The objective of this study was to determine whether dietary fat supplementation and level of undegradable intake protein (UIP) could affect daily milk output and composition by influencing nutrient supply to the mammary gland. Three lactating Holstein cows (60, 68, and 74 d postpartum) were used in an incomplete 4 x 4 Latin square design (2 x 2 factorial) and fed diets (15.9% CP and 19.5% ADF) with 0% or 2.5% partially hydrogenated tallow and 33% or 41% UIP. A 5:2.5:1 mixture of dried brewer's grains, corn gluten meal, and blood meal was substituted for soybean meal to raise dietary UIP from 33% to 41% UIP. Despite similar DM intake across treatments, cows produced 9% more milk per day when fed 2.5% supplemental fat, 41% UIP, or the combination of 2.5% fat and 41% UIP when compared to the control diet. Fat supplementation depressed milk protein percentage but not daily milk protein output. Mammary blood flow was estimated using the Fick principle at 6-hr intervals for 24 h. Concentration of individual nutrients in arterial (carotid) and venous (abdominal vein) blood and corresponding blood flows were used to calculate nutrient uptakes by the mammary gland. Calculated carbon uptake was 95 to 101% of output when using estimated carbon content of nutrients, and 100 to 106% when using an elemental analyzer to determine actual carbon output in milk. Uptake of glucose, β-hydroxybutyrate, lactate, pyruvate, acetate, and O₂ were not affected by dietary treatment. Triacylglycerol concentration in arterial blood and uptake of long-chain fatty acids were elevated by fat supplementation, resulting in milk fat with a higher percentage of 18-carbon fatty acids and a lower ratio of saturated to unsaturated fatty acids. Arterial essential and total amino acid (AA) concentrations in plasma and whole blood were elevated when cows were fed 41% versus 33% UIP. However, mammary arteriovenous differences, extraction percentages, and uptakes of most AA were not significantly affected by dietary treatments. Across treatments, peptide AA accounted for ~10% of AA in arterial whole blood but no net uptake of peptide AA by the lactating gland was detected. Results indicated that dietary fat supplementation at two levels of UIP can increase milk production by altering mammary lipid metabolism, thereby improving the efficiency of milk synthesis. However, depression of milk protein percentage in response to dietary fat supplementation was not alleviated by elevating arterial essential and total AA through higher dietary UIP. / Ph. D.

Forty ewe lambs were grouped into four treatment groups: A) fed a standard, high-energy diet, ad libitum; G) fed as group A, but treated with GH (.1 mg/kg bodyweight/d); R) fed the standard diet in restricted amounts to a target weight gain of 120 g/d; S) fed a ration including 30% of a protected fat supplement, ad libitum. Rations were formulated to be isonitrogenous and isocaloric and were fed from approximately seven to 22 weeks of age. Growth rates differed in the order S>A = G> R, although final weights did not differ among ad libitum fed groups. Lambs in group S had heavier mammary glands, with greater amounts of parenchyma and fat pad and higher content of dry, fat-free parenchymal tissue compared to the mean of the remaining groups. Total gland weight was lower in group R, although weight of parenchyma was similar to groups A and G. Parenchyma made up a higher percent of total udder weight in lambs of group R compared to any other group. Parenchymal DNA content was not different by treatment, but glands from group G had twice the total DNA of groups A and R, and group S had 50% more than the latter groups. Volume of mammary glands occupied by parenchyma was increased by more than 50% in group S, compared to the other groups which were similar. Concentrations of prolactin receptors in mammary parenchyma and of GH receptors in liver were increased in lambs of group S. Percent Iinoleic acid in mammary parenchymal lipid of lambs in group S was increased relative to other groups. Unsaturated acids also made up a greater percentage of total fatty acids in group S. Feeding the protected fat supplement resulted in increased unsaturated fatty acid, especially linoleic acid, percentage in mammary fat. This effect was associated with increased mammary growth compared to lambs fed a standard ration. Lambs treated with GH showed some indications of increased mammary growth, but groups A and R were similar except for the increase in percent of gland occupied by parenchyma in group R. In a second study, mammary growth in prepubertal mice increased with increasing dietary energy intake. Differences in ductal growth persisted at 18 weeks of age, and effects of exogenous steroids at this time were not significant. Prepubertal mammary growth in mice is not sensitive to inhibition by high plane of nutrition as is the case in ruminants. / Ph. D.

