Submitted by (edna.saturno@ufrpe.br) on 2016-10-18T17:21:44Z No. of bitstreams: 1 Maria Juliana C D S Teixeira.pdf: 3669007 bytes, checksum: 05acdce0c853db1f30e76e43a1a72023 (MD5) / Made available in DSpace on 2016-10-18T17:21:44Z (GMT). No. of bitstreams: 1 Maria Juliana C D S Teixeira.pdf: 3669007 bytes, checksum: 05acdce0c853db1f30e76e43a1a72023 (MD5) Previous issue date: 2007-02-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The major incidence and the high level of mortality associated to the huge diversity of the cancer phenotypic require numerous strategies for its diagnosis. In this research, the aim was to evaluate the mutation relation in the gene Tp53 with the breast cancer in female dogs, through the immunohistochemistry expression of the protein p53 and of the gene polymorphism in the breast tissues. In the Experiment 1, was studied the immunohistochemistry expression of the protein p53 in relationship to the morphologic aspects of healthy mammary tissues, to classification and grade of tumors and to the ipsilateral inguinal lymph-nodes tissue, metastatic or not. For this purpose 19 breasts from healthy female dogs were used (Group 1- control), 29 samples of breasts with clinical diagnosis of tumor (Group 2), 29 contralateral breasts (Group 3) and 29 lymphnodes (Group 4). The material was analyzed by conventional techniques of microscopy (staining HE) for tissue’s characterization and by immunohistochemistry (estreptavidina-biotina peroxidase method) to evaluate the expression of the protein p53 in the cellular compartment. The contralateral breasts and the lymph-nodes that showed metastasis were also marked, as well as the tissues that still showed normality. There weren´t statistical differences between the staining intensity and the number of cells stained with malignity grade. In the Experiment 2 the aim was to detect mutations in sequences of the exon 8 of the gene Tp53 through the PCR-RFLP technique with specific restriction enzymes to associate with band patterns obtained to the breast tumor type or normal tissues in female dogs. Were used 19 healthy female dogs (control) and 50 female dogs submitted to the tumor exerese from wich samples of tumor tissue and of healthy contralateral mammary glands were obtained. The DNA extraction was done through the fenol-cloroformic method and the PCR-RFLP technique was used for amplifiation and digestion of the exon 8 of the Tp53 with 6 restriction endonucleases, SmaI, DdeI, Rsa, AvaI, BsobI e AluI. The amplified DNA showed only one side of 200 pb in the agarose gel (0,8%). The PCR product showed on average 94% of homology with the known sequence of the gene (GeneBank –AAB42022.1). Among the 6 chosen enzymes, only 4 showed polymorphism (AluI, BsobI, DdeI e SmaI) in the tumor sequence of the exon 8 as to the numbers of pairs of the base (pb) or the quantity of cleavages. The band patterns of each enzyme showed polymorphism among the different groups, but not inside each group (normal female dogs, tumor mammary glands and contralateral breasts). There was´nt any difference among the breast tissues with tumor and the opposite laterals of the same animal, for the enzymes AluI, BsoBI e SmaI, reinforcing the idea that the tissues that didn´t showed visible alterations were already changed at the molecular level and therefore were potentially capable of developing tumors over the time. It is concluded that the gene Tp53 is strongly involved in the breast cancer etiology, and it could be used as a parameter to analyse these tumors´ behavior. The analysis of the protein p53 expression associated to the genetic markers that identify the mutations in the gene sequence could propitiate the early diagnosis of this pathology, allowing the treatment in time of avoiding metastasis and risks of the animal´s survival. / A incidência e o nível de mortalidade associada a diversidade dos fenótipos de câncer requerem estratégias para seu diagnóstico. Neste trabalho, objetivou-se avaliar a relação das mutações no gene Tp53 com o câncer de mama em cadelas, através da expressão imunoistoquímica da proteína p53 e do polimorfismo do gene nos tecidos mamários. No Experimento 1 foi verificada a expressão imunoistoquímica da proteína p53 associando-a ao aspecto morfológico de tecido glandular mamário sadio, a classificação e gradação dos tumores mamários e ao tecido linfonodal inguinal ipsilateral, (com ou sem metástase). Para isso foram utilizadas 19 mamas normais (Grupo 1 - controle), 29 amostras de mamas com diagnóstico clínico de tumor (Grupo 2), 29 mamas contra laterais (Grupo 3) e 29 linfonodos (Grupo 4). O material foi