Source: Masters Abstracts International, Volume: 48-02, page: . Advisers: Mary E. Edgerton; Michael W. Ross. Includes bibliographical references.

Ductal carcinoma in situ (DCIS); is thought directly to precede invasive breast cancer (IBC). Screening mammography has driven the incidence of this key precursor lesion to >65,000 cases per year. However, little is known about the factors controlling the natural history or risk for recurrence following treatment of a particular patients DCIS. Though the heterogeneity of the disease is well established, no histologic or demographic criteria have been able to stratify DCIS for treatment. We hypothesize that at initial diagnosis there exist biologically distinct subsets of DCIS with associated prognoses that may be recognized by molecular markers. Molecular approaches have been limited by technical design issues related to the types of tissue available for analysis, namely degraded formalin-fixed paraffin embedded (FFPE) specimens and small core biopsy samples. However, new technologies promise to overcome these issues. In the first phase of our investigation, we aimed a) to pilot feasibility studies on the use of FFPE DCIS for molecular analyses including gene expression microarray and b) to pilot feasibility study of selective, high throughput sequencing through the use of "exon capture" on small input material that simulated expected DCIS core biopsy amounts. The results of this work offer specific technical guidelines for the molecular study of DCIS. Moreover, they have enabled the initiation of the second phase of this study, which aims to assess molecular profiles of DCIS recurrence and progression.

Tse, Ka Yan Agnes. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 106-113). / Abstract also in Chinese. / Table of Contents --- p.i / List of Figures and Tables --- p.iii / List of Abbreviations --- p.vi / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Breast cancer overview --- p.1 / Chapter 1.2 --- General mechanism of breast carcinoma --- p.5 / Chapter 1.3 --- Ductal carcinoma in situ (DCIS) --- p.7 / Chapter 1.3.1 --- Clinical features of DCIS --- p.7 / Chapter 1.3.2 --- Classification of DCIS --- p.8 / Chapter 1.3.3 --- Molecular markers for DCIS --- p.14 / Chapter 1.3.4 --- Progression of DCIS --- p.19 / Chapter 1.4 --- Aim of project --- p.20 / Chapter Chapter 2: --- Materials and Methods --- p.21 / Chapter 2.1 --- Categorization of DCIS samples using Van Nuys Grading System --- p.21 / Chapter 2.2 --- "RNA extraction and reverse transcription of formalin-fixed, paraffin embedded (FFPE) DCIS samples" --- p.22 / Chapter 2.2.1 --- "Formalin-fixed, paraffin embedded (FFPE) DCIS sample" --- p.22 / Chapter 2.2.2 --- Tissue Microarray (TMA) --- p.24 / Chapter 2.2.3 --- RNA extraction --- p.25 / Chapter 2.2.4 --- First strand cDNA synthesis --- p.30 / Chapter 2.3 --- PCR screening for potential DCIS markers --- p.31 / Chapter 2.4 --- Statistical analysis of PCR expression pattern --- p.36 / Chapter 2.4.1 --- Chi-square lest --- p.36 / Chapter 2.4.2 --- Logistic regression --- p.37 / Chapter 2.5 --- Real-time RT-PCR analysis of the expression pattern of the potential DCIS marker --- p.39 / Chapter 2.5.1 --- Real-time RT-PCR for FFPE samples --- p.39 / Chapter 2.5.2 --- Real-time RT-PCR in the study --- p.40 / Chapter 2.6 --- Statistical analysis of real-time RT-PCR results --- p.41 / Chapter 2.7 --- Immunohistological analysis of the expression pattern of the potential DCIS marker --- p.42 / Chapter 2.8 --- Evaluation of immunohistological staining --- p.44 / Chapter 2.9 --- Statistical analysis of immunohistological results --- p.44 / Chapter Chapter 3: --- Analysis of expression pattern of potential DCIS markers --- p.45 / Chapter 3.1 --- Construction of DCIS data base from FFPE samples --- p.45 / Chapter 3.2 --- Analysis of expression of potential DCIS marker from PCR --- p.46 / Chapter 3.2.1 --- First strand cDNA synthesis and PCR screening --- p.46 / Chapter 3.2.2 --- Statistical analysis of PCR results --- p.60 / Chapter 3.3 --- Conclusion --- p.66 / Chapter Chapter 4: --- Real-time RT-PCR analysis of expression of selected potential DCIS markers --- p.67 / Chapter 4.1 --- Expression of ETV6 in tumor and adjacent normal tissues in different DCIS grades --- p.69 / Chapter 4.2 --- Expression of Erbb2 in tumor and adjacent normal tissues in different DCIS grades --- p.71 / Chapter Chapter 5: --- Analysis of Protein Expression Pattern of Potential DCIS Markers --- p.73 / Chapter 5.1 --- Chi square test --- p.73 / Chapter 5.1.1 --- Immunohistochemical study of ETV6 in tumor and adjacent normal DCIS --- p.74 / Chapter 5.1.2 --- Immunohistochemical study of Erbb2 in tumor and adjacent normal DCIS tissues --- p.77 / Chapter 5.2 --- Conclusion --- p.80 / Chapter Chapter 6: --- General discussion --- p.81 / References --- p.106