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

La morphogenèse de la glande mammaire résulte de la coordination de différentes voies, incluant l'apoptose, la prolifération, la différenciation et la dynamique des cellules souches/progénitrices. La transition épithéliale-mésenchymateuse (EMT) semble être impliquée dans ces voies de signalisation. Ainsi, nous nous sommes concentrés sur le facteur de transcription Slug, un gène clé régulant l'EMT, et son implication dans la morphogenèse de la glande mammaire. Dans un premier temps, en utilisant un modèle de souris transgéniques Slug-Lacz, nous avons localisé Slug dans une sous-population couvrant 10 à 20% des cellules basales du tubule et des cap cells du bourgeons terminal, coexprimant les marqueurs P-cadhérine, CK5, CD49f. Ensuite, nous avons montré par des expériences in vitro de perte et de gain de fonction, que Slug régulait la différenciation et la prolifération des cellules épithéliales mammaires. De plus, nous avons trouvé que Slug inhibait l'apoptose, promouvait la motilité cellulaire, et permettait l'émergence et la croissance de mammosphères clonales. Ce dernier point montre l'implication de Slug dans les cellules souches, qui est renforcé par le fait que des cellules primaires déficientes pour Slug étaient incapables de donner des mammosphères secondaires. Par ailleurs, nous avons pu observer in vivo que les souris déficientes pour Slug présentaient un retard de développement de la glande mammaire, possédant moins de cellules en prolifération, et une surexpression des marqueurs des cellules luminale CK8/18, GATA3 et ER. D'autres gènes régulant l'EMT sont retrouvés surexprimés, suggérant un mécanisme de compensation, qui peut expliquer le fait que le retard de développement de la glande mammaire est rattrapé à l'âge adulte. Les glandes mammaires Slug-knockout présentaient également des branchements excessifs, évoquant une différenciation précoce, similaire aux glandes mammaires de souris déficientes pour la P-cadhérine, exprimée dans les cellules basales. Sachant cela, nous avons constaté que la P-cadhérine était diminuée dans les glandes mammaires Slug-knockout, et dans les cellules CommaDβ traitées par siRNA ciblant Slug. Nous avons alors trouvé que Slug se liait directement au promoteur de la P-cadhérine et l'activait, et que cette dernière intervenait dans certains effets fonctionnels de Slug, tels que la croissance de mammosphères, la différenciation et la migration cellulaire. Ainsi, nous avons montré l'importance d'une nouvelle voie de signalisation Slug/P-cadhérine dans les capacités souches/progénitrices des cellules épithéliales mammaires, intégrant la différenciation et la motilité cellulaire, et nous avons maintenant une meilleure compréhension de son rôle dans l'agressivité de certains cancers du sein. / Mammary gland morphogenesis results from the coordination of different pathways, including apoptosis, proliferation, differentiation, and stem/progenitor cell dynamics. Epithelial-mesenchymal transition (EMT) appears to be involved in these signalling pathways. Thus, we focused on transcription factor Slug, a key gene regulating EMT, and its involvement in mammary gland morphogenesis. First, using a Slug–LacZ transgenic mice model, we located Slug in a subpopulation covering about 10–20% basal duct cells and cap cells of terminal end bud, coexpressed with basal markers P-cadherin, CK5 and CD49f. Then, we have shown by in vitro experiments of loss and gain of function that Slug regulated the differentiation and proliferation of mammary epithelial cells. Moreover, we found that Slug inhibited apoptosis, promoted cell motility, and allowed the emergence and growth of clonal mammospheres. This last point shows the involvement of Slug in stem cells, which is reinforced by the fact that primary cells deficient for Slug were unable to give secondary mammospheres. Furthermore, we observed in vivo that mice deficient for Slug showed delayed development of the mammary gland, with less proliferating cells, and overexpression of markers of luminal cells CK8/18, GATA3 and ER. Other genes regulating EMT are found overexpressed, suggesting a compensatory mechanism, which can explain the fact that the delayed development of the mammary gland is caught up in adulthood. The Slug-knockout mammary glands also showed overbranching, evoking an early differentiation, similar to the mammary glands of mice deficient in P-cadherin, expressed in the basal cells. Knowing this, we found that P-cadherin was decreased in Slug-knockout mammary glands, and in CommaDβ cells treated with siRNA targeting Slug. We then found that Slug binds directly to the promoter of the P-cadherin and activated it, and that P-cadherin was involved in some functional effects of Slug, such as mammospheres growth, differentiation and cell migration. Thus, we have shown the importance of a new signalling pathway Slug/P-cadherin in the capacity of mammary epithelial stem/progenitor cells, integrating differentiation and cell motility, and we now have a better understanding of its role in the aggressiveness of some breast cancers.

