A neoplasia mamária é uma afecção que acomete freqüentemente as fêmeas da espécie canina sendo responsável por grande parte das neoplasias que atingem esses animais. Esta pesquisa visou comparar, no momento pré-operatório, o exame tomográfico contrastado ao exame radiográfico para pesquisa de metástases na cavidade torácica de cadelas com neoplasias mamárias malignas, de forma que, nesta oportunidade, ateve-se à análise descritiva das imagens radiográficas e tomográficas. Para tanto foram realizados exames radiográficos e de tomografia computadorizada contrastada da cavidade torácica de vinte fêmeas da espécie canina portadoras de neoplasias mamárias malignas atendidas pelos Serviços de Obstetrícia e Ginecologia e de Cirurgia do Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo. Constaram do grupo experimental, somente animais que não apresentaram evidências de portarem nódulos pulmonares. Dos vinte animais estudados nesta pesquisa 20% apresentaram quadros pulmonares (10% quadro intersticial - difuso, 5% quadro intersticial - nódulo suspeito e 5% quadro misto) e um animal (5%) apresentou efusão pleural aos exames radiográficos. Já aos exames tomográficos foram observados os seguintes achados: quadro alveolar em 25% dos animais, quadro brônquico em 30% e em 45% quadro misto; em 25% dos animais foram visibilizados nódulos pulmonares e em 75% linfonodos mediastinais. Apenas um animal (5%) apresentou tamanho de linfonodo mediastinal maior que 10mm, 20% apresentaram tamanho entre 6 e 10mm e 50 % dos animais apresentaram linfonodos que mediam até 5mm. Em 65% dos animais os linfonodos axilares foram visibilizados pelos exames tomográficos, sendo que, em 25% dos animais eles mediam até 5mm, em 35% eles mediam de 6 a 10mm e em apenas uma cadela o linfonodo media mais que 10mm. Em 30 % dos exames foi possível, através das imagens tomográficas, observar alterações em fígado e em 40% alterações em pele e/ou subcutâneo. Também foram relacionados aspectos relativos à técnica tomográfica com a finalidade de possibilitar a reprodução das técnicas utilizadas. O exame tomográfico mostrou-se um importante complemento do exame radiográfico na pesquisa de metástases de neoplasias mamárias em cadelas especialmente no que se refere à avaliação do interstício pulmonar e dos linfonodos mediastinais e axilares. O exame tomográfico mostrou-se superior ao exame radiográfico nos detalhes da avaliação de campos pulmonares, mediastino e espaço pleural. O exame tomográfico possibilitou, de forma suplementar, o estudo dos linfonodos mediastinais e axilares, das alterações presentes em pele, subcutâneo e em fígado, que não puderam ser avaliados pelo exame radiográfico. / Mammary gland tumors affect frequently female dogs, accounting for the majority of neoplasias that occur in these animals. This research was performed in bitches with malignant mammary gland tumors, to compare contrast computed tomographyc and survey radiographc techniques in detecting thoracic metastasis at preoperatory moment. Twenty bitches with malignant mammary gland tumors were examined at the Obstetric and Ginecology and Surgery Services of the Veterinary School Hospital of the Faculdade de Medicina Veterinária e Zootecnia of the University of São Paulo. Only animals with no evidence of pulmonary nodules were studied. Of the 20 studied animals, 20% presented pulmonary patterns (10% diffuse intersticial pattern, 5% suspected nodule and 5% presented mixed pattern). At the tomographic exam, 25% presented alveolar pattern, 30% bronquial pattern and 45% mixed pattern; 25% of the animals presented pulmonary nodules and in 75% mediastinal lymph nodes were seen. Only one animal (5%) had lymph node bigger than 10mm, 20% between 6 and 10 and 50% of the animals had lymph nodes measuring up to 5mm. Axilary lymph nodes were seen in 65% of the cases, 25% of the animals presented axilary lymph nodes measuring up to 5mm, 35% had had lymph nodes measuring between 6 to 10mm and only one had lymph node bigger than 10mm. In 30% of the exams hepatic findings could be determined, also in 40% of the cases, skin and subcutaneous alterations were seen. Aspects related to the tomographic technique were also registered to make the techniques reproduction possible. The tomographic exam showed up as an important complement of the radiographic exam, in searching for mammary gland metastasis in female dogs, specialy concerning the evaluation of the pulmonary intersticium and the axilary and mediastinal lymph nodes. The tomographic exam was considered superior in details than the radiographic exam in the evaluation of the pulmonary fields, mediastinal structures and pleural space. Different from the radiographic examinations, the tomographic technique allowed the study of mediastine and axilary lymph nodes, skin, subcutaneous and liver.

