Three studies were conducted to determine the effects of ovarian steroids on bovine mammary epithelial cell proliferation. In a first study, estrogen (E), progesterone (P), or E+P were administered to prepubertal beef heifers and biopsied mammary parenchyma taken before and following treatment were compared for growth by evaluation of histoautoradiographic incorporation of thymidine. Estrogen increased epithelial cell growth by 24 h, and fibroblasts to a lesser magnitude by 48-96 h. Estrogen and P was less effective and P was ineffective in increasing proliferation in all cell types studied. Proliferation of adipocytes was not altered. A second study characterized hormone responsive proliferation of Mac-T cells, a recent clonal bovine mammary epithelial cell strain. Mac-T cells responded to all hormones tested as would be expected in vivo. Additionally, passage, harvesting, quantification, freezing, and co-culture techniques were modified to facilitate uncomplicated, timely, inexpensive, effective testing of growth responsiveness to hormones or growth factors. In a third study E and P alone, together, with or without serum were unable to increase Mac-T cell proliferation. Serum from prepubertal Holstein heifers after E treatment did not increase growth of Mac-T cells over serum before treatment. Conditioned media from Mac-T or Fib-T (mammary bovine fibroblast cell line) with or without steroids were tested for ability to increase Mac-T cell proliferation. Growth of Mac-T cells was greatest in Fib-T + E conditioned media followed by Fib-T, then Mac-T and lastly fresh media. Steroid exposure did not enhance the ability of Mac-T cell conditioned media to increase Mac-T cell proliferation. In conclusion, E appears to be the primary ovarian steroid involved in initiating bovine mammogenesis. However, estrogen’s action is not direct and may be caused by paracrine release of growth factors. / Master of Science

NASEM (2021) recently made strides in characterizing effects of 5 individual EAA on milk protein production. However, there are 15 other AA that are incorporated into milk protein, and as such, these AA likely also play significant roles in driving milk protein synthesis, but lack of data prevents their incorporation into current models. A greater supply of AA to the mammary glands does not always mirror AA absorption—the process by which absorbed AA convert into milk protein is variable, and this may be linked to the way the udder regulates AA uptake to preserve intracellular balance. AA transporters housed within the cellular membranes of mammary epithelial cells (MEC), the mammary glands' constituents, are responsible for mediating this intracellular balance. Thus, the objectives of this dissertation were to investigate how AA transport is affected by various AA concentrations using both in vitro and in vivo approaches. In study 1, we evaluated effects of valine and a group of NEAA (AQG; Ala, Gln, and Gly) on exchange transport rates of AA in bovine MEC. High AQG concentrations stimulated Leu, Phe, and Val influx rate parameters, demonstrating that AQG likely increased transport activity for these substrates through exchange transporters. Additionally, high Val concentrations decreased Ile and Leu net uptakes, which occurred via efflux stimulation and transamination downregulation. In study 2, we aimed to identify the effects of 10 EAA and 2 Tyr (CDENSPY) on transport rates and transporter regulation (mRNA expression and protein abundance). Within the physiological AA concentrations used, we were able to measure differential effects of AA on each AA transporter. For example, His stimulated SLC38A2 and SLC38A2 mRNA expression at a decreasing rate; the apex for this curve was reached at a concentration very close to mean plasma concentrations in lactating dairy cows. Therefore, we determined that these transporters may be transcriptionally regulated to regulate intracellular His concentrations. Additionally, all EAA and NEAA groups were involved in significant 2-way interactions on transporter expression and activity. Furthermore, we measured transport rates and rate constants (free of mass influence) of 12 AA to determine important AA on influx, efflux, transamination, irreversible loss, and protein synthesis. We demonstrated competitive inhibition among several AA that share transport systems such as between BCAA. Furthermore, we again demonstrated that NEAA can stimulate transport activity