Orientador: Maria Cecília Rui Luvizotto / Coorientador: Heitor Flávio Ferrari / Banca: Daniela Bernadete Rozza / Banca: Livia Castanhas Breganó / Resumo: O tumor mamário canino (TMC) é a neoplasia que mais comumente acomete cadelas idosas não castradas. Na análise histopatológica, 41 a 53% dos TMCs são classificados como malignos. Os receptores de estrógeno α (REα) são receptores nucleares importantes na transcrição de fatores e transdução de sinais hormonais, considerados um dos fatores prognósticos para o tumor mamário humano, participam de forma ativa na carcinogênese mamária. Durante o desenvolvimento do TMC, macrófagos teciduais compõe o microambiente tumoral, com a função de estimular a angiogênese, aumentando a possibilidade de metástases, sendo fundamental para o prognóstico de neoplasia mamária na cadelae na mulher. O objetivo deste trabalho foi pesquisara expressão e a provável correlação de REα com a distribuição de macrófagos tumorais (TAMs) em TMCs por meio de imunohistoquímica. Foram analisados 40 tumores mamários malignos, classificados histologicamente em quatro grupos distintos e um grupo controle composto por 08 glândulas mamárias caninas sem alteração anatomopatológica e seus respectivos linfonodos regionais. Os resultados mostraram que a imunomarcação positiva ao REα variou de um a três casos dentre os grupos histológicos estudados, havendo nestes, menor número de metástase para linfonodo. A positividade para os TAMs mostrou associação com o tipo histológico, variando de moderada a acentuada nos carcinomas mamários de alto grau histológico que apresentaram necrose, invasão linfática e formação tubular. Não ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The canine mammary tumor (CMT) is the neoplasia that most commonly affects uncastrated elderly female dogs. On histopathological analysis, 41 to 53% of CMTs are classified as malignant. Estrogen receptors α (REα) are important nuclear receptors for transcription factors and signal transduction of hormones, considered prognosis factor for breast cancer in human, and recognized as actively involved in breast carcinogenesis. During the development of mammary cancer in canine species, macrophages composes the tumor microenvironment, stimulating angiogenesis, facilitating the local dissemination of neoplastic cells and increasing the possibility of metastasis, being fundamental for mammary neoplasia prognosis in dogs and women. This study aimed to evaluate the expression and the probable correlation of REα with the distribution of tumor associated macrophages (TAMs) in CMTs by immunohistochemistry. Forty CMTs were analyzed histologically in four distinct groups and a control group composed of eight canine mammary glands without alteration anatomopathological and their respective regional lymph nodes. The results showed that the REα positive immunostaining varied from one to three cases among the histological groups studied, and there was less lymph node metastasis. The positivity for TAMs showed a positive correlation with the histological type, ranging from moderate to severe in mammary carcinomas of high histological grade that presented necrosis, lymphatic invasion and tubular ... (Complete abstract click electronic access below) / Mestre

Orientador: Guilherme de Paula Nogueira / Resumo: O objetivo desse estudo foi comparar as concentrações séricas de cálcio, progesterona e fator de crescimento semelhante à insulina tipo 1 (IGF-I) e a ocorrência de crescimento microbiano no leite e de retenção de placenta antes da secagem e após o parto de vacas de alta produção tratadas ou não com cabergolina. Foram utilizadas 48 vacas multíparas, das raças holandesas, girolandas ou jersolandas no final da lactação (aproximadamente 60 dias antes do parto), com produção acima de 14 L/dia. Os animais foram divididos em grupo controle e grupo tratado, recebendo 5,6 mg de cabergolina via I.M. As coletas de sangue para mensuração das concentrações de progesterona, IGF-I e de cálcio sérico foram realizadas no dia da secagem (antes da aplicação da cabergolina), um dia após a secagem, sete dias após a secagem, de quinze a sete dias antes do parto e sete dias após o parto; no dia do parto e um dia após o parto foi feita apenas a dosagem de cálcio. Para análise microbiológica foi coletado leite sete dias antes da secagem, no dia da secagem e no dia do parto e foi observada a ocorência de retenção de placenta. Não foi observada diferença significativa entre os grupos (p > 0,05) na ocorrência de infecção da glândula mamária, nas concentrações séricas de cálcio e hormônios, exceto nas amostras coletadas uma semana após o parto, quando a concentração de cálcio foi superior no grupo tratado quando comparado ao controle (11,10 ± 1,751 vs. 9,610 ± 2,212 mg/dL, p = 0,03). A ocorrência de rete... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to compare the serum concentrations of calcium, progesterone, insulin-like growth factor-1 (IGF-I), occurrence of milk microbiological growth and of retained placenta before drying off and after parturition in high production dairy cows treated or not with cabergoline. A total of 48 multiparous Holstein, Gyr x Holstein and Jersey x Holstein cows at the end of lactation period (approximately 60 days before partum) were used, with production above 14L/day and sorted into control and treated group receiving 5.6 mg of cabergoline via I.M. Blood samples for serum concentrations of progesterone, IGF-I and calcium were drawn on the day of drying off (before cabergoline injection), one day after drying off, seven days after drying off, between fifteen and seven days before partum and seven days postpartum; on the day of parturition and one day postpartum only calcium was measured. For microbiological analysis, milk samples were collected seven days before drying off, on the day of drying off and on the day of partum. In addition, occurrence of retained placenta were recorded. No significant difference was observed between the groups (p > 0.05) in the occurrence of mammary gland infection, calcium and hormones serum concentrations, except in the samples collected one week after calving, when the calcium concentration was higher in the treated group compared to the control group (11.10 ± 1.751 vs. 9.610 ± 2.212 mg/dL, p = 0.03). The occurrence of placent... (Complete abstract click electronic access below) / Mestre

Os objetivos dos experimentos (I e II) foram avaliar os efeitos da inclusão de 2% de fontes de ácidos graxos poliinsaturados (óleo de soja, de linhaça e de peixe) e doses crescentes de óleo de soja (0, 30, 60 e 90 g/dia) sobre o consumo, digestibilidade dos nutrientes no trato digestório total, produção e composição do leite, perfil de ácidos graxos arterial e no leite, concentração arterial e retenção de metabólitos pela glândula mamária das cabras em lactação. Dois meses antes do início do experimento, os animais foram preparados cirurgicamente para exteriorização subcutânea de ambas as artérias carótidas a fim de facilitar a colheita do sangue arterial. Os animais foram alojados em baias individuais onde receberam ração com 40% de silagem de milho e 60% de concentrado, com base na matéria seca. No experimento 1, foram utilizadas quatro cabras Saanen multíparas, produzindo 2,4 kg/dia e com 35 dias de lactação e foram distribuídas em Quadrado Latino 4 x 4 com período experimental de 112 dias. Cada subperíodo teve a duração de 24 dias de adaptação e 4 dias de colheita. Os tratamentos do experimento I foram: a) ração sem adição de óleo (controle); b) controle mais 2% de óleo de soja (OS); c) controle mais 2% de óleo de linhaça (OL) e d) controle mais 2% de óleo de peixe (OP). No experimento 2, foram utilizadas quatro cabras Saanen multíparas, produzindo 3,7 kg/dia e com 35 dias de lactação e foram distribuídas em Quadrado Latino 4 x 4 com período experimental de 112 dias. Cada período teve a duração de 24 dias de adaptação e 4 dias de colheita. Os tratamentos do experimento 2 foram: Ração controle mais o fornecimento oral de doses de óleo de soja (0, 30, 60 e 90 g). No experimento l, a inclusão dos óleos não influenciou o consumo da matéria seca (CMS), da matéria orgânica (CMO), da fibra em detergente neutro (CFDN) e da proteína bruta (CPB), embora tenha havido efeito quando o CMS e o de CMO foram relacionados com o peso metabólico. O CEE foi influenciado pelos óleos. As digestibilidades da MS, MO, FDN, PB e do EE no trato digestório total não foram influenciadas. Não houve efeito na produção de leite e nos seus componentes (gordura, proteína, lactose, sólidos totais e extrato seco desengordurado), embora tenha havido efeito no perfil de ácidos graxos. O óleo de peixe promoveu elevação (350%) na concentração do ácido vacênico (C18:1 t11) no leite, alem de maiores elevações na concentração arterial do ácido vacênico (376%) e na relação C18:1 t11, C18:2 c9, t11 (1111%). A adição dos óleos nas rações, não influenciou a concentração arterial e a retenção de metabólitos (glicose, ß- hidroxibutirato) pela glândula mamária. No experimento 2, as doses de óleo de soja alteraram o CMS e o CMO, assim como a digestibilidade da MS e do EE. Não houve efeito na produção de leite, porém a concentração de gordura diminuiu com as doses de óleo. Os perfis de ácidos graxos no leite e no sangue arterial foram influenciados pelas doses de óleo de soja. O aparecimento do C18:2 t10, c12 no leite culminou com a redução de gordura (558%). A dose de 90 g propiciou a elevação do ácido vacênico (810%), ao contrário do C18:2 c9, t11, que diminuiu. A concentração arterial e a retenção mamária de glicose e ßhidroxibutirato não foram influenciadas pelas doses de óleo de soja. / The objectives of these studies were to evaluate the effects of adding 2% palyunsaturated fatty acid sources (soybean, linseed and fish oils) or soybean oil doses (O, 30, 60 or 90 g/day) on dry matter intake, apparent