Deux tiers des cancers du sein expriment le récepteur à l’estrogène alpha (REα) et leur croissance dépend des estrogènes alors que l’expression de REα induit la différenciation et la sénescence des cellules humaines mammaires épithéliales normales. Le développement embryonnaire et la différenciation de la glande mammaire adulte sont contrôlés par des facteurs de transcription, dont beaucoup sont aussi impliqués dans la tumorigenèse. Plusieurs études d'association pan-génomiques ont identifié le putatif facteur de transcription TOX high mobility group box family member 3 (TOX3) comme un nouveau gène de prédisposition au cancer du sein.L’objectif de cette étude était la caractérisation fonctionelle de TOX3 dans l’épithélium mammaire normal et le cancer du sein.J’ai examiné l’expression de TOX3 dans des tumeurs du sein humaines et dans le sein normal par analyse des données d’expression issues de puces à ADN. L’effet de TOX3 sur la différenciation des cellules épithéliales mammaires était analysé par des assays CFC et FACS. De plus, j’ai effectué une analyse microarray des cellules luminales cancéreuses MCF-7 exprimant TOX3 afin d’identifier les gènes cibles de TOX3 ainsi que la méthode de purification d’affinité en tandem (TAP) combinée à la spectrométrie de masse afin d’identifier des protéines interactants de TOX3. Finalement, j’ai étudié le potentiel oncogénique de TOX3 dans un modèle de xénogreffe. / Two thirds of breast cancers express the estrogen receptor alpha (ERα) and are estrogen-dependent for growth. In contrast, expression of ERα induces differentiation and senescence in normal human mammary epithelial cells. Both embryonic development and lineage commitment in the adult mammary gland are governed by transcriptional regulators, many of which have also been implicated in tumourigenesis. Genome-wide association studies have identified the previously uncharacterised putative transcription factor TOX high mobility group box family member 3 (TOX3) as a new candidate breast cancer susceptibility gene.In the present study, I aimed to characterise TOX3 function in the normal human mammary epithelium and in breast cancer.I have examined TOX3 expression in primary breast tumours and in the normal mammary gland using micorarray data. The influence of TOX3 expression on lineage commitment was investigated using the colony forming cell (CFC) assay and FACS analysis. I further carried out microarray analysis of luminal breast cancer cells ectopically expressing TOX3 to identify TOX3 target genes as well as tandem affinity purification of TOX3 to identify TOX3 interacting proteins. Finally, the oncogenic potential of TOX3 was investigated in a human-in-mouse xenograft model.

Maspina é uma proteína supressora de tumor e metástase e sua localização subcelular está relacionada ao prognóstico do câncer de mama. Nosso grupo mostrou em MCF-10A que quando fosforilada maspina se acumula no citoplasma. Porém, esta correlação ainda não foi relatada in vivo. Aqui investigamos a expressão, fosforilação e localização subcelular de maspina ao longo do desenvolvimento da glândula mamária murina. Maspina foi detectada no estágio mais tardio da gestação, na lactação e na involução. Os níveis de fosforilação de maspina são maiores no período de lactação do que na involução. Interessantemente, a porcentagem de células que apresenta maspina no núcleo é maior na fase de involução do que na fase de lactação Estes dados mostram que a correlação entre níveis de fosforilação de maspina e localização subcelular também é observada in vivo e que esses processos são reguladas ao longo do desenvolvimento na glândula mamária murina. / Maspin is a protein with tumor and metastasis suppressing activity and its subcellular localization is related to breast cancer prognosis. Using MCF-10A cells as a model system, our group demonstrated a correlation between maspin phosphorylation and cytoplasmic accumulation. Here we investigated maspin expression, phosphorylation levels and subcellular localization in vivo during the murine mammary gland development. Maspin was detected in late pregnancy, during lactation and involution. Maspin phosphorylation levels is higher during lactation than during involution. Interestingly, the percentage of cells which present nuclear maspin is higher in the involution than in lactation. These data show that the correlation between maspin phosphorylation and subcellular localization is also observed in vivo and these processes are regulated during murine mammary gland development.