for AA involved in exchange transport. In study 3, we investigated the effects of jugular Lys, Ile, Val, or AQG infusion on mammary AA metabolism and production in lactating dairy cows. Interestingly, Val decreased DMI and milk protein production along with net uptakes of several AA, while the remaining treatments had little metabolic effects. In study 4, we demonstrated that both high protein and starch concentrations independently stimulated milk protein production, but glucose precursor partitioning (lactate, propionate and other) was only affected by starch. In conclusion, we anticipate that nutrition models estimating milk protein production will eventually incorporate up to 20 AA and multiple 2-way interactions; additionally, extremely high concentrations of AA should be prevented to combat negative impacts caused by AA imbalances. However, much more work is required to take steps in this direction. / Doctor of Philosophy / Overfed protein can pose a significant health and environmental risk. Unabsorbed amino acids (AA), the building blocks of protein, are released by dairy cattle into the environment as various nitrogen products. One specific risk is excess runoff of nitrates from dairy farms into nearby water bodies. This contamination can result in serious water quality issues, including eutrophication, which depletes O2 levels in aquatic ecosystems with algae overgrowth, causing dead zones where aquatic life cannot survive. Furthermore, high nitrate levels in drinking water can decreased oxygen availability in humans, in which pregnant women and babies are the most at risk. Finally, the volatilization of N compounds also contributes to air pollution and the formation of greenhouse gases like nitrous oxide, a potent climate-altering compound with global warming potential. Theoretically, feeding an AA profile to precisely match dairy cow requirements would minimize these losses. However, the udder does not take up all available AA. Thus, this research aimed to better understand different AA profiles on AA transport, the route in which AA are taken up by the milk-producing cells in the cow's udder, to ultimately increase efficiency of milk production. In our first study, we demonstrated that non-essential AA (Ala, Gln, and Gly), which can be synthesized in the mammary glands, stimulated Leu, Phe, and Val transport activity within mammary epithelial cells, which could mean that supplemental non-essential AA could increase essential AA transport efficiency. Interestingly, high Val had a negative effect on net uptake (entry minus exit) of Leu and Ile. The second study sought to understand how varying concentrations, within ranges observed in the blood of lactating dairy cows, of 10 different essential AA and 2 non-essential AA groups affected AA transporter expression and activity. We observed greater protein presence and mRNA expression levels of several transporters in response to low availability of their AA transport substrates. Additionally, some AA were involved in stimulating transporter expression and activity when present at high concentrations, such as Leu. There was a plethora of 2-way interactions among AA on transporter protein quantity, mRNA expression, and activity that indicated that the relationship between certain AA will need to be incorporated into future nutrition models. In our third study, we observed that high Val supply decreased the amount that cows ate as well as their milk protein production. This demonstrated that excessive concentrations of certain AA may negatively affect cow metabolism. Lastly, we wanted to investigate the relationship between protein and glucose production in dairy cows, as energy availability is another driver of milk protein production. Our fourth study revealed that dietary protein and starch independently increased milk protein production, yet only starch affected glucose formation. Our findings urge caution against excessive AA concentrations in diets, as imbalances can have negative effects. Overall, we have demonstrated that AA transporters are differentially affected by changes in individual AA supply and various 2-way interactions. This work unveiled the almost unlimited AA interactions that must be further explored to better integrate this knowledge into practical dietary formulations for dairy cows.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis--Amsterdam. / Summary in English and Dutch. Includes bibliographical references.