nutrient digestibilities, milk yield, milk composition, arterial and milk fatty acid profiles, and metabalite uptake by the mammary gland in lactating goats. Animais were housed in individual tie stalls and received diets containing 40% com silage and 60% concentrate (dry matter basis). In Experiment \"four Saanen goats producing 2.4 kg/day and 38 days in milk were used in a 4 x 4 latin Square design during 112 days. Each period lasted 28 days (24d for adaptation and 4d for sampling). Four experimental diets were used: a) Control diet (CO, without additional oil); b) CO plus 2% soybean ail (OS); c) CO plus 2% linseed oil (Ol); ar d) CO plus 2% fish oil (OP). Experiment 11, four Saanen goats producing 3.7 kg/day and 32 days in milk were used in a 4 x 4 latin Square design during 112 days. Each period lasted 28 days (24d for adaptation and 4d for sampling). Gaats were fed a control diet plus an oral daily dose (O, 30, 60 or 90 g) of soybean oi!. In experiment I, ail sources did not influence intakes of dry matter, organic matter, or neutral detergent fiber. Hawever, an oil effect was observed when expressed on the basis of metabolic weight. The OM, OM, NOF, CP, and EE apparent digestibilities were not affected by treatment. Production of milk components (fat, protein, lactose, total solids, and non-fat salids) was nat different among treatments, although an effect in fatty acid profile was observed. Fish oil increased arterial and milk vaccenic acid t11 C18:1 concentrations by more than 350%, and increased the t11 C18:1: c9,t11 C18:2 ratio in the arterial blood by 1111 %. Gil addition did not influence arterial blood concentration and mammpry gland uptake of glucase or l3-hydroxybutyrate. Experiment li showed that supplementation of saybean oil resulted in changes of OM, OM, and EE intakes, but intake of the NOF and CP were not affected. Oigestibility of OM and EE were influenced by soybean ai!. Oaily milk production was not influenced, but milk fat was reduced by soybean oi!. Milk and arterial fatty acids profiles were influenced by saybean oi!. As the concentration of t10,c12 C18:2 increased in arterial blood, a reduction (558%) in milk fat was observed and also a decrease in ô9-desaturase activity was detected. The 90 9 supplement of soybean oil resulted in an elevation (810%) in the milk vaccenic concentratian; conversely (c9,t11 C18:2), decreased. The. arterial blood concentration and mammary gland uptake of glucose and l3-hydroxybutyrate were not affected by soybean oil.

Com o objetivo de avaliar a relação entre o exame físico da glândula mamária de ovelhas com o leite, 292 ovelhas da raça Santa Inês foram examinadas. As amostras de leite foram submetidas ao exame bacteriológico, ao CMT e a CCS (automática e microscópica). Variações especificamente identificadas por inspeção da mama apresentaram alterações de celularidade observadas pelo CMT (p<0,002), na CCS automática (p<0,006), e na CCS microscópica diferencial para células mononucleares (p<0,03). Mamas pendulosas puderam ser associadas a maior isolamento bacteriológico (p<0,0006) e maior celularidade nas CCS automática (p<0,01) e microscópica (p<0,05). Na inspeção do teto foram encontradas diferenças no exame da CCS automática (p<0,002), no CMT (p<0,004) e na CCS microscópica diferencial para células mononucleares (p<0,005). Nos tetos com soluções de continuidade, foram observadas diferenças apenas no exame bacteriológico (p<0,03). Quanto à palpação da mama, foram observadas diferenças no exame bacteriológico (p<0,001), no CMT (p<0,01) e na CCS microscópica total (p<0,02) e diferencial para células polimorfonuleares (p<0,02) e para células mononucleares (p<0,0002). Quanto à palpação do teto, foram encontradas diferenças no CMT (p<0,01) e na CCS microscópica total (p<0,002) e diferencial para células polimorfonucleares (p<0,002) e para células mononucleares (p<0,002). Associando-se a inspeção à palpação das metades mamárias, observou-se diferenças na CCS automática (p<0,0002) e na CCS microscópica total (p<0,04) e diferencial para células mononucleares (p<0,01). No exame do fundo escuro, foram observadas diferenças no CMT (p<0,0001), na CCS automática (p<0,0001), e na CCS microscópica total (p<0,0001) e diferencial para células polimorfonucleares (p<0,0001) e mononucleares (p<0,0001). Quando associadas duas categorias do exame físico da glândula mamária, a inspeção e a palpação, e o exame do fundo escuro, foram observadas diferenças no CMT (p<0,0001), na CCS automática (p<0,0001) e na CCS microscópica total (p<0,0001) e diferencial para células mononucleares (p<0,0001). Nas ovelhas acompanhadas durante a lactação foram observadas diferenças na inspeção da mama (p<0,0001) e do teto (p<0,0001), na palpação da mama (p<0,005) e do teto (p<0,003), na inspeção e palpação das metades mamárias (p<0,04), na inspeção, palpação e fundo escuro das metades mamárias (p<0,03), na CCS automática (p<0,0001) e na CCS microscópica