regions and sub-compartments. Previous work has shown that genes in both cis and trans can make specific contacts with each other. I hypothesise that such a preferred juxtaposition may impact the propensity for specific cancerinitiating chromosomal translocations to occur. In this thesis, I describe how I have extended and developed a ligation based proximity assay known as enriched 4C. I have coupled this technique with high throughput sequencing to determine genomic regions that spatially co-associate with the proto-oncogenes MLL, ABL1 and BCR. In addition to further developing the laboratory protocol, I have created bioinformatics tools used in the analysis of the sequencing data. I find that the association profiles of the three genes show strong correlation to the binding profile of RNA polymerase II and other active marks, suggesting that transcribed genes have a propensity to associate with other transcribed regions of the genome. Each gene also exhibits a unique repertoire of preferred associations with specific regions of the genome. Significantly, I find that the most frequent trans association of BCR is telomeric chromosome 9, encompassing its recurrent translocation partner gene ABL1. Interestingly, ABL1 is not at the maximum point of interaction. I use DNA-fluorescence in-situ hybridisation to validate the e4C association. My data supports a hypothesis that gene transcription has a direct role on genome organisation. I suggest that preferred co-associations of genes at transcription factories may promote the occurrence of specific chromosomal translocations.

The process of oncogenic transformation in vitro has been examined in an attempt to define the molecular mechanisms of carcinogenesis. Transformation in Ki-MSV-infected rat adrenal cortex cells appears to be a multistep process (Auersperg et al., 1981), as does the process of transformation in other nonestablished cells (Land et at., 1983b). The Ki-MSV-infected adrenal cortex cells initially express a partially transformed phenotype and after further passaging progress to a highly transformed phenotype (Calderwood and Auersperg, 1984). Examination of Ki-MSV-infected adrenal cortex cells indicated that progression to a highly transformed phenotype could occur in the absence of significant changes in the level of the expression of the viral ras oncogene. These results indicated that an oncogenically activated ras gene could be expressed in these nonestablished cells in the absence of transformation. Since ras and myc cooperate to transform primary fibroblasts the effect of the co-introduction of myc on Ki-MSV-induced transformation of adrenal cortex cells was examined. It could be demonstrated that myc and ras cooperate to transform the adrenal cortex cells more efficiently than either oncogene alone, but that the infected cultures initially only express some of the phenotypes associated with transformation. The appearance of a fully transformed phenotype, as monitored by growth in soft agar, was not expressed until several passages after infection. An analysis of the Ki-MSV/MMCV-infected cultures indicated that some of the phenotypes associated with activated oncogenes in immortalized cell lines appeared to be suppressed in the coinfected adrenal cortex cells. Transformation by ras and myc appears to require a further cellular change resulting in a loss of the suppression of oncogene action. The emergence of transformed cultures from the Ki-MSV-infected rat adrenal cortex cells was correlated with the reduced expression of a novel ras-related protein of 27000 Mr. Transformation induced by src and myc was also examined. These two oncogenes appeared to cooperate in a two step pathway of transformation that was not susceptible to cellular suppression. The transformed phenotype did not appear to be entirely free of external influence as the growth rate of the transformed cells could be modified by culture conditions. The ability of myc to cooperate with src and ras in the transformation of the early passage adrenal cortex cells provides further support for mutistep carcinogenesis. The effect of oncogenes on steroidogenesis was examined in the Y-1 adrenocortical tumour cell line. The effect of the virally borne oncogenes on growth and morphology of the Y-1 cells was relatively subtle. The oncogenes appear to stimulate the production of fluorogenic steroids, each in a distinct fashion. A model of transformation can be derived in which the roles of the oncogenes and their interaction with the cell can be evaluated. The differences in the pathways of transformation for the two combinations of oncogenes illustrates the potential complexity of the transformation process and provides an in vitro model system for further study. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In classification problems, it is always important to use the suitable combination of features that will be employed by classifiers. Generating the right combination of features usually results in good classifiers. In the situation when the problem is not well understood, data items are usually described by many features in the hope that some of these may be the relevant or most relevant ones. In this study, we focus on one such problem related to genes implicated in ovarian cancer (OC). We try to recognize two important OC-related gene groups: oncogenes, which support the development and progression of OC, and oncosuppressors, which oppose such tendencies. For this, we use the properties of promoters of these genes. We identified potential “regulatory features” that characterize OC-related oncogenes and oncosuppressors promoters. In our study, we used 211 oncogenes and 39 oncosuppressors. For these, we identified 538 characteristic sequence motifs from their promoters. Promoters are annotated by these motifs and derived feature vectors used to develop classification models. We made a comparison of a number of classification models in their ability to distinguish oncogenes from oncosuppressors. Based on 10-fold cross-validation, the resultant model was able to separate the two classes with sensitivity of 96% and specificity of 100% with the complete set of features. Moreover, we developed another recognition model where we attempted to distinguish oncogenes and oncosuppressors as one group from other OC-related genes. That model achieved accuracy of 82%. We believe that the results of this study will help in discovering other OC-related oncogenes and oncosuppressors not identified as yet.

