Master of Science

The growth of many breast cancers is stimulated by the action of the hormone estrogen. Hormonal therapy used to treat these estrogen-dependent breast cancers acts by interfering with the action of estrogen. Current treatments, such as tamoxifen, are not consistently useful due to development of resistance to these drugs. Tamoxifen treatment can also lead to the development of other gynecological cancers, therefore the discovery of novel treatment options for breast cancer is critical. Vitamin B6 is well documented for its role as a modulator of steroid hormones. Pyridoxal phosphate (PLP), the active form of Vitamin B6, may interfere with the action of the estrogen receptor (ER) by blocking the hormone-binding and/or DNA-binding site of the ER. The objective of this study was to examine the effects of Vitamin B6 supplementation on cell proliferation and estrogen-dependent gene expression in breast cancer cells. To accomplish this, estrogen-dependent (MCF-7 and T-47D) and estrogen-independent (BT-20) breast cancer cells were grown in medium supplemented with 0,100, or 300 µM pyridoxal (PL) in the absence or presence of 0.01µM estradiol. Cell counts and [3H]-thymidine incorporation into DNA were assessed in all cell lines. The expression of pS2, an estrogen-sensitive gene, was performed using RNA extracted from MCF-7 cells. PL supplementation was found to significantly decrease total cell numbers and DNA synthesis in both the estrogen-dependent (ER+) and -independent (ER-) breast cancer cells, but did not alter the expression of pS2. These results indicate that PL significantly impairs growth of breast cancer cells and may be exerting its effects via a steroid-independent mechanism. / Master of Science

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.

Vitamin D3 must be metabolically activated by the liver to 25-hydroxyvitamin D3 (25OHD3) and then by the kidney 1alphahydroxylase (1alphaOHase) to become 1,25dihydroxyvitamin D 3 (1,25(OH)2D3). 1,25(OH)2 D3 is a potent inhibitor of tumor growth in vitro and in vivo. Recent studies indicate that metabolic activation of 1,25(OH) 2D3 also occurs in cancer cells such as breast cancer. Consequently, the major objective of this project was to determine if tumoral 25OHD 3-1alphahydroxylase modulates any or all of the stages of breast tumor progression without inducing the hypercalcemic side effects of 1,25(OH) 2D3. For this purpose we used the PyVMT breast cancer mouse model in which the oncoprotein, polyomamiddle T antigen (PyMT) is under the control of mouse mammary tumor virus LTR (MMTV LTR). Mice exhibited tumors restricted to the mammary epithelium progressing to the various stages of breast cancer. Animals were treated with either vehicle, 25OHD3 (2000 pM/24h) or 1,25(OH)2D3 (12pM/24h). Mice treated with the vitamin D precursor, 25OHD3, exhibited a marked reduction in tumor onset and growth comparable to the 1,25(OH)2D3 treated group. Furthermore, biomarkers of tumor progression were markedly reduced in 25OHD3 and 1,25(OH)2D3 animals as compared to vehicle-treated animals. However, mean circulating calcium concentrations remained unchanged in 25OHD3 treated animals but increased significantly in 1,25(OH)2D3 treated animals as compared to controls. Tumoral levels of 1,25(OH)2D3 in mice treated with 25OHD3 were increased 79% in comparison to vehicle control mice. Additionally, 25OHD3 and 1,25(OH)2D 3 treated animals had a significant decrease in the mean number of lung metastases per animal as compared to vehicle treated control animals. This study therefore suggests an important autocrine role of 1alphaOHase expression in breast tumor cells. Furthermore, accumulation of intra-tumoral 1,25(OH) 2D3 in response to 25OHD3 administration strongly suggests that locally produced 1,25(OH)2D3 plays a significant role in restraining tumor growth without inducing the hypercalcemic side effects associated with 1,25(OH)2D3.

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

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

Programa de Apoio aos Planos de Reestruturação e Expansão das Universidades Federais / Cancer is one of the main causes of death in dogs and cats and the mammary neoplasm is one of the most frequent in these species. Although some studies have been performed, there is little data on the epidemiology of this disease in cats. Thus, this study has, as primary goal, to determine the most prevalent tumors in cats and to associate the mammary tumors to some prognostic factors. The files from the Veterinary Pathology Laboratory of the Universidade Federal de Santa Maria were reviewed. Between the years 2000 and 2011, a total of 1427 feline cases including necropsies and biopsies were found. Based on the collected data from these cases, a relation between mammary tumors and some factors such as sex, age, breed, reproductive status, use of contraceptives, number and location of affected glands, presence of ulcerations, size of the tumor, and presence and location of metastases, was studied. Mammary tumors were the second most prevalent tumors (39,57%), after skin neoplasms (41,49%). All feline tumors were in females, mostly older and Domestic Shorthaired cats. Malignant neoplasms were the more frequently diagnosed (72,35%), followed by non-neoplastic tumors (22,58%), and benign neoplasms (5,06%). The smaller tumors were mostly carcinomas. Ulcers were observed in malignant tumors (92%) and non-neoplastic tumors as well (8%). Distant metastases were found mainly in the lungs and skin. / Câncer é uma das principais causas de morte em cães e gatos, sendo o neoplasma mamário um dos mais frequentes. Embora estudos tenham sido realizados nesta área, há, ainda, poucos dados epidemiológicos na espécie felina. Logo, esta dissertação teve como objetivos determinar os tumores mais prevalentes em gatos e relacionar os tumores mamários a alguns de seus fatores prognósticos. Os arquivos do Laboratório de Patologia Veterinária (LPV) da Universidade Federal de Santa Maria (UFSM) foram revisados e um total de 1.427 protocolos de biopsias e necropsias de felinos, entre 2000 e 2011, foi encontrado. Com base nas informações dos arquivos, foi estabelecida a relação entre os tumores mamários e alguns fatores como sexo, idade, raça, estado reprodutivo, uso de contraceptivos, número e localização das glândulas afetadas, ulcerações, tamanho do neoplasma, metástases distantes e para os linfonodos. Assim, observou-se que os tumores de mama foram o segundo diagnóstico (39,57%) mais prevalente, após os tumores de pele (41,49%). Todos os gatos com tumores mamários eram fêmeas, sendo os sem raça definida e os idosos os mais afetados. Os neoplasmas malignos foram diagnosticados com maior frequência (72,35%), seguidos pelos tumores não neoplásicos (22,58%) e pelos neoplasmas benignos (5,06%). Os tumores menores eram, na sua maioria, carcinomas (82,97%). Ulcerações estavam presentes não só em neoplasmas malignos (92%), mas também em alterações não neoplásicas (8%). Metástases distantes foram encontradas principalmente nos pulmões e na pele.

La recherche sur la fonction de lactation est indispensable pour la filière laitière. La glande mammaire est un organe composé de cellules épithéliales, de cellules myoépithéliales, de fibroblastes, d’adipocytes et de cellules endothéliales. L’identification et l’évolution en dynamique des populations cellulaires dans la glande mammaire bovine au cours la lactation reste encore à ce jour méconnues. Les objectifs de cette thèse ont été 1) l’obtention d’un panel des biomarqueurs pour identifier les différentes cellules dans la glande mammaire bovine et 2) le suivi de leur évolution au cours d’un cycle de lactation. Les biomarqueurs de surface (CD49f, EpCAM, CD24 et CD10) ont permis de phénotyper deux lignées mammaires bovines. Nous avons également utilisé ces biomarqueurs pour phénotyper les cellules dans la glande mammaire bovine au cours d’un cycle de lactation.Dans cet objectif, des biopsies ont été prélevées sur 5 vaches laitières primipares à quatre étapes durant la lactation. Après la digestion des biopsies, les cellules obtenues ont été marquées et phénotypées par cytométrie en flux. L’analyse des résultats montre des corrélations positives entre les populations cellulaires CD49f+ et CD49f+/CD24- avec la production laitière. Nous avons mis en évidence une population cellulaire CD49f-/EpCAM- qui augmente au cours de la lactation. Ces résultats suggèrent qu’à partir de l’utilisation de biomarqueurs il est possible d’identifier et phénotyper l’évolution de différentes cellules pendant la lactation dans la glande mammaire bovine. / Research of lactation function of the bovine mammary gland remains essential in dairy farming. The mammary gland is an organ composed of epithelial cells, myoepithelial cells, fibroblasts, adipose cells and endothelial cells. The identification and evolution of cell populations in the bovine mammary gland during lactation is currently unknown. The objectives of this thesis were 1) to obtain a panel of biomarkers capable of identifying different cells within the mammary gland 2) to follow the evolution of these cells throughout a lactation cycle. The cell surface biomarkers (CD49f, CD24, CD10 and EpCAM) allowed us to phenotype two bovine mammary cell lines. We also used these cell biomarkers to phenotype cell populations during lactation.For this objective mammary gland explants were obtained by biopsies taken on 5 primiparous lactating cows at four different times during lactation. After the biopsies were digested, the cells obtained were phenotyped by flow cytometry. Analysis of the results revealed a positive correlation between the CD49f+ and CD49f+/CD24- cell populations and milk yield. We also found evidence that one cell population (CD49f-EpCAM-) was increased during lactation. In general these results suggest that biomarker expression can be utilized to identify the phenotype and the evolution of different cell types during lactation in the bovine mammary gland.

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