total (p<0,02) e diferencial para células polimorfonucleares (p<0,02) e para células mononucleares (p<0,02). Foi possível concluir que há relação entre o exame físico e o perfil celular e bacteriológico, mas que a inflamação da mama foi melhor identificada pelo CMT, através da inspeção da mama e do teto, da palpação da mama e do teto, do exame do fundo escuro e da avaliação da inspeção, palpação e fundo escuro das metades mamárias. A inflamação também foi identificada pela avaliação da CCS automática e/ou da CCS microscópica total e/ou diferencial através da inspeção da mama e do teto, pendulosidade da mama, palpação da mama e do teto, inspeção e palpação das metades mamárias, exame do fundo escuro e a inspeção, palpação e fundo escuro das metades mamárias. O processo infeccioso mamário pôde ser identificado através da pendulosidade mamária, presença de soluções de continuidade no teto e pela palpação da mama. A maior celularidade ocorreu no início da lactação. No final da lactação, houve maiores proporções de alterações na inspeção da mama e do teto e na palpação do teto. Nas fases intermediárias da lactação, as alterações na inspeção e palpação associadas e na inspeção, palpação e fundo escuro associados foram menores. / The aim of this work is to evaluate the relationship between the mammary gland physical examination of ewes and their milk. 292 ewes of Santa Inês breed were evaluated, and the milk samples were submitted to bacteriological examination, to CMT (California Mastitis Test), to automatic SCC (somatic cell count) and microscopic SCC. Specifically identified variations during the mama inspection had cellular alterations, observed in CMT (p<0,002), in automatic SCC (p<0,006) and in differential microscopic count for mononuclear cells (p<0,03). Pendulous mamas could be associated to the largest bacteriological isolation (p<0,0006) and the largest cellular in automatic SCC (p<0,01) and microscopic SCC (p<0,05). In the teat inspection, differences were found in the automatic SCC (p<0,002) in the CMT (p<0,004) and in differential microscopic count for mononuclear cells (p<0,005). In the teat with lesion, differences were observed only in bacteriological examination (p<0,03). In mamma palpation, there were observed differences in bacteriological examination (p<0,001), in CMT (p<0,01), in total microscopic count (p<0,02), differences to polymorphonuclear cells (p<0,02) and to mononuclear cells (p<0,0002). In teat palpation, differences were found in CMT (p<0,01), in total microscopic count (p<0,002) and differences to polymorphonuclear cells (p<0,002) and to mononuclear cells (p<0,002). Associating the inspection and palpation of mammary gland, differences were observed in automatic SCC (p<0,0002) and in total microscopic count (p<0,04) and differences to mononuclear cells (p<0,01). In tamis exam, differences were observed in the CMT (p<0,0001), in automatic SCC (p<0,0001), and in total microscopic count (p<0,0001) and differences to polymorphonuclear cells (p<0,0001) and mononuclear cells (p<0,0001). When the two categories of mammary gland physical exam, the inspection, the palpation, and the tamis exam were associated, there were observed differences in the CMT (p<0,0001), in the automatic SCC (p<0,0001) and in the total microscopic count (p<0,0001), and differences in the microscopic SCC to mononuclear cells (p<0,0001). In the ewes followed during the lactation there were differences in mamma inspection (p<0,0001), in teat (p<0,0001), in mamma palpation (p<0,005) and in teat (p<0,003), in the inspection and palpation of mammary gland (p<0,04), in inspection, palpation and tamis exam of mammary gland (p<0,03), in automatic SCC (p<0,0001) and in total microscopic count (p<0,02), and differences for polymorphonuclear cells (p<0,02) and to mononuclear cells (p<0,02). It can be concluded that there is a relationship among the physical exam and the cellular and bacteriological profile, but the mamma inflammation was better identified by the CMT, through inspection of mamma and teat, and mamma and teat palpation, and tamis exam and the evaluation by inspection, palpation and macroscopic assessment of the milk by mammary glands. The inflammation was also identified by the evaluation of automatic SCC and/or total microscopic count through inspection of mamma and teat, pendulous mamma, palpation of mamma and teat, inspection and palpation of mammary gland, tamis exam and the inspection, palpation and tamis exam of mammary gland and milk. The infection in mammary gland can be identified by pendulous mamma, lesion in teat and by palpation of mamma. The biggest cellular was found in early lactation. And in late lactation, there were found more alterations in mamma and teat inspection and palpation of teat. In the intermediate phases of lactation, the alterations in inspection and palpation associated and the inspection, palpation and the tamis exam associated were smaller.

Para produção de biofármacos protéicos, como a eritropoietina recombinante humana (EPOrh), são necessárias alterações pós-traducionais adequadas que garantam a sua especificidade e atividade biológica. Essas características são obtidas apenas em biorretores baseados em células eucarióticas, como as da glândula mamária. Sistemas baseados nesse tipo celular, tanto in vivo quanto in vitro, já são utilizados para produção estratégica e viável de proteínas recombinantes biologicamente ativas. Assim, tanto o estabelecimento de novas linhagens de células mamárias que apresentem boa expressão protéica quanto o desenvolvimento de sistemas in vivo que utilizem a estrutura da glândula mamária para essa produção de proteínas recombinantes são de grande valia. O presente trabalho teve como objetivo comparar dois métodos de estabelecimento de uma cultura de células de glândula mamárias ovinas, enzimático e não enzimático, e verificar sua capacidade de expressão das proteínas do leite &beta;-lactoglobulina, &alpha;-caseína, &beta;-caseína e &kappa;-caseína mediante o tratamento com SFB (soro fetal bovino) ou SOL (soro de ovelha lactante), na presença ou não de Matrigel. Para isso, foi realizado um experimento in vitro, no qual foi estabelecido o cultivo celular até a passagem 12 (P12) de duas linhagens celulares: digerida (LD) e não digerida (LND). Para a LD na P12 foi observado apenas um tipo celular, o qual era positivo para a marcação com vimentina. Essa linhagem apresentou expressão gênica de &beta;-caseína e &beta;-lactoglobulina apenas quando tratada com meio de cultivo acrescido de SFB, sendo a expressão inferior (P=0,001) ao grupo da LND submetido ao mesmo tratamento. Já a LND, quando tratada com meio adicionado com SFB expressou &kappa;-caseína além da &beta;-caseína e &beta;-lactoglobulina. A troca do SFB do meio de cultivo por SOL aumentou a expressão gênica de &beta;-lactoglobulina (P=0,001) para ambas linhagens. Foi realizada a curva de crescimento para LD e LND na P12 com o meio de cultivo acrescido com SFB ou SOL. Para a LND observou-se o efeito do meio na velocidade de crescimento celular, sendo que foi maior para o grupo tratado com SFB (P&lt;0,05). Para a LD, não ocorreu o efeito do meio na velocidade de crescimento celular (P&gt;0,05), não sendo observada diferença com a LND tratada com SOL (P&gt;0,05). A LND apresentou marcação positiva para a presença de vimentina e citoqueratina. Este trabalho visou, ainda, estabelecer um sistema de produção da EPOrh no leite de ovelhas não transgênicas pela técnica de infusão intra-mamária in vivo de dois plasmídeos diferentes e verificar a secreção qualitativa desta proteína por Western-blotting. Assim, foi feito um experimento in vivo no qual glândulas mamárias de ovelhas foram transfectadas com dois plasmídeos diferentes: ALAC (n=2), BGL (n=2) e controle negativo (n=2). Após a infusão dos plasmídeos, foi realizada a eletroporação de cada teto (3 choques de 500 volts com a duração de 15ms cada, sendo realizada a inversão da polaridade). Os animais foram ordenhados durante 20 dias após a transfecção, porém não foi possível detectar a presença de EPOrh nas amostras de leite analisadas. O limiar de detecção do teste utilizado foi de 67,5pg de EPOrh (Eritromax&reg;) em leite controle negativo de ovelha. Concluindo, foi possível estabelecer o cultivo in vitro das LD e LND com capacidade de expressar proteínas do leite, sendo a expressão da &beta;-lactoglobulina aumentada pelo tratamento com SOL. Ambas as linhagens apresentaram marcação positiva para vimentina, mas apenas LND para citoqueratina. Ainda, para o experimento in vivo, não foi possível detectar a expressão de EPOrh no leite das ovelhas transfectadas com os plasmídeos ALAC e BGL. / Some post-translational modifications are necessary for the production of biopharmaceutical proteins, such as recombinant human erythropoietin (rhEPO), with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors, in vivo or in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression and the development of systems for production of recombinant proteins by the mammary gland in vivo are essential studies. One of the main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as &beta;-lactoglobulin, &alpha;-casein, &beta;-casein and &kappa;-casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel&reg;. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed &beta;-lactoglobulin and &beta;-casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0,001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed &beta;-lactoglobulin, &beta;-casein and &kappa;-casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of &beta;-lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P&lt;0,05). However, the medium did not have effect on growth speed of LD (P&gt;0,05) and no difference was observed at the NDL treated with LOS (P&gt;0,05). The NDL was positive for staining with vimentin and cytokeratin. The second main objective of this study was to establish an in vivo system for the production of rhEPO in milk of non-transgenic ewes by the intra-mammary infusion of two different plasmids and verify the qualitative milk secretion of this protein by western-blotting. In this way, in the in vivo experiment ovine mammary glands were transfected with two different plasmids: ALAC (n=2), BGL (n=2) and negative control (n=2). Each half udder was filled with plasmid solution and three 3 electric pulses of 500 volts were applied for 15ms each, followed by another three pulses with reversed polarity. The three animals were milked for 20 days after transfection, nevertheless it was not possible to identify rhEPO in any milk sample. The test threshold to identify rhEPO (Eritromax&reg;) in milk from a negative control animal was 67,5pg. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of &beta;-lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. In the in vivo experiment, rhEPO secretion was not detected in the milk from ewes transfected with ALAC and BGL plasmids.

As serino proteases participam de diversos processos fisiológicos (tal como o de coagulação) e patológicos. Essas enzimas estão amplamente distribuídas entre as espécies, são também toxinas dos venenos de serpentes, sendo denominadas SVSPs (snake venom serine proteases). Essas SVSPs são multifuncionais e contêm uma tríade catalítica formada pelos aminoácidos HDS. Algumas SVSPs são comercialmente disponíveis, sendo indicadas para o tratamento de infarto do miocárdio, tromboses e embolia pulmonar. No veneno de Crotalus durissus terrificus estão descritas até o momento, apenas duas SVSPs sendo que a mais estudada é a giroxina que representa cerca de 2,5% do veneno total. No presente estudo foi reportado a clonagem de sete serino proteases amplificadas a partir de uma biblioteca de cDNA de glândula de veneno de um único espécime adulto de Crotalus durissus terrificus. Estes clones foram analisados com relação à organização do cDNA, estrutura e prováveis funções. A construção do modelo tridimensional da giroxina permitiu verificar as similaridades com tripsina, trombina e outras SVSPs. A glicosilação e a presença de muitas pontes dissulfetos dificultam a obtenção das SVSP recombinantes na forma solúvel e com atividade, por expressão em E.coli. Assim, neste trabalho foi abordada a expressão em células de mamífero (que realiza as modificações pós-traducionais) com resultados promissores. Para tanto, o peptídeo sinal de Igk, a seqüência madura e a região 3 UTR da giroxina foram clonados no vetor pED, originando um novo vetor (pED-Giro). Este vetor carrega o peptídeo sinal de Igk, o que possibilitou a secreção da giroxina para o meio de cultura. O vetor pED-Giro foi transfectado em células CHO DXB11 dhfr e COS-7. A giroxina foi detectada no extrato total das células COS-7 por western blot e, em seguida, purificada do meio de cultura com coluna de afinidade (Benzamidina Sepharose) e demonstrado sua integridade pelo ensaio de atividade esterásica. / The serine proteases affect several physiological processes (such as the coagulation cascade) and pathological ones. These enzymes are widely distributed beyond the species; they are also toxins from snake venoms and are called SVSPs (snake venom serine proteases). These SVSPs are multifunctional and have a catalytic triad formed by HDS amino acids. Some of them are commercially available for use in clinical treatment for heart attack, tromboses and pulmonary embolism. So far, in Crotalus durissus terrificus venom only two SVSPs are described and gyroxin is considered the most studied SVSP which represents about 2,5% of the total venom. In the present study was reported the cloning of seven serine proteases amplified from a cDNA library of a venomous gland of a single adult specimen from Crotalus durissus terrificus venom. These clones have been analyzed in relation to the cDNA organization, structure and probable functions. The three-dimensional model of the gyroxin made possible the analysis of similarities with trypsin, thrombin and other SVSPs. The glycosylation and many disulfide bonds of the SVSPs make difficult the expression in E.coli to obtain the soluble recombinant toxin with activity. The expression in mammalian cells is very promising, because it is possible to make pos translation modification and to obtain the recombinant toxin secreted to the culture medium. The IgK signal peptide, the mature sequence and 3\'UTR region of gyroxin were cloned in the pED expression vector resulting in a new vector (pED-Giro). This vector carries the Igk signal peptide, which allows the secretion of the protein to the culture medium. The pED-Giro vector was transfected in CHO DXB11 dhfr and COS-7 cells. The gyroxin was detected in COS-7 total extract by western blot and after, purified from the medium culture and its integrity was confirmed by esterase activity assay.