Orientador: Humberto Tonhati / Resumo: A mastite é a enfermidade que mais causa prejuízos de ordem quantitativa e qualitativa nos sistemas de produção de leite no mundo. A prevenção e controle da doença precisam envolver atividades práticas e eficientes que auxiliem a tomada de decisões rápidas na fazenda. Os métodos de triagem para o diagnóstico da mastite aplicados às búfalas leiteiras são aqueles desenvolvidos especificamente para as vacas. Assim, o objetivo geral da tese foi estudar a mastite subclínica em búfalas e avaliar os métodos de triagem que auxiliam o diagnóstico da doença na fazenda. O trabalho se divide em quatro capítulos que tratam de: revisar a literatura sobre as temáticas (I); caracterizar o leite de quartos mamários com e sem infecção intramamária, e avaliar a associação entre a presença de infecção intramamária e as características microbiológicas do leite de quartos mamários de búfalas, posição dos tetos e os resultados dos métodos de triagem (II); e avaliar os testes rápidos California Mastitis Test , Somaticell e condutividade elétrica como métodos de triagem para auxiliar o diagnóstico da mastite subclínica em búfalas diretamente na fazenda (III). O estudo foi realizado em um rebanho de búfalas no estado do Rio Grande do Norte. Foram colhidas 533 amostras de leite de quartos mamários que foram submetidas à contagem eletrônica de células somáticas (CCS), aos testes de triagem (California Mastitis Test – CMT, teste Somaticell - SO e condutividade elétrica – CEL) e ao exame microbiológico pa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mastitis is the disease that causes the most quantitative and qualitative losses in milk production systems in the world. Disease prevention and control need to involve practical and efficient actions that may help on decision-making on farm. Screening methods applied for mastitis diagnosis in dairy buffaloes are those developed specifically for cows. Thus, the objective of the thesis was to study subclinical mastitis in buffaloes and to evaluate the screening methods that help the disease diagnosis in farm. The work is divided into four chapters that deal with: reviewing the literature on the themes (I); to characterize breast milk with and without intramammary infection and to evaluate the association between the presence of intramammary infection and microbiological characteristics of buffalo milk, teat position, and the results of screening methods (II); and evaluate the California Mastitis Test (CMT), Somaticell (SO) and electrical conductivity (EC) rapid tests as a screening method to assist the diagnosis of subclinical mastitis in buffaloes on farm (III). The work was developed in a buffaloes commercial herd in the Rio Grande do Norte state. A total of 533 randomly selected milk samples were obtained and submitted to electronic somatic cell count (SCC), screening tests (CMT, SO and EC) and microbiological examination for diagnosis of subclinical mastitis (IMM: positive/negative), infection classification (negative, contagious, environmental, mixed and opportunistic) an... (Complete abstract click electronic access below) / Doutor

We have investigated phase II activation of the food-derived mutagen 2- hydroxyamino-1-methyl-6-phenyl[4,5-b]pyridine (N-OH-PhIP) by cytosolic acetyltransferase, sulfotransferase, and tRNA synthetase/kinase enzymes from human breast tissue. Cytosol from homogenates of mammary gland tissue obtained from breast-reduction surgery or mastectomy was incubated with and without enzyme-specific cofactors, and mutagen binding to calf thymus DNA was quantified by 32P-postlabeling. In addition, microsomal fractions of mammary epithelial cells from some individuals were examined for prostaglandin H synthetase activation of N-OH-PhIP. Our results show that all four enzymes can participate in activating N-OH-PhIP, thus inducing PhIP-DNA adduct formation in human mammary cells. However, not all individuals exhibited all these activities; instead each individual showed a combination of one or more activation pathways. The present findings demonstrate that the human mammary gland has the capacity to metabolically activate a dietary mutagen by several enzyme systems, including acetyltransferase, sulfotransferase, tRNA synthetase/kinase, and prostaglandin hydroperoxidase catalysis.

The cJUN NH2-terminal kinase (JNK) pathway responds to environmental stresses and participates in many cellular processes, including cell death, survival, proliferation, migration, and genome maintenance. Importantly, genes that encode components of the JNK signaling pathway are frequently mutated in human breast cancer, but the functional consequence of these mutations in mammary carcinogenesis is unclear. Anoikis – suspension-induced apoptosis – has been implicated in oncogenic transformation and tumor cell metastasis. Anoikis also contributes to lumen formation during mammary gland development and epithelial cell clearance during post-lactational involution. JNK is known to contribute to certain forms of cell death, but the role of JNK during anoikis was unclear. I examined the requirement of JNK in anoikis and discovered that JNK promotes cell death by transcriptional and post-translational regulation of pro-apoptotic BH3-only proteins. This conclusion suggested that JNK signaling may contribute to mammary gland remodeling during involution. Indeed, JNK deficiency in mammary epithelial cells disrupted the remodeling program of gene expression and delayed involution. Finally, I sought to understand the importance of JNK in mammary carcinogenesis. I found that JNK loss in the mammary epithelium was sufficient for genomic instability and tumor formation. Moreover, JNK loss in a model of breast cancer resulted in significantly accelerated tumor development. Collectively, these studies advance our understanding of the JNK pathway and breast biology, and provide insight that informs the design of therapeutic approaches that target the JNK signal transduction pathway.

No description available.

Mammary gland (MG) is a dynamic organ that is essential for the production and secretion of breast milk. During pregnancy the MG undergoes a critical phase of remodeling, which is accompanied by the differentiation of mammary epithelial cells (MECs). During lactation, the MG requires high level of energy for proper folding of proteins in the secretory pathway, which takes place in the endoplasmic reticulum (ER). Limited evidence has been reported on the impact of cannabis or its components, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on the differentiation of MECs. However, both THC and CBD have been reported to induce ER stress in various cell types, resulting in impacting cellular function. Furthermore, consequences of cannabis use in the perinatal period on breast milk composition have not been reported. Using the HC11 cell line, we investigated whether THC and CBD evoke ER stress in MECs resulting in the impaired cellular function. We also reported on the effects of cannabis use during pregnancy and lactation on the levels of macronutrients and bioactive factors in the breast milk of cannabis users. Relative to control, 10μM THC and 10μM CBD reduced mRNA levels of milk proteins (CSN2 and WAP) and lipid synthesizing enzymes (FASN, FABP4, PLIN2 and LPL), as well as whey acidic protein and lipid levels. In addition, 10μM THC, 10μM CBD, and the combination of 10μM THC + 10μM CBD significantly induced the expression of ER stress genes in HC11 cells. Furthermore, in the milk of women who used cannabis during pregnancy and lactation, the levels of secretory immunoglobulin A (SIgA) were decreased, relative to non-users. Altogether, our findings indicate that cannabis use in the perinatal period may have implications on breast milk composition and infant health. / Thesis / Doctor of Philosophy (Medical Science) / Breast milk is crucial to the nutrition and immunity of the newborn. It is produced by the mammary gland (MG), which is composed of mammary epithelial cells (MEC) that play a key role in producing and secreting proteins and factors into the milk. During pregnancy, the MG undergoes remodeling which is accompanied by differentiation of the MECs (change from unspecialized to a specialized stage that allows the MECs to produce milk proteins and other factors). The disruption in MEC differentiation can result in altered milk production and composition. Cannabis is used by women during pregnancy and breastfeeding. In this study, we investigated the impact of cannabinoids on the MEC differentiation and examined the effect of cannabis use in the perinatal period on the breast milk composition in humans. We reported that cannabinoids reduced the differentiation of MECs and maternal cannabis use during breastfeeding reduced levels of an essential immune factor.