analisado por técnicas convencionais de microscopia (coloração HE) para caracterização dos tecidos e por imunoistoquímica (método da estreptavidina-biotina peroxidase) para avaliar a expressão da proteína p53 nos compartimentos celulares. As mamas do Grupo 2, pela análise histopatológica, apresentaram 65,52% de tumores malignos e 13,79% de tumores benignos. Foram observadas metástases em 6,9% das mamas contra laterais (Grupo 3) e em 31,03% dos linfonodos (Grupo 4). A marcação imunoistoquímica, para expressão de p53, não foi observada em tecidos de animais normais, mas foi evidente tanto em células de tumores malignos quanto benignos localizando-se no núcleo, no citoplasma ou em ambos compartimentos celulares. As mamas contra laterais e os linfonodos que apresentaram metástase também foram marcados, bem como tecidos que ainda apresentavam padrão de normalidade. Não houve diferenças estatísticas entre a intensidade da marcação e do número de células marcadas com o grau de malignidade. No Experimento 2, o objetivo foi detectar mutações em seqüências do exon 8 do gene Tp53 através da técnica PCR-RFLP com enzimas específicas de restrição para associar o padrão de bandas obtidas ao tipo de tumor de mama ou tecido de glândula mamária normal de cadelas. Foram utilizadas 19 cadelas sadias (controle) e 50 cadelas submetidas à retirada de tumor das quais foram obtidas amostras dos tecidos tumorais e de glândulas mamárias sadias contra laterais. A extração de DNA foi realizada pela técnica de fenol-clorofórmio e a PCR-RFLP foi utilizada para amplificação e digestão do exon 8 do Tp53 com 6 endonucleases de restrição, SmaI, DdeI, Rsa, AvaI, BsobI e AluI. O DNA amplificado apresentou uma única banda de 200 pb no gel de agarose 0,8%. O produto de PCR apresentou em média 94% de homologia com a seqüência conhecida do gene (GeneBank –AAB42022.1). Das 6 enzimas selecionadas, apenas 4 apresentaram polimorfismo (AluI, BsobI, DdeI e SmaI) na seqüência do exón 8 do tumor quanto ao número dos pares de base nucleotídeos (pb) ou quantidade de clivagens. O padrão das bandas de cada enzima apresentou polimorfismo entre os grupos diferentes, mas não dentro do seu grupo (cadelas normais, glândulas mamárias com tumor e mama contra lateral ao tumor). Não houve diferença entre os tecidos de mamas com tumor e as contra laterais do mesmo animal, para as enzimas AluI, BsoBI e SmaI, reforçando a idéia de que os tecidos que ainda não apresentam alteração visível, já se encontram alterados no nível molecular e portanto são potencialmente capazes de desenvolver tumores ao longo do tempo.Conclui-se que o gene Tp53 está fortemente envolvido na etiologia das neoplasias de mamas em cadelas, podendo ser utilizado como parâmetro para analisar o comportamento destes tumores. A análise da expressão da proteína p53 associada a marcadores genéticos que identificam mutações na seqüência do gene, pode propiciar o marcadores genéticos que identificam mutações na seqüência do gene, pode propiciar o diagnóstico precoce da patologia, permitindo o tratamento em tempo de evitar metástases.

BACKGROUND:Tumor cell lines that can be tracked in vivo during tumorigenesis and metastasis provide vital tools for studying the specific cellular mechanisms that mediate these processes as well as investigating therapeutic targets to inhibit them. The goal of this study was to engineer imageable mouse mammary tumor cell lines with discrete propensities to metastasize to bone in vivo. Two novel luciferase expressing cell lines were developed and characterized for use in the study of breast cancer metastasis to bone in a syngeneic mouse model.RESULTS:The 4 T1.2 luc3 and 66c14 luc2 cell lines were shown to have high levels of bioluminescence intensity in vitro and in vivo after orthotopic injection into mouse mammary fat pads. The 4 T1.2 luc3 cell line was found to closely model the sites of metastases seen in human patients including lung, liver, and bone. Specifically, 4 T1.2 luc3 cells demonstrated a high incidence of metastasis to spine, with an ex-vivo BLI intensity three orders of magnitude above the commercially available 4 T1 luc2 cells. 66c14 luc2 cells also demonstrated metastasis to spine, which was lower than that of 4 T1.2 luc3 cells but higher than 4 T1 luc2 cells, in addition to previously unreported metastases in the liver. High osteolytic activity of the 4 T1.2 luc3 cells in vivo in the bone microenvironment was also detected.CONCLUSIONS:The engineered 4 T1.2 luc3 and 66c14 luc2 cell lines described in this study are valuable tools for studying the cellular events moderating the metastasis of breast tumor cells to bone.