Integrins have the ability to impact major aspects of epithelial biology including adhesion, migration, invasion, signaling and differentiation, as well as the formation and progression of cancer (Hynes 2002; Srichai and Zent 2010; Anderson et al. 2014). This thesis focuses on how integrins are regulated and function in the context of mammary epithelial biology and breast cancer with a specific focus on the α6 integrin heterodimers (α6β1 and α6β4). These integrins function primarily as receptors for the laminin family of extracellular matrix (ECM) proteins and they have been implicated in mammary gland biology and breast cancer (Friedrichs et al. 1995; Wewer et al. 1997; Mercurio et al. 2001; Margadant and Sonnenberg 2010; Muschler and Streuli 2010; Nistico et al. 2014). The first project investigates how alternative splicing of the α6 subunit impacts the genesis and function of breast cancer stem cells (CSCs). This work revealed that the α6Bβ1 splice variant, but not α6Aβ1, is necessary for the function of breast CSCs because it activates the Hippo transducer TAZ (Zhao et al. 2008a), which is known to be essential for breast CSCs (Cordenonsi et al. 2011). My work also led to the discovery that laminin (LM) 511 is the specific ligand for α6Bβ1 and that autocrine LM511, which is mediated by TAZ, is needed to sustain breast CSCs by functioning as a ‘ECM niche’. An important aspect of this study is the finding that surface-bound LM511 characterizes a small population of cells in human breast tumors with CSC properties. The second project of my thesis concentrated on identifying transcription factors that regulate expression of the β4 subunit. The expression of the α6β4 integrin is repressed during the epithelial-mesenchymal transition (EMT) (Yang et al. 2009) but the contribution of specific transcription factors to this repression is poorly understood. This study revealed that Snai1 is a transcriptional repressor of β4, which is responsible for establishing the PRC2 (Polycomb complex 2)- associated repressive histone mark H3K27Me3. However, I also found that the ability of Snai1 to repress transcription is abrogated by its interaction with Id2. Specifically, I identified the biochemical mechanism for how Id2 regulates Snai1. Id2 binds the SNAG domain of Snai1 that is the docking site for several corepressors (Peinado et al. 2004; Lin et al. 2010b; Dong et al. 2012a). One important consequence of Id2 interacting with Snai1 on the β4 promoter is that it prevents repressive epigenetic modifications. This finding may explain why some epithelial cells express Snai1 and β4 because they also express Id2 (Vincent et al. 2009; Bastea et al. 2012). The repression of the α6β4 integrin during the EMT is consistent with data indicating that this integrin is not expressed in CSCs (Mani et al. 2008; Goel et al. 2012; Goel et al. 2013; Goel et al. 2014). An important question going forward is to understand how the α6β4 integrin contributes to tumor formation. In summary, my thesis provides novel insights into the biology of the α6 integrins that has important implications for the function of these integrins in mammary gland biology and breast cancer, especially our understanding of breast CSCs.

A dose na paciente é uma consideração importante em mamografia e sua medida possibilita avaliar o risco para a paciente, associado à realização desse exame. Para o cálculo da Dose Glandular Média (DGM) que é definida como sendo a dose média absorvida no tecido glandular no interior de uma mama comprimida no exame de mamografia, esta é a grandeza que melhor caracteriza o risco carcinogênico induzido pela radiação ionizante.Valores da DGM podem ser obtidos por métodos que se baseiam na medida do kerma incidente (Ki), associado a fatores de conversão tabelados que dependem da camada semiredutoras, da composição glandular da mama e da espessura da mama comprimida.Visando estes aspectos foi desenvolvido um objeto teste com bolinhas de chumbo ou seja radiopacas inseridos no mesmo, que colocado no momento da irradiação acima da bandeja de compressão, juntamente com espessuras conhecidas de simuladores de mama, após a exposição e processamento da imagem possibilitou a medida da distância entre os pontos na imagem radiológica. Foram desenvolvidos retas de calibrações para que através destas retas possamos chegar ao valor real da espessura do simulador, que representa uma mama comprimida. A metodologia desenvolvida mostrou que através das retas de calibrações podemos chegar aos valores de espessuras conhecidas, portanto chegarmos ao valor de espessura da mama comprimida. / The dose in the patient is an important consideration in mammography and its measure enables evaluating the risk to patients, associated with this examination. For calculating the Average Glandular Dose (AGD) which is defined as the average absorbed dose in the glandular tissue within a compressed breast in a mammogram, this is the greatness that best characterizes the carcinogenic risk of radiation-induced ionizante.The DGM can be obtained by methods based on the measurement of the incident kerma (Ki) associated with the tabulated conversion factors that depend on the half-value layer of the composition of the glandular breast and breast thickness compressed. Aiming these aspects has baen developed a test object with radiopaque objects included in this test object placed upon irradiation above the tray compression, along with known thicknesses of simulators breast after exposure and image processing allowed the measurement of the distance between points in the radiological image. Calibration curve was developed that through these lines can reach the actual value of the thickness of the simulator, which represents a compressed breast. The methodology has shown that through the straight calibration can reach values of thicknesses known, so we get the amount of compressed breast thickness.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Segundo dados do Cadastro Nacional de Estabelecimentos de Saúde (CNES), o Estado de Minas Gerais possui aproximadamente 477 mamógrafos em uso. Destes, estima-se que em torno de 200 são digitais utilizando, principalmente, sistemas de radiografia computadorizada (CR) ou radiografia direta (DR). A mamografia é insubstituível no diagnóstico e detecção precoce do câncer de mama, principal causa de morte entre mulheres no mundo. Um alto padrão de qualidade da imagem, aliada com uma dose tão baixa quanto razoavelmente exequível, são essenciais para esta detecção precoce de forma segura, levando a necessidade de controle de qualidade através do processo de otimização dos procedimentos. Dentro deste contexto este estudo teve como objetivo determinar a dosimetria e a qualidade da imagem em 72 combinações mamógrafo/CR e DRs em 68 serviços de mamografia do Estado de Minas Gerais, o que corresponde aproximadamente 14% do total de mamógrafos atualmente em uso no Estado de Minas Gerais e a cerca de 34% dos mamógrafos digitais em uso (CRs e DRs). O teste que avalia a linearidade da resposta do detector mamógrafico atendeu aos critérios de desempenho especificados pela Sociedade Espanhola de Física médica em 85% dos equipamentos avaliados. Porém dos serviços que apresentaram não conformidade nesse quesito apenas dois apresentaram menos de quatro anos de uso do mamógrafo. Possivelmente o tempo de utilização dos mamógrafos esteja relacionado a não conformidade apresentada por esses serviços. A relação contraste ruído mostrou-se ser um ponto crítico na cadeia de produção da imagem em sistemas digitais, principalmente para maiores espessuras de acrílico. Mais da metade dos serviços avaliados nessa pesquisa obtiveram um bom desempenho na relação sinal ruído (SNR), e na avaliação da imagem através do simulador de contraste e detalhe CDMAM. Em relação ao ruído da imagem a maioria dos serviços ficaram não conformes. Na avaliação da dose glandular média realizada com simuladores de mama, a média geral dos valores de doses obtidos ficaram dentro dos limites considerados aceitáveis na literatura, para a maioria das espessuras simuladas de mama. Porém os resultados obtidos nesse teste são preocupantes, pois a maioria dos sistemas digitais avaliados ficaram não conformes nesse quesito em pelo menos uma espessura de acrílico avaliada, e além disso houve um aumento considerável da dose glandular média em comparação a medidas anteriores realizadas no Estado de Minas Gerais. Já na comparação da dose glandular média entre sistemas CR e DR os sistemas DR apresentaram doses consideravelmente menores para maiores espessuras, em comparação com os sistemas CR. Em relação à comparação entre avaliações subsequentes a amostra seguiu o comportamento geral dos serviços avaliados nesta pesquisa, com exceção da SNR que apresentou um pior desempenho entre as avaliações subsequentes e o ruído, que apesar do baixo índice de conformidade obteve uma melhora entre uma avaliação e outra. Apesar dos potenciais benefícios que podem ser alcançados com o uso do sistema CR e DR, o emprego dessa tecnologia precisa ser revisto e otimizado para que possa oferecer imagens de qualidade utilizando a menor dose possível de radiação. / According to the National Registration of Health Establishments (CNES), there are approximately 477 mammographs operating in the state of Minas Gerais, of which, roughly estimating, 200 are digital apparatus using mainly computerized systems of radiography (CR) or direct radiography (DR). Mammography is irreplaceable in the diagnosis and early detection of breast cancer, the main cause of death in women worldwide. A high standard of image quality coupled with a dose as low as reasonably practicable, are essential for early detection of this disease with safely, leading to the need for quality control through optimization of process procedures. Within this context, this study aimed to determine dosimetry and image quality on 72 mammography combinations / CR and DR in 68 mammography services in the State of Minas Gerais, which represents approximately 14% of mammography units currently in use in the State of Minas Gerais and about 34% in use of digital mammography (CRs and DRs). The test evaluates the linearity of response of the detector mammographic met the performance criteria specified by the Spanish Society of Medical Physics in 85% of evaluated equipment. However, the services they showed non-compliance in this regard, only two were less than four years of use of mammography. Possibly the time of use of mammography is related to noncompliance by these services. The contrast noise ratio proved to be a critical point in the production chain image in digital systems, especially for thicker acrylic. More than half of the services evaluated in this study showed good performance in signal noise, and image evaluation by the phantom contrast and detail CDMAM. Regarding the image noise, most services were non-compliance. In evaluating the average glandular dose with breast phantom, the overall average dose values obtained were within acceptable limits in the literature for most of the simulated breast thicknesses. However the results of that test are troubling, since most digital systems evaluated were non-compliant in this regard at least one thick acrylic evaluated, and in addition there was a considerable increase in the average glandular dose compared to previous measurements performed in the State of Minas Gerais. In comparison between the average glandular dose in CR and DR systems, the DR systems showed significantly lower doses for greater thickness as compared with the CR systems. Regarding the comparison between subsequent reviews, the sample followed the general behavior of the services evaluated in this study, except the SNR had a worse performance between subsequent evaluations and noise, which despite the low level of compliance achieved an improvement between evaluation and other. Therefore, despite the potential benefits that can be attained with the use of the CR and DR systems, the employment of this technology has to be revised and optimised so that a better quality image can be achieved and the radiation dose reduced as much as possible.

Orientador: Gisele Fabrino Machado / Banca:Daniela Bernadete Rozza / Banca: Rosemeri de Oliveira Vasconcelos / Resumo: As neoplasias mamárias em gatas representam o terceiro tipo neoplásico mais frequente nesta espécie. A maioria destas neoplasias são malignas, e exibem alto potencial metastático. Devido a isto, e a escassez de trabalhos referentes ao prognóstico de gatas com câncer de mama, foram avaliados em 32 amostras de neoplasia mamária de gatas, a expressão imuno- histoquímica de alguns marcadores de prognóstico. As amostras foram segregadas em grupos seguindo os parâmetros de classificação da organização mundial de saúde em carcinomas tubulopapilares, cribriformes e sólidos e de acordo com o método de Nottighan modificado em graus I, II e III. Os cortes histológicos foram submetidos a processamento imuno-histoquímico para avaliação da expressão da metaloproteinase 9 (MMP-9), do fator de crescimento endotelial vascular (VEGF), da proteína de proliferação celular Ki- 67 e de linfócitos TCD3+, CD79+ e macrófagos, afim de se avaliar os diferentes padrões de classificação. Os resultados da marcação imuno- histoquímica foram avaliados com auxílio de um sistema de análise de imagem computadorizada para determinar a porcentagem da área imunomarcada. Em relação à classificação morfológica observou-se diferença significativa entre a densidade de linfócitos TCD3+ entre os grupos. Durante a análise nas amostras segregadas de acordo com a gradação histológica verificou-se diferença significativa entre a imunomarcação da MMP-9, entre a intensidade de infiltração de células TCD3+ e uma tendência de variação na proteína Ki-67. Observamos também correlação positiva entre os níveis de expressão da MMP-9 e da Ki-67, da MMP-9 e células TCD3, e da proteína Ki-67 com as células TCD3. Desta forma concluímos que classificação histológica dos carcinomas de mama em gatas não é um bom parâmetro para determinação do prognóstico da doença... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The mammary tumors in cats represent the third most common tumor type in the species. Most of these are malignant neoplasms exhibit a high metastatic potential. Because of this and the lack of studies regarding the prognosis for cats with breast cancer were evaluated 32 samples of breast cancer in cats. These samples were grouped according to morphological and histological classification, and the sections were submitted to immunohistochemical processing to evaluate the expression of metalloproteinase 9 (MMP-9), the vascular endothelial growth factor (VEGF) protein of cell proliferation Ki-67 and CD3 + T lymphocytes, and CD79 + macrophages in order to evaluate differences in the different grading standards. The results of immunohistochemical marking were evaluated by using a system of computerized image analysis to assess the percentage of area immunomarked. Regarding the morphological classification showed a significant difference between the amount of T lymphocytes CD3 + between the groups. During analysis the samples segregated according to histologic grading there was significant difference between the immunostaining of MMP-9, between the intensity of infiltration of T cells CD3 + and a tendency of variation in the Ki-67 protein. We also observed a positive correlation between the expression levels of MMP-9 and Ki-67, MMP-9 and TCD3 cells, and Ki-67 protein in cells T CD3+. Thus we conclude that histologic classification of breast carcinomas in cats is not a good parameter for determining the prognosis of the disease when evaluated together with the levels of immunohistochemical expression of MMP- 9, VEGF and Ki67. The best best parameters for determining the prognosis was observed when the histological grade was considered . The higher was the proliferative index determined by the positivity of Ki-67 protein from neoplastic cells, the greater... (Complete abstract click electronic access below) / Mestre

Orientador: Maria cecilia Rui Luvizotto / Banca: Gisele Fabrino Machado / Banca: Sandra Helena Penha de Oliveira / Resumo: Os tumores mamários são as mais freqüentes neoplasias em cadelas, representando cerca de 50% dos tumores diagnosticadas nesta espécie e tem maior incidência em fêmeas de meia idade a idosas. Têm sido utilizadas como modelo de estudo para neoplasias mamárias de mulher devido às várias características comuns como as similaridades histológicas e imuno- histoquímicas. Na última década, estudos baseados nos perfis moleculares das neoplasias mamárias, tornaram possível a identificação de algumas células neoplásicas com características de células tronco - as células tronco tumorais (CTT). Uma das moléculas putativas para marcação das CTTs é o CD44. A telomerase, que vem sendo relatada como marcador tumoral, é potencialmente expressa também em CTTs. Estudos recentes estabeleceram um elo crucial entre a transição epitelial-mesenquimal (EMT) e a aquisição de propriedades moleculares e funcionais de células-tronco. Por este motivo analisamos a expressão das proteínas CD44, Citoqueratinas AE1/AE3 e Vimentina, e da enzima telomerase, em tumores mamários de cadelas a fim de investigar o potencial de marcação de CTTs e sua relação com a EMT. Para tal foram utilizados métodos de imuno-histoquímica em tecidos parafinizados tanto in vivo quanto in vitro, fazendo-se uso de técnicas como Tissue MicroArrays (TMA), citoinclusão de tecidos tumorais e cultivos celulares. A expressão da telomerase nas células de tumores mamários cultivadas foi analizada por PCR em tempo real. Nas amostras de tecido parafinizado in vivo, a imunomarcação da citoqueratina demonstrou diferença significativa entre tumores benignos e malignos (p˂0,05), mostrando-se mais intensa em tumores malignos. Já a vimentina apresentou maior intensidade de marcação em tumores benignos, porém não apresentando diferença significativa (p˂0,05)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract:Mammary tumors are the most frequent cancers in dogs, representing about 50% of tumors, and have a higher incidence in females of middle aged and elderly. These tumors have been used as a model for breast cancer in women due to several common characteristics such as histological and immunohistochemical similarities. In the last decade, studies based on molecular profiles of breast cancer, made possible the identification of some neoplastic cells with characteristics of stem cells - cancer stem cells (CSC). One of the putative molecules of CSCs is CD44. Telomerase, which has been reported as a tumor marker, is potentially also expressed in CSCs. Recent studies have established a crucial link between the epithelial- mesenchymal transition (EMT) and the acquisition of molecular and functional properties of stem cells. For that reason we analyzed the expression of proteins CD44, Cytokeratins AE1/AE3 and Vimentin, and the telomerase enzyme, in dogs mammary tumors, to investigate the potencial for CSC markers, and its relation with the EMT using immunohistochemistry in paraffin embedded tissues in both in vivo and in vitro, making use of techniques such as Tissue MicroArrays (TMA), cell blocks from tumors and cell cultures. Besides that, we analyzed the telomerase expression in mammary tumor cultivated cells by quantitative real time PCR. Immunostaining of cytokeratin had no significant difference between benign and malignant tumors (p ˂ 0,05), being more intense in malignant tumors. However vimentina showed higher staining intensity in benign tumors, but with no significant difference (p ˂ 0,05). The expression of CD44 was higher in malignant tumors that have greater proliferative and metastatic potencial, however its relation with EMT was not detected in the analyzed tumors. Telomerase expression was positive in the neoplastic cultivated cells, however without significant difference... (Complete abstract click electronic access below) / Mestre

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