Estrogenic chemicals are common pollutants in wastewater and current methods used in wastewater treatment processes are not typically effective in removing these compounds. Thus, many estrogenic chemicals, as well as other pharmaceuticals, are detected in drinking water supplies, contributing to human exposures. This concerning public health situation has led some green chemists to investigate the feasibility and efficacy of tetra-amido macrocyclic ligands (TAML), which have been proposed to be environmentally friendly catalysts that can be used to treat wastewater. TAML works by catalyzing the oxidation of micropollutants, breaking pollutants down into substances that can be assimilated by bacteria. These breakdown products are anticipated to no longer pose an environmental or health concern. Prior to their use in environmental matrices, TAML must first be evaluated to ensure that these compounds pose a low environmental and human-health hazard. Here, we exposed adult female mice to one of three doses of New-TAML 7 (NT7), or an estrogen receptor agonist (ethinyl estradiol, EE2) or an androgen receptor antagonist (flutamide), for two weeks prior to mating, throughout pregnancy, and throughout the lactational period. Male mice used for breeding purposes, the F0 males, were also exposed to the drinking water during the 2-week mating period. Following this acute exposure, the F0 males were necropsied and evaluated for signs of overt toxicity and repro-endocrine disruption. There was a statistically significant decrease in the F0 males spleen weight. Male offspring, the F1 generation, were evaluated at weaning (postnatal day 21), in puberty (postnatal day 32), and in adulthood. We first evaluated organs known to be sensitive to toxic effects. In male mice developmentally exposed, we have discovered that all treatments decreased spleen size at postnatal day (PND) 21 but not in early adulthood. At PND 16, no significant effects were observed. At PND21, no effects were observed on the weight of the kidney, liver, testes, or seminal vesicles; however, spleen weight was significantly decreased in the positive controls and the mid-NT7 group. In early adulthood, no effects have been observed on organ weights of the flutamide group; however, the size of both testes is significantly affected by the mid-NT7 dose and the EE2 treatment. By six months of age, there were no statistically significant effects, although this is likely in part due to inadequate statistical power. We also evaluated the effect of NT7, EE2, and flutamide on the male mouse mammary gland. Because of the conserved role hormones play in mouse development, a mammary gland is a valuable tool for identifying endocrine disruptors. We found that EE2 increased the size and number of branching points starting at PND21 and continuing until nine weeks of age. TAML had little effect on the morphology of the male mammary gland, with the exception of increased ductal branching at PND21. This, along with the change in testes weight, may suggest TAML can affect estrogen-mediated outcomes; many of our observed effects warrant additional study.

In the first part of this thesis, we utilize the mouse model to evaluate the environmental chemical Butyl Benzyl Phthalate. Due to lack of research on female exposure to BBP, this thesis focuses on quantifying the effects of gestational exposure on the female mammary gland Here male and female parental mice were exposed before mating and through pregnancy and lactation to one of three doses of BBP or the control via oral ingestion. After weaning, offspring were sacrificed at puberty or early adulthood and evaluated for altered mammary gland morphology or hormonal receptor expression. Results indicate a persistent statistically significant increase in weight among the highest BBP dose group. Additionally, the high-dose adult treatment group demonstrated a statistically significant decrease in terminal ends. Finally, the mid-dose adult group demonstrated significantly higher expression of the progesterone receptor compared to the low and high-dose BBP groups. There were no significant findings in pubertal female outcomes. In the second part of this thesis, we evaluate the existing OECD Extended One-Generation Reproductive Toxicity Guidelines (TG 443). First by summarizing the endpoints and outcomes evaluated in studies that implement these guidelines, and additionally discussing the current OECD recommendations for mammary gland evaluation. We conclude with outlining the remaining questions to be evaluated and further research necessary to establish that the mammary gland should be added to TG 443.