Obesity increases breast cancer risk and progression in postmenopausal women. The Akt/mTOR signaling pathway is activated in tumors in response to increased levels of obesity-related growth factors, including insulin-like growth factor (IGF)-1. Hence, we evaluated energy balance modulation as a mechanism for breast cancer prevention through modulation of Akt/mTOR. Studies suggest that dietary calcium can decrease weight gain, although an exact mechanism is not yet identified. Therefore, we investigated the effects of low-fat (10 kcal % fat) or high-fat (45 kcal % fat) diets containing either calcium phosphate (dairy) or calcium carbonate (supplement) on body weight in ovariectomized (OVX) C57BL/6 mice to determine if dietary calcium could overcome the effects of a high-fat diet. We showed that dairy decreased body weight, with no effect on food consumption. However, it is not known if restoration of normal weight can reverse mammary tumor progression and/or Akt/mTOR pathway activation. To evaluate this, mice were fed a control diet, a calorie restricted regimen, or a diet-induced obesity (DIO) regimen for 17 weeks, after which the DIO mice were switched to the control diet, and this resulted in a 20% weight loss and mice of equal weight to control mice. MMTV-Wnt-1 mammary tumor cells were orthopically injected at week 20, following weight loss. At week 22, mice began placebo or RAD001, an mTOR inhibitor, treatment by oral gavage. Tumor growth and Akt/mTOR signaling were enhanced in formerly obese mice, despite reduction in weight, adiposity and serum hormone levels. RAD001 decreased tumor growth in the CR and control group, but was less effective in the formerly obese mice. In an additional study, we added a DIO gourp which was not switched to the control diet, and found that circulating IGF-1 levels remained significantly elevated in formerly obese mice relative to control and were comparable to levels in the DIO mice. We found that the mechanism of tumor progression was through enhanced Akt/mTOR signaling in both obese and formerly obese mice. Based on the Akt/mTOR activation in MMTV-Wnt-1 tumor growth and progression, we next investigated the anticancer effects of ursolic acid (UA), a pentacyclic triterpene. It was previously shown that UA can affect Akt signaling. Our results showed that UA was effective decreasing tumor growth and Akt/mTOR signaling. Taken together, our findings show that the growth-enhancing effects of obesity on mammary tumor may persist even after weight loss and suggest that a combination of dietary and pharmacologic interventions targeting IGF-1/Akt/mTOR may be an effective strategy in the treatment of postmenopausal breast cancer. / text

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Made available in DSpace on 2014-12-17T14:13:50Z (GMT). No. of bitstreams: 1 ElielS_DISSERT_partes_autorizadas.pdf: 70324 bytes, checksum: 39ba330935bd00fb14cab6f93f0af7ab (MD5) Previous issue date: 2010-10-17 / OBJECTIVE: Evaluating the kit-Bh performance in carrying out of breast biopsies. METHODS: They were randomly selected a sample of 30 patients with breast cancer undergoing mastectomy, based on the results of a pilot study from February 2008 to April 2010. They were excluded women with had not palpable, stone-hard consistency tumors, previous surgical manipulation or that contains liquid. Using the helicoid biopsy Kit (kit Bh) and an equipment Core biopsy with cannula and needle and 14 gauge respectively, it was collected a fragment of sound equipment in the area and in tumors in each specimen, totaling 120 fragments for histological study. For data analysis, it was defined a 95% confidence level and used the SPSS-13 version, the Kappa index and the parametric Student t test. RESULTS: Mean age of patients was 51.6 years (? 11.1 years). The infiltrating ductal carcinoma showed a higher incidence, 26 cases (86.7%). The Core biopsy had a sensitivity of 93.3%, specificity of 100% and accuracy 96.7%, while the helicoid biopsy had a sensitivity of 96.7%, specificity of 100% and accuracy 98.3%. By comparing the histology of tumors and the fragments of biopsies, there was high degree of agreement in diagnoses (kappa of 0.93 with p <0.05) CONCLUSION: Both devices provided the histological diagnosis of lesions with high accuracy. Results of this study showed that the helicoid biopsy is a reliable alternative in 22 the preoperative diagnosis of breast lesions. Further studies in vivo better will define the role of Kit Bh in the diagnosis of these lesions / OBJETIVO: avaliar o desempenho do Kit Bh na realiza??o de biopsias mam?rias. M?TODOS: de fevereiro de 2008 a abril de 2010, com base nos resultados de um estudo piloto, selecionou-se aleatoriamente uma amostra composta de 30 pacientes portadoras de c?ncer de mama submetidas a mastectomia. Exclu?ram-se as mulheres portadoras de tumor que tivesse consist?ncia p?trea, n?o palp?vel, com manipula??o cir?rgica pr?via ou que contivesse l?quido. Utilizando-se o Kit de biopsia helic?ide ( Kit Bh ) e um equipamentos de Core biopsy com c?nula e agulha de 14 gauge respectivamente, coletou-se um fragmento por equipamento em ?rea s? e nos tumores, em cada pe?a cir?rgica, totalizando 120 fragmentos para estudo histol?gico. Para a an?lise dos dados definiu-se um n?vel de confian?a de 95% e utilizou-se o software SPSS-vers?o 13, o ?ndice de concord?ncia Kappa e o teste param?trico t de Student. RESULTADOS: a m?dia das idades das pacientes foi de 51,6 anos (? 