Array comparative genomic hybridization (array-CGH) was used in this study to analyze the chromosomal aberrations in 9 gastric cancer cell lines. Our results showed good concordance with those of conventional CGH. We correlated the results from array-CGH with expression profiling and found some novel and independent target genes which deserved further confirmation. / Gastric cancer is one of the most common malignancies worldwide and is the second most frequent cause of cancer related death. A variety of genetic and epigenetic aberrations underlie development abnormality of gastric cancer. / Taken together, our findings supported YAP1 is a functional oncogene in gastric cancer. We provided the first evidence that YAP1 exerted the oncogenic function by enhancing the capacity to activate the early response gene pathway. YAP1 could be a prognostic biomarker and potential therapeutic target for gastric cancer. / The study was focused on the putative oncogene Yes-associated Protein 1 (YAP1) located in 11q22.1. Up-regulation of YAP1 was observed in 92.3% of gastric cancer by immunohistochemistry (IHe) on gastric cancer tissue microarrays. YAP1 nuclear accumulation correlated with cancer specific survival. In addition, multivariate Cox regression showed that YAP1 was an independent predictor of short disease specific survival time for patients with early stage gastric cancer (P=0.042) in addition to T stage ( P=O.038). Knockdown YAP1 in gastric cancer cell lines MKN1 and AGS resulted in a significant reduction in proliferation, anchorage-dependent colony formation, cell invasion and cell motility. Ectopic YAP1 expression in MKN45 cells promoted anchorage-independent colony formation, induced a more invasive phenotype and accelerated cell growth both in vitro and in vivo. Microarray analysis highlighted the alteration of MAPK pathway by YAP1. We confirmed a constitutive activation of RAF/MEKJERK in YAP1-expressing MKN45 cells and further demonstrated that YAP1 enhanced serum/EGF induced c-Fos expression in gastric cancer cells. Furthermore, we demonstrated that ectopic MST1 promoted phosphorylation and cytoplasmic translocation of YAP1 and subsequently quenched the oncogenic function of YAP1 in the nucleus. / Kang, Wei. / "December 2010." / Adviser: To Kai-fai. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 175-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

A infecção por HPV de alto risco é considerada um dos principais fatores de risco para o desenvolvimento do carcinoma do colo uterino, um das neoplasias mais freqüentes em mulheres de todo o mundo. As oncoproteínas E6 e E7 de HPV-16 são capazes de induzir a degradação dos genes supressores de tumor p53 e pRb, respectivamente. Mais do que isso, a expressão dessas oncoproteínas está relacionada a alterações na via de PI3K/AKT/mTOR. A proteína quinase mTOR apresenta importante papel no controle da tradução de proteínas e é considerada o principal mediador entre crescimento celular e proliferação. A ativação de mTOR é correlacionada à fosforilação das proteínas eIF4G1 e 4EBP1, aumentando assim a taxa de síntese de proteínas. A Rapamicina é um inibidor específico de mTOR e seus análogos apresentam potente atividade antiproliferativa em um grande número de células tumorais e tumores gerados em animais. Uma vez que as proteínas E6 e E7 são capazes de interagir com diversas proteínas da via que controla a atividade de mTOR optamos por investigar o efeito da rapamicina na proliferação de culturas organotípicas de queratinócitos expressando esses genes. Também avaliamos o efeito dos genes E6 e E7 na atividade de mTOR após o tratamento com essa droga. Para geração de culturas organotípicas de queratinócitos infectamos essas células com vetores retrovirais recombinantes contendo os genes E6 e E7 de HPV-16 em conjunto ou separadamente. Nós também avaliamos o papel da degradação de p53 e pRb na resposta à rapamicina através da utilização de mutantes de E6 e E7 incapazes de induzir a degradação dessas proteínas celulares. Após a infecção dos queratinócitos, os mesmos foram semeados em uma matriz de colágeno. Após 6 dias as culturas foram tratadas com 100ng/ml de rapamicina e permaneceram 60h em contato com a droga. Para análise por imunohistoquímica os tecidos foram fixados em formalina tamponada e emblocados em parafina. A reação de imunohistoquímica foi realizada utilizando os anticorpos contra BrdU, p-4EBP1 (ser 65), p-eIF4G1 (ser 1188) e pAKT (ser 473). Os resultados obtidos ilustram que a rapamicina apresenta efeito antiproliferativo em culturas de queratinócitos contendo o vetor vazio. Por outro lado, culturas contendo o gene E7 são resistentes ao efeito antiproliferativo dessa droga. Essa resistência parece estar relacionada à capacidade de E7 induzir a degradação da proteína pRb, uma vez que em queratinócitos expressando o mutante de E7, incapaz de induzir a degradação dessa proteína, não foi observada resistência. Além disso, a fosforilação de eIF4G e 4EBP1 indica que a expressão de E7 impede que a rapamicina seja capaz de inibir a atividade de mTOR. Esses resultados mostram, pela primeira vez, que o efeito antiproliferativo da rapamicina pode ser superado pela expressão de uma proteína viral, no caso a proteína E7 de HPV-16. / High-risk HPV infection has a major etiologic role in development and progression of cervical cancer, one of the most frequent forms of cancer among women worldwide. HPV-16 E6 and E7 oncoproteins are able to induce degradation of p53 and pRb tumor suppressor proteins respectively. Moreover, the expression of these oncoproteins is related to alterations in the PI3K/AKT/mTOR pathway. The cellular kinase mammalian target of Rapamycin (mTOR) is an important regulator of the cellular protein synthesis machinery and has emerged as a principal mediator of cell growth and proliferation. mTOR activation has been shown to stimulates eIF4G1 and 4EBP1 phosphorylation, thus increasing the rate of protein synthesis. Rapamycin is a specific inhibitor of mTOR signaling pathway and its analogues have demonstrated impressive activity against a broad range of human cancer derived cell lines in culture and in human tumor xenograft models. Since E6 and E7 target several proteins controlling the mTOR pathway we aimed to investigate the effect of Rapamycin in the proliferation of organotypic raft cultures expressing these genes. We also evaluated the effect of E6 and E7 genes in mTOR activity after rapamycin treatment. To generate organotypic culture of keratinocytes we infect these cells with recombinant retroviruses containing HPV-16 E6 and E7 together or separately. We also analyzed the role of p53 and pRb degradation in rapamycin responsiveness by using E6 and E7 mutants lacking the hability to inactivate these cellular proteins. After infection, keratinocytes were seeded on to a collagen matrix. After 6 days, these cultures were treated with 100ng/ml of Rapamycin for 60 hours. BrdU was added in the last 12 hours to evaluate proliferation. For immunohistochemistry analysis tissues were fixed in buffered formalin and embedded in paraffin. Immunohistochemistry reactions against BrdU, p-4EBP1 (ser 65), p-eIF4G1 (ser 1188) and p-AKT (ser 473) were performed The results show that proliferation of organotypic cultures of keratinocytes transduced with empty vector is inhibited by Rapamycin. On the other hand, cultures generated with keratinocytes transduced with E7 gene were completely resistance to the antiproliferative effect of Rapamycin. Moreover, we found that this antiproliferative effect was dependent of Rb degradation since the cells transduced with E7 mutant unable do induce Rb degradation were sensitive. In addition, eIF4G and 4EBP1 phosphorylation indicates that E7 expression impairs mTOR inhibition by rapamycin. AKT phosphorilation indicates that rapamycin resistance could be dependent of Rb inactivation induced by E7 expression. These results show for the first time that the Rapamycin antiproliferative effect is bypassed by the expression of a viral oncogene, in this case the HPV-16 E7. Moreover, E7 expression impairs rapamycin to inactivate mTOR.