Orientador: Jose Andres Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T17:40:29Z (GMT). No. of bitstreams: 1 Silva_AriostoSiqueira_D.pdf: 3970975 bytes, checksum: 7d1f1e04224af1b835486da199d18d00 (MD5) Previous issue date: 2008 / Resumo: Neste trabalho criou-se uma ferramenta de simulação e modelos computacionais para estudo da carcinogênese a fim de se responder a perguntas biológicas pertinentes ao tratamento desta doença. Os modelos computacionais se basearam no modelo teórico de Evolução Somática e Invasão Mediada por Acidez, proposto por Gatenby e Gillies, e foi implementado em uma ferramenta desenvolvida pelo autor deste trabalho no âmbito deste projeto, o Tissue Simulator (TSim, www.i-genics.com). O modelo teórico de invasão mediada por acidez propõe que células tumorais possuem maior resistência a acidez, assim como produzem quantidade de ácido lático, originado da glicólise anaeróbica, suficiente para acidificar o meio extracelular, causando assim morte do tecido saudável por apoptose induzida por acidez, e facilitando a invasão do tecido saudável pelo tumor. Estudos experimentais, na literatura, mostraram que a administração de bicarbonato de sódio na água em ratos portadores de tumores reduz o número de metástases, o que seria uma indicação de que a hipótese sobre a importância da acidez na invasividade tumoral é válida. Neste estudo, criou-se um primeiro modelo computacional para testar se o aumento da concentração de bicarbonato no sangue poderia influenciar no gradiente de acidez entre o tumor (micrometástases) e o tecido saudável, e também identificaram-se as características físico-químicas de um tampão ideal a ser usado com esse propósito. O modelo teórico de Evolução Somática, adotado neste projeto, propõe que para que um tumor epitelial se torne invasivo, é necessário que suas células adquiram três fenótipos: hiperplasia, hiperglicólise e resistência a acidez. O segundo modelo criado neste trabalho consistiu na identificação de quais seriam os valores mínimos de hiperglicólise e resistência à acidez para o aparecimento da característica de invasividade em um tumor em desenvolvimento dentro de um duto epitelial (DCIS). Uma vez identificados os fenótipos mínimos para a invasão tumoral de um DCIS, restaria saber quais as mutações em enzimas ou transportadores específicos dessa via metabólica para que o dito fenótipo seja atingido. A título de exemplo de um estudo para responder perguntas desse tipo, fez-se uma análise comparativa da robustez do fluxo glicolítico em duas células distintas: uma levedura (S. Cerevisiae) e uma célula humana especializada (célula beta pancreática), cujas enzimas principais são reguladas por estratégias distintas. Este estudo foi implementado em uma ferramenta computacional disponível na literatura (Jarnac e Matlab) e resultou na publicação de um artigo. No todo, os resultados desta tese mostram que, com o uso de ferramentas computacionais e dados quantitativos da literatura, é possível criar modelos teóricoquantitativos que podem ser usados para validar teorias sobre fenômenos biológicos assim como extrapolar novas hipóteses e testá-las, integrando-se assim a modelagem computacional no processo de pesquisa científica / Abstract: In this work, we created a piece of software and computer models for studying carcinogenesis, in order to answer biological questions related to the treatment of this disease. The computer models were inspired in the theory of Somatic Evolution and Acid Mediated Tumor Invasion, proposed by Gatenby and Gillies, and were implemented in a tool developed by the author under the scope of this thesis, Tissue Simulator (TSim, www.i-genics.com). The theory of Acid Mediated Tumor Invasion proposes that cancer cells are more resistant to toxicity of an acidic environment that they help create by producing excess of lactic acid through anaerobic glycolysis. Acidification of the extra-cellular environment causes death of healthy tissue through acid-induced apoptosis and ultimately facilitates tumor invasion. Experimental studies, from literature, showed that administration of sodium bicarbonate in water to mice bearing tumors reduced the number of metastases, thus supporting the importance of acidity in tumor invasion. In this study a computer model was built to test if an increase in concentration of bicarbonate in blood serum could alter the pH gradient between the tumor (micrometastases) and halthy tissue, as well as to identify the chemical properties of and ideal buffer with this purpose. The theory of Somatic Evolution, proposes that epithelial tumor cells are submitted to environmental barriers and are selected for three main phenotypes: hyperplasia, hyperglycolysis and acid resistance. A second computer model was created in order to identify the minimum values of these phenotypes that allowed a DCIS to change into an invasive tumor. Once the minimum phenotypic values identified, one can study how mutations on specific enzymes can alter the flux of a metabolic pathway, such as glycolysis, to produce the altered phenotype. As an example of this, we performed a comparative study of robustness of glycolytic flux in two different cells: yeast (S. cerevisiae) and pancreatic human beta-cell, whose enzymatic regulatory strategies differ. This computer model was implemented on Matlab and Jarnac. Overall, our results show that the use of computational tools and quantitative data may be used to create theoretical-quantitative models that help adressing theories about biological systems, as well as to extrapolate and tes new hypothesis, integrating the approach of computational modeling in the scientific research process / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular