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.

The cutaneous sweat loss of calcium, copper, iron, magnesium, manganese, potassium, sodium and zinc of preadolescent boys on diets containing 30, 60, and 90 g protein and various mineral concentrations were studied. Arm sweat collections were made using the arm bag technique. Mineral and nitrogen concentrations were determined by atomic absorption spectrophotometry and the micro-Kjeldhl procedure, respectively. Total body mineral loss was extrapolated from arm sweat mineral and nitrogen loss and total body nitrogen loss. The influence of protein and mineral intake on mineral excretion in sweat was also studied. Statistical analysis (α= 0.05) showed no effect of protein and mineral intake on cutaneous mineral excretion. Arm sweat results are in accordance with those found by other researchers working with children but total body mineral losses are much lower than those found in research done with adults. / M.S.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Post-natal mammary gland development is a complex process in which epithelial proliferation and branching of lactiferous ducts is followed by extensive formation of lobuloalveolar units that produce milk. Classical nuclear factor-kappa B (NF-κB) p65/p50 transcription factors are dynamically induced in the mammary gland during pregnancy, and inhibitor of NF-κB-alpha (IκB-α) deficiency leads to hyperplasia of the mammary epithelium. To further elucidate the role of NF-κB factors in mammary development, we examined NF-κB subunit expression in the mammary glands of transgenic mice expressing the IκB-α S32/36A super-repressor (SR) protein under control of the mouse mammary tumor virus (MMTV)-long terminal repeat promoter, in which mammary gland development is transiently delayed, but not completely blocked. Developmental recovery correlated with induction of RelB/p52 NF-κB complexes, which failed to interact with an IκB-α fusion protein and potently induced cyclin D1 and c-myc promoter activities. Activation of IκB-α kinase alpha (IKKα) and NF-κB inducing kinase (NIK) was detected by day 5.5, and were hypothesized to be responsible for the induction of ReIB/p52. In support of this hypothesis, we found that constitutively active IKKα induced p52, RelB, and cyclin D1 in untransformed mammary epithelial cells. Moreover, mammary tumors induced by high-dose 7,12-dimethylbenz(a)anthracene (DMBA) treatment in wild type FVB/N mice, displayed increased RelB/p52 binding activity. These results implicate activation of RelB/p52 complexes by the alternative NF-κB signaling pathway in branching of lateral ducts and alveolar development during mammary gland development, and in mammary carcinogenesis. / 2999-01-01

Massas adrenais clinicamente silenciosas, diagnosticadas ao acaso ("incidentalomas"), são frequentemente encontradas em avaliações radiológicas devido ao constante progresso dos métodos de imagem. Na revisão das principais fontes científicas até 2004, analisado o grau de evidência concluiu-se: a maioria dos "incidentalomas" são não hipersecretores, mas a avaliação endócrina demonstrou que é comum o achado de hiperfunção hormonal discreto; "incidentaloma" acima de 6cm sugerem malignidade e entre 4-6cm devem ser analisadas por suas características de imagem; adrenalectomia deve ser indicada em massas adrenais funcionantes; massas adrenais não operadas devem ser acompanhadas pelo prazo de 2 anos / Introduction: Clinically silent adrenal masses, incidentally diagnosed during imaging methods performed for other clinical conditions (“incidentalomas") have been more frequently detected due to the constant improvement in imaging methods. There are several causes, diagnoses and treatments for these masses. Thus, whenever a physician comes across such lesion, it is necessary to define whether this mass is hormonally active and whether there is a risk of being malignant. Nevertheless, the methods for clarifying these issues have yet to be defined. Objective: To evaluate the best diagnosis, treatment and follow up of the incidental adrenal lesion. Methods: The main scientific literature available until October 2004 was reviewed, taking evidence into account. Results: Two studies which selected and reviewed articles until September 2003 were found. Fourty-three other studies included in a systematic review until October 2004 were added to this study. Conclusions: In general, “incidentalomas" are non-functioning, but endocrinological evaluation has shown that subclinical hormonal hyperfunction is not unusual, thus stressing the need for measuring substances such as with metanephrine assay, dexamethasone suppression test in low dosage and establishing the upright plasma aldosterone/plasma renin activity ratio. Non-functioning “incidentalomas" smaller than 4 cm should be followed carefully; those between 4 and 6 cm should be analyzed for its imaging characteristics; for those greater than 6 cm adrenalectomy is indicated. Functioning “incidentalomas" must undergo adrenalectomy. Nonoperated adrenal masses must be followed for two years through imaging and function testing.