11,1 anos). O Carcinoma ductal infiltrante apresentou maior incid?ncia, 26 casos (86,7%). A Core biopsy apresentou sensibilidade de 93,3%, especificidade de 100% e acur?cia de 96,7%, enquanto a Biopsia helic?ide teve sensibilidade de 96,7%, especificidade de 100% e acur?cia de 98,3%. Na compara??o entre a histologia dos tumores e dos fragmentos de biopsias houve alto grau de concord?ncia nos diagn?sticos ( Kappa igual a 0,93 com p<0,05) CONCLUS?ES: ambos os equipamentos proporcionaram o diagn?stico histol?gico das les?es com alta acur?cia.Os resultados deste estudo demonstraram que a biopsia helic?ide ? uma alternativa confi?vel no diagnostico pr?-operat?rio de les?es mam?rias. Estudos mais aprofundados in vivo, definir?o melhor o papel do Kit Bh no diagn?stico dessas les?es

Les cancers sont l’une des causes majeures de mortalité dans le monde. Les mécanismes en jeu dans la croissance tumorale sont qualitativement connus, mais on se sait pas à l’heure actuelle prédire précisément quel sera le développement d’une tumeur donnée, ni estimer de façon certaine le protocole thérapeutique optimal pour chaque patient. Il est entendu que la modélisation mathématique pourrait apporter des éléments de réponse à ces questions. Durant cette thèse on s'est alors intéressé à la construction de formalismes mathématiques pour décrire la croissance tumorale et l’action de traitement anti-cancéreux. En particulier, on s'est intéressé à la prise en compte des mécanismes aussi bien moléculaires que cellulaires et tissulaires, par la construction d’un modèle continu, multi-échelles, de croissance de tumeur solide et d’angiogenèse. A partir de ce modèle, nous a pu envisager de façon qualitative un protocole optimal de combinaison entre un anti-angiogénique et une chimiothérapie.Le modèle multi-échelles inclut une représentation mathématique des voies de signalisation du VEGF dont on détaille la construction.Dans une autre approche, on a considéré un modèle discret, cellule-centré, reproduisant le développement de sphéroïdes de cellules épithéliales mammaires telles qu’observées lorsque ces cellules sont cultivées in vitro. On a pu mettre en évidence les différents mécanismes cellulaires impliqués dans la morphogenèse de structures composées de cellules saines, et celles composées de cellules mutées.Ces contributions montrent l’intérêt du formalisme multi-échelles adopté pour intégrer les connaissances et données sous-jacentes à l’étude du traitement des tumeurs. / Cancer is one of the leading causes of death in Europe. The mechanisms involved in tumour growth are qualitatively known, but we are still unable to precisely predict how a given tumour will evolve, nor estimate with certainty the optimal therapeutic protocol for each patient.It is well understood that mathematical modelling could give part of the answer to these questions. That is why during this thesis we considered the building of mathematical formalisms to describe tumour growth and the action of anti-cancer treatments. In particular, we investigated the molecular to tissular mechanisms of cancer development and angiogenesis through the building of a continuous multi-scale model. We were able to reproduce the effect of anti- angiogenesis treatments on tumour growth, and qualitatively study an optimal therapeutic protocol of anti-angiogenic combined with cytotoxic drugs. This multi-scale model integrates a mathematical representation of the signalling pathways of VEGF (Vascular Endothelial Growth Factor). We detail the development of this model which is based solely on information available in the literature and dedicated databases. In another approach, we considered a discrete, cell-based model to reproduce the development of spheroid structures of mammary epithelial cells. This model considers the behaviour of these cells when observed while grown in vitro in an appropriate medium. We were able to highlight the different mechanisms involved in the morphogenesis of wild and mutated cells structures.This work shows the importance of the multi-scale formalism we used to integrate the knowledge and data related to the study of cancer treatment.