Thesis (Ph.D.)--Ohio University, March, 2009. / Release of full electronic text on OhioLINK has been delayed until April 1, 2010. Includes bibliographical references (leaves 70-91)

Thesis (Ph.D.)--Ohio University, March, 2009. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until April 1, 2010. Includes bibliographical references (leaves 70-91)

Thesis (Ph.D.)--Ohio University, November, 2004. / Includes bibliographical references (leaves 115-128)

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains xi, 323 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 191-211).

Thesis (Ph.D.)--Memorial University of Newfoundland, Faculty of Medicine, / Typescript. Bibliography: leaves [199]-[223].

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 2001. / Typescript. Bibliography: leaves 123-144.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, / Typescript. Bibliography: leaves 184-211.

Submitted by Nelci Antunes de Moura (nelcimoura@gmail.com) on 2016-05-20T00:29:22Z No. of bitstreams: 1 tese Nelci final.pdf: 3449766 bytes, checksum: 82850e9746cb84286bf3b3204344f1e9 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-05-23T19:39:07Z (GMT) No. of bitstreams: 1 moura_na_dr_bot.pdf: 3449766 bytes, checksum: 82850e9746cb84286bf3b3204344f1e9 (MD5) / Made available in DSpace on 2016-05-23T19:39:07Z (GMT). No. of bitstreams: 1 moura_na_dr_bot.pdf: 3449766 bytes, checksum: 82850e9746cb84286bf3b3204344f1e9 (MD5) Previous issue date: 2015-12-18 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O ferro heme presente na carne vermelha está associado ao aumento da incidência do câncer colorretal (CCR). O heme pode catalisar a formação de compostos nitrosos e a peroxidação lipídica no lúmen intestinal. No entanto, os efeitos pró-carcinogênicos do heme podem ser inibidos por alguns compostos como os sais de cálcio, clorofila entre outros. Sabe-se que o indol-3-carbinol (I3C), presente nas plantas da família das Brassicas e os simbióticos são compostos promissores na prevenção do câncer de cólon, atuando em via de proliferação, apoptose e modulação da microbiota intestinal. Dessa forma, o objetivo desse estudo foi o de avaliar os efeitos da ingestão de simbiótico (prebiótico inulina associado ao probiótico Bifidobacterium lactis bb-12) e de I3C, isolados ou em associação sobre o processo de carcinogênese de cólon induzido pela 1,2-dimetilhidrazina (DMH) em ratos Wistar alimentados ou não com dieta suplementada com heme. Os animais foram alocados em 9 grupos, os grupos 1 a 8 (n=12) receberam quatro doses de DMH (40 mg/Kg) nas duas semanas iniciais do experimento. Os grupos 1 e 9 (n=12 e 5) receberam ração basal até o final do experimento e os grupos 2 a 8 receberam ração basal suplementada com heme, heme+I3C, heme+simbiótico, heme+I3C+simbiótico, I3C, simbiótico e I3C+simbiótico, respectivamente. A eutanásia ocorreu ao final da 25ª semana. Neste momento foi realizada a coleta do cólon com os respectivos tumores e amostras de fezes do ceco. Em seguida, procedeu-se a medida dos tumores e coleta de amostras para biologia molecular. Após a fixação em formalina tamponada e a retirada dos tumores, realizou-se a contagem de focos de criptas aberrantes (FCA) pela coloração de azul de metileno. Realizou-se a análise histológica dos tumores e a análise da expressão de 95 genes relacionados a via da carcinogênese colônica, pela técnica Taqman Low Density Array, e a expressão proteica da E-caderina, TGFB1 (Transforming growth factor beta 1) e RAF1 (Serine/threonine-protein kinase) por Western Blotting. Foram analisados os índices de proliferação celular e apoptose pelo PCNA (Proliferating cell nuclear antigen) e caspase 3-clivada, respectivamente, tanto nos cólons como em tumores, e a expressão de β-catenina e E-caderina nos tumores, por imunoistoquímica. Células da linhagem Caco-2 foram incubadas com água fecal extraída das fezes do ceco e submetidas a testes de citotoxicidade e genotoxidade pelos testes do MTT (mitochondrial tetrazolium test) e Cometa, respectivamente. Os dados foram comparados utilizando-se o software Sigma Stat 3.5 e Expression Suíte para expressão gênica. Foi observado aumento significativo no número de criptas aberrantes (CA) no grupo que recebeu heme (G2) quando comparado ao grupo que recebeu apenas ração basal (G1). Redução significativa no número de CA foi observada no grupo que recebeu heme+I3C (G3) e heme+simbiótico (G4) quando comparado ao grupo que recebeu heme (G2). O número de FCA totais com ≥ 9 criptas aberrantes foi significativamente menor no grupo que recebeu heme+simbiótico (G4) quando comparado ao grupo que recebeu heme (G2). Entretanto, aumento significativo no número de tumores com mais de 60 mm3 foi observado no grupo suplementado com heme+I3C+simbiótico (G5), quando comparado ao grupo que recebeu heme (G2). Além disso, foi observado aumento significativo na incidência de tumores invasivos no grupo que recebeu heme+I3C+simbiótico (G5) quando comparado ao grupo que recebeu heme (G2). Os tumores do grupo suplementado com heme+I3C+simbiótico (G5) apresentaram baixa expressão dos genes Cdh1, Tgfb1, Appl1 e alta expressão do Raf1, já os tumores do grupo suplementado com heme +I3C (G3) apresentaram baixa expressão do Cdh1. A água fecal do grupo que recebeu heme (G2) apresentou significativamente maior citotoxicidade e genotoxicidade quando comparado ao grupo que recebeu ração basal (G1). Com relação aos tratamentos, a água fecal do grupo que recebeu heme+I3C (G3) e heme e simbiótico (G4) apresentaram água fecal significativamente com menor potencial genotóxico quando comparada ao grupo que recebeu heme (G2). No entanto, o grupo que recebeu heme+I3C+simbiótico (G5) apresentou aumento significativo na genotoxicidade da água fecal. Dessa forma, concluímos que o heme associado a uma dieta com níveis normais de cálcio não é um potente indutor de FCA, mas aumenta a citotoxicidade e genotoxicidade da água fecal. No entanto, tanto o I3C como o simbiótico reduzem os efeitos citotóxicos/genotóxicos da ingestão de heme. Contudo, a associação do heme+I3C+simbiótico apresentou efeito promotor da carcinogênese de cólon. / Colorectal cancer (CRC) is the third most common type of cancer worldwide. Hemin iron, which is found in red meat, catalyzes the formation of carcinogenic N-nitroso compounds and lipid peroxidation end-products in the colon lumen. The procarcinogenic effect of hemin is known to be inhibited by molecules, such as calcium, chlorophyll and others. However, the preventive effect of indole 3-carbinol and synbiotics on colon carcinogenesis remains uninvestigated. The aim of this study was to assess the modifying effects of a synbiotic (inulin+ Bifidobacterium lactis) and/or I3C against dimethylhidrazine (DMH)-induced colon carcinogenesis in hemin-fed male Wistar rats. Nine groups of animals were evaluated. Groups 1–8 received a total of four s.c. DMH injections (40 mg/kg b.w.) over 2 weeks, whereas group 9 was given EDTA solution (vehicle). Two weeks after DMH-initiation, G1 and G9 were fed a basal diet while groups G2, G3, G4, G5, G6, G7 and G8 received a basal diet containing hemin, hemin+I3C, hemin+synbiotic, hemin+I3C+synbiotic, I3C, synbiotic and I3C+synbiotic, respectively, during 23 weeks. At 25 week, all animals were killed and their colons were removed. Cecal contents were collected to determine fecal water cytotoxicity and genotoxicity (DNA damage) in Caco-2 cells. Colon tumors were measured and samples were collected and stored at -800C. The colons were fixed flat in 10% buffered formalin for 24 h and stained with 1.0% methylene blue for classical ACF analysis and quantification. Tumor incidence and multiplicity were assessed after histopathological analysis. Gene and protein expression were determined in tumor samples alone. The total number of aberrant crypts (AC) was significantly higher (p= 0.03) in the hemin group (G2) than in the group fed basal diet (G1). AC number in both hemin+I3C (G3) and hemin+synbiotic (G4) groups was also significantly lower than in the group fed hemin (G2). Tumor volume was higher in the hemin+I3C+ synbiotic (G5) group and invasive adenocarcinoma was more frequent in the hemin+I3C+synbiotic group (G5) than in the group fed hemin (G2). Colon tumor expression analysis showed that in comparison with the group fed hemin (G2), Cdh1, Tgfb1 and Appl1 were downregulated while Raf1 was upregulated in the group hemin+I3C+synbiotic (G5), and Cdh1 was down-regulated in the group hemin+I3C (G3). Fecal water cytotoxicity in the hemin group (G2) was higher than in groups fed basal diet (G1) and hemin+I3C (G3). Fecal water genotoxicity was also significantly higher in the group fed hemin alone (G2) than in the basal diet group (G1), as well as, in groups fed hemin+I3C (G3) and hemin+synbiotics (G4). However, when compared to hemin alone (G2), fecal water from group hemin+I3C+ synbiotics (G5) presented the highest DNA damage levels. Our results suggest that although hemin in a regular-calcium diet was not a powerful ACF promoter, it increased fecal water citotoxicity and genotoxicity. On the other hand, hemin associated with either I3C or synbiotics prevented ACF promotion. Nonetheless, a synergistic interaction among hemin, I3C and synbiotic did promote DMH-induced tumorigenesis. / FAPESP: 2011/23699-4

No description available.

Cervical malignancy is the fourth most common cause of cancer-related mortality in women worldwide; infection with high-risk human papillomavirus (HRHPV) is responsible for over 500,000 cases of cervical carcinoma each year, approximately 90% of which are squamous cell carcinomas (SCCs). Over half of all HPV-positive cervical SCCs are caused by the deregulated expression of HPV16 oncogenes E6 and E7 in proliferating basal cells of the cervical squamous epithelium. The major risk factor associated with cervical neoplastic progression is integration of HRHPV into the host genome, which is detected in $~$85% of HPV16-positive cervical carcinomas. The work presented in this doctoral thesis sought to provide insights into our understanding of the process of HPV16 integration as well as to elucidate mechanisms that deregulate both virus and host gene expression following integration. The W12 cell model system used in this project is a polyclonal cervical keratinocyte line generated by explant culture of a low-grade cervical squamous intraepithelial lesion (LSIL) that arose following natural infection with HPV16. The W12 clones were isolated in the absence of selective pressure, and as such represent the range of integration events that occur in a pre-malignant lesion at the early stages of carcinogenesis, prior to integrant selection. Despite identical genetic backgrounds, expression levels of oncogenes E6 and E7 varied up to 16-fold between the W12 clones. Expression of HPV oncogenes is ultimately determined by transcription factor binding to the non-coding long control region (LCR) of the viral genome. The initial result of this study found that genomic mutations affecting transcription factor binding at the LCR of the W12 clones was not a cause of differential viral expression, concluding that epigenetic control may be at play. Higher levels of virus expression per template were associated with increased levels of histone post-translational modification (PTM) hallmarks of transcriptionally active chromatin and reduced levels of repressive hallmarks. There was greater abundance of the active/elongating form of the RNA polymerase-II enzyme (RNAPII-Ser2P), together with CDK9, the component of positive transcription elongation factor-b (P-TEFb) responsible for the Ser2 phosphorylation. The changes observed were functionally significant, as cells with higher HPV16 expression per template showed greater sensitivity to depletion and/or inhibition of histone acetyl transferases and CDK9, as well as reduced sensitivity to histone deacetylase inhibition. Employing next generation sequencing data available for five representative W12 clones, the sites of HPV16 host integration were identified. The three-dimensional (3D) structure of the nucleus and physical interactions between stretches of the genome over long distances (i.e. enhancer and promoters) are known to exert an additional level of gene regulation. Identification of 3D virus-host interactions in the W12 clones employing the newly developed and unique 'Sequence Capture of Regions Interacting with Bait Loci Hi-C' (SCRiBL-Hi-C) protocol showed that both short- ($~$50 kb), and long-range ($~$1 Mb) interactions occur during the early stages of carcinogenesis. Together, the data in this thesis indicate that transcription and subsequent expression of the HPV16 genome is controlled by multiple layers of epigenetic regulation.