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

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

Massas adrenais clinicamente silenciosas, diagnosticadas ao acaso ("incidentalomas"), são frequentemente encontradas em avaliações radiológicas devido ao constante progresso dos métodos de imagem. Na revisão das principais fontes científicas até 2004, analisado o grau de evidência concluiu-se: a maioria dos "incidentalomas" são não hipersecretores, mas a avaliação endócrina demonstrou que é comum o achado de hiperfunção hormonal discreto; "incidentaloma" acima de 6cm sugerem malignidade e entre 4-6cm devem ser analisadas por suas características de imagem; adrenalectomia deve ser indicada em massas adrenais funcionantes; massas adrenais não operadas devem ser acompanhadas pelo prazo de 2 anos / Introduction: Clinically silent adrenal masses, incidentally diagnosed during imaging methods performed for other clinical conditions (“incidentalomas”) have been more frequently detected due to the constant improvement in imaging methods. There are several causes, diagnoses and treatments for these masses. Thus, whenever a physician comes across such lesion, it is necessary to define whether this mass is hormonally active and whether there is a risk of being malignant. Nevertheless, the methods for clarifying these issues have yet to be defined. Objective: To evaluate the best diagnosis, treatment and follow up of the incidental adrenal lesion. Methods: The main scientific literature available until October 2004 was reviewed, taking evidence into account. Results: Two studies which selected and reviewed articles until September 2003 were found. Fourty-three other studies included in a systematic review until October 2004 were added to this study. Conclusions: In general, “incidentalomas” are non-functioning, but endocrinological evaluation has shown that subclinical hormonal hyperfunction is not unusual, thus stressing the need for measuring substances such as with metanephrine assay, dexamethasone suppression test in low dosage and establishing the upright plasma aldosterone/plasma renin activity ratio. Non-functioning “incidentalomas” smaller than 4 cm should be followed carefully; those between 4 and 6 cm should be analyzed for its imaging characteristics; for those greater than 6 cm adrenalectomy is indicated. Functioning “incidentalomas” must undergo adrenalectomy. Nonoperated adrenal masses must be followed for two years through imaging and function testing.

Orientador: Luiz Paulo Kowalski / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-04T05:01:41Z (GMT). No. of bitstreams: 1 Perez_DanyelEliasdaCruz_D.pdf: 1930321 bytes, checksum: 307f3509cc4d84642154bbe8c9644f16 (MD5) Previous issue date: 2005 / Resumo: Introdução: O objetivo deste estudo foi avaliar as características clínicas, histopatológicas e imunohistoquímicas de 129 casos de carcinoma adenóide cístico (CAC) de cabeça e pescoço, a fim de identificar fatores prognósticos importantes associados a este tumor. Material e métodos: Entre os anos de 1955 e 1997, cento e vinte e nove casos de CAC foram tratados no Departamento de Cirurgia de Cabeça e Pescoço do Hospital do Câncer A. C. Camargo, São Paulo. Os dados clínicos dos pacientes foram obtidos dos prontuários médicos e resumidos em uma ficha clínica padronizada. As lâminas histológicas foram revisadas para confirmação do diagnóstico e os tumores classificados em tipos histológicos cribriforme, tubular ou sólido. Reações imunohistoquímicas contra proteína p53, antígeno nuclear de proliferação celular (PCNA), Ki-67, antígeno carcinoembrionário (CEA), c-erbB-2 e bcl-2 foram realizadas utilizando a técnica estreptavidina-biotina-peroxidase. Resultados: Setenta e um pacientes eram homens (55%) e 58 (45%) mulheres, com uma idade média de 51,5 anos (10-96 anos). As glândulas salivares menores intra-bucais e as parótidas foram as mais acometidas e 96 pacientes (74,4%) foram diagnosticados com tumores em estádios clínicos III ou IV. O tipo histológico mais comum foi o tipo cribriforme (54,2%), seguido pelos tipos tubular (25,2%) e sólido (20,6%). Cirurgia foi a principal forma de tratamento (75,9%) e 42,7% dos pacientes foram submetidos à radioterapia pós-operatória. As taxas de sobrevida global aos 5 e 10 anos foram 56,5% e 32,5%, respectivamente, e a sobrevida livre de doença foi 42.7% e 29.2% para os mesmos períodos. Análise univariada da sobrevida revelou que pacientes com idade superior a 45 anos (p=0.04), tempo de queixa inferior a 18 meses (p=0.007), presença de parestesia (p=0.04), estádio T (p=0.01), estádio N (p=0.04), estádio M (p<0.001), estádio clínico (p=0.01), tipo histológico sólido (p<0.001), presença de tumor residual após a cirurgia (p<0.001) e aumento da expressão de p53 (p=0.08) correlacionaram com pobre prognóstico. A análise multivariada de sobrevida mostrou que estádio clínico avançado, tipo histológico sólido e expressão de p53 foram fatores prognósticos significantes independentes em pacientes com CAC. Conclusões: Após análise dos dados, concluímos que estádio clínico avançado, tipo histológico sólido e expressão de p53 foram os fatores prognósticos mais significantes associados ao CAC de cabeça e pescoço / Abstract: Introduction: The aim of this study was to analyze the clinical, histological and immunohistochemical prognostic factors of a large series of adenoid cystic carcinoma treated in a single institution, using univariate and multivariate survival analyses. Methods: All cases of head and neck ACC treated between 1955 and 1997, at the Department of the Head and Neck Surgery and Otorhinolaryngology, Hospital do Cancer A. C. Camargo, Sao Paulo, were selected for the study. The clinical data were obtained from the medical records and the histopathological slides reviewed. Immunohistochemical reactions against p53 protein, proliferation cell nuclear antigen (PCNA), Ki-67, carcinoembryonic antigen (CEA), c-erbB-2 and bcl-2 were performed using the streptavidin-biotin-peroxidase method. Results: Of 129 cases, seventy-one were male (55%) and 58 female (45%), with mean age of 51.5 years (10-96 years). The palate and parotid gland were the most common sites. TNM stage revealed 96 (74.4%) clinical stages III + IV tumors, and the most common histological type was the cribriform type (54.2%), followed by tubular (25.2%) and solid type (20.6%). Surgery was the main treatment modality (75,9%) and 42,7% of the patients were submitted to postoperative radiotherapy. The overall survival rates at 5 and 10 years were 56.5% and 32.5% respectively, and the free disease survival rates were 42.7% and 29.2% for the same periods. Univariate survival analysis revealed that age older than 45 years (p=0.04), period of complaints inferior to 18 months (p=0.007), presence of paresthesia (p=0.04), T stage (p=0.01), N stage (p=0.04), M stage (p<0.001), clinical stage (p=0.003), solid histological type (p<0.001), presence of residual tumor (p<0.001) and increased expression of p53 (p=0.08) correlated with a poor prognosis. In the multivariate survival analyses, clinical stage, solid histological subtype and increased expression of p53 were independent significant prognostic factors. Conclusions: According to our findings, clinical stage, solid growth pattern and expression of p53 were the most important prognostic factors in patients with ACC / Doutorado / Patologia / Doutor em Estomatopatologia