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Master of Science / Department of Animal Sciences and Industry / Barry J. Bradford / Two experiments were conducted to explore potential cellular and biochemical mechanisms by which zinc may impact milk production and mammary health of dairy cattle. For the first experiment, twelve lactating Holstein cows were enrolled in a replicated 3 x 3 Latin square, and provided a balanced diet supplemented with either 30 ppm zinc as ZnSO₄ (30-ZS), 60 ppm zinc as ZnSO₄ (60-ZS), or 60 ppm zinc as a mixture of ZnSO₄ and Zn methionine (60-ZM). Blood and milk were collected and analyzed for markers of blood-milk leak and dairy food properties of milk. Milk cells were also isolated and analyzed for RNA abundance of genes related to zinc-mediated apoptosis. Aside from a tendency for 60-ZS to increase feed intake and 60-ZM to increase milk fat compared to 30-ZS, no effects were observed on performance, markers of blood-milk leak, transcript abundance of selected genes, or dairy food properties of milk. A cell culture study was also conducted using immortalized bovine mammary epithelial (MAC-T) cells. Messenger RNA abundance of GPR39—a G protein-coupled receptor shown to regulate cellular activity in response to extracellular zinc concentrations in other epithelial cell lines—was knocked down using RNA interference. Following GPR39 knockdown, cells were treated with 0 or 100 μM zinc. Phosphorylation of the kinases ERK1/2 and AKT was measured in GPR39-knockdown and control cells treated with either 0 or 100 μM zinc. No effect of zinc treatment or GPR39 knockdown was detected on kinase phosphorylation. Interestingly, the 100 μM zinc treatment showed a tendency to increase GPR39 mRNA abundance in control cells. There remain many questions about the cellular mechanisms whereby zinc can impact milk production in dairy cattle.

Orientador: Silvia Regina Rogatto / Banca: Maria Inês de Moura Campos Pardini / Banca: Carlos Eduardo Bacchi / Resumo: O câncer de mama (CM) ocupa o primeiro lugar em incidência e o segundo em mortalidade entre as mulheres no mundo. É considerada uma doença heterogênea com alterações em diversas vias de sinalização molecular. Os fatores prognósticos atuais são incapazes de predizer a evolução da totalidade dos pacientes, assim como os marcadores preditivos não conseguem evitar que grande parte das pacientes com CM sejam submetidas à tratamento desnecessário. A busca de outros marcadores tumorais, tanto prognósticos quanto preditivos tem sido alvo de inúmeras pesquisas, que buscam predizer a evolução clínica de um número maior de pacientes. Dois marcadores tumorais com possíveis implicações clínicas são o TGFB1 e o seu receptor TGFBR2. No entanto, o papel do TGFB1 na carcinogênese e na progressão do câncer de mama ainda não é totalmente conhecido. Desta forma, o objetivo deste estudo foi avaliar a expressão do TGFB1 e do TGFBR2 em tumores de mama e correlacionar com os dados clínicos e histopatológicos. Para tanto, foram analisadas 49 amostras de carcinomas ductais invasivos primários e sete amostras de mamas normais como controles pelas técnicas de RT-PCR quantitativa em tempo real (qRTPCR) e imunohistoquímica (IHQ). Os resultados mostraram que baixos níveis de expressão protéica de TGFB1 nas células tumorais estavam associados a menor sobrevida livre de doença. Os tumores apresentaram acentuada diminuição da expressão do transcrito TGFBR2 quando comparados ao controle normal. Além disso, foram observados baixos níveis dos transcritos TGFB1 e TGFBR2 nos tumores com fenótipos mais agressivos (alto índice proliferativo e estadio avançado). Esses dados sugerem que o TGFB1 atua como supressor tumoral em CMs e que a diminuição da expressão TGFB1 e TGFBR2 é um evento importante na carcinogênese mamária. A detecção dos níveis de expressão protéica... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Breast cancer (BC) is the leading female cancer in incidence and the second one in mortality worldwide. BC is considered a heterogeneous disease, that presents abnormalities in several molecular pathways. The known prognostic markers are not capable to predict the outcome of all the patients, as well as the predictive markers are not capable to avoid unnecessary treatments. It is necessary to search for other tumor markers, even prognostic and predictive, regarding the prediction of a higher number of patient's outcome. Two tumor markers with possible clinical implications are TGFB1 and its receptor TGFBR2. However the role of TGFB1 in carcinogenesis and in the BC progression is not totally understood yet. In this way, the aim of this study was to analyze the expression of TGFB1 and TGFBR2 in breast tumors and to correlate it with clinical and histopathological data. For this, it were analyzed 49 ductal invasive carcinomas and 7 normal breast tissues as controls with quantitative real time PCR (qRT-PCR) and immunohistochemistry (IHC) technologies. The results showed that low expression levels of TGFB1 protein in tumor cells were associated with a lower disease-free survival. Tumors presented highly diminished TGFBR2 transcript expression when compared with normal controls. Moreover, low levels of TGFB1 and TGFBR2 transcripts were observed in tumors with aggressive phenotypes (high proliferation index and advanced stage). These results suggests that TGFB1 acts like a tumor supressor in BCs and that the TGFB1 and TGFBR2 lowering expression is an important event in mammary carcinogenesis. The detection of tumor TGFB1 protein expression levels can be helpful in clinical practice like a prognostic marker in mammary carcinomas. / Mestre

Worldwide, more than 1,000,000 women develop breast cancer each year, and more than 400,000 die because of it. Basal-like breast cancers, which account for 8% to 20% of all breast cancer cases, represent the most aggressive of breast cancers with the majority resistant to existing targeted therapies. Accumulating lines of evidence implicate the Notch pathway in the aetiology of these basal-like breast cancers; while current work in our lab supports the notion that signalling through the Ectodermal Dysplasin Receptor (EDAR) pathway is also important. Notch signalling functions in normal development to control cell fate decisions and is mediated primarily, although not exclusively, through the CBF1 / RBP-Jĸ transcription factor. Aberrant Notch signalling leads to mammary tumourigenesis in mice; however at the outset of this work it was unclear whether signalling through RBP-Jĸ and / or through alternative pathways is required. This thesis presents novel data showing that elevated Notch signalling through the RBP-Jĸ-dependent pathway alone in murine mammary glands causes a number of developmental defects, including reduced ductal outgrowth, increased ductal side branching at puberty and, most significantly, is sufficient to induce mammary tumourigenesis. The data presented also provide supporting evidence that Notch signalling through RBP-Jĸ likely contributes to tumourigenesis, at least in part, via the suppression of apoptosis. At the outset of this thesis far less was known regarding the role(s) of the EDAR pathway within the mammary gland. Despite its recognised function in the development of ectodermal appendages it has been predominantly studied in the context of hair and tooth development. We show here that elevated Edar signalling affects the morphology of numerous ectodermally-derived glands, including the mammary gland, where in general, it results in glands that are enlarged or more elaborately branched. Most significantly, we show that elevated Edar signalling causes mammary tumourigenesis in mice, and provide data to support the hypothesis that elevated EDAR signalling might also be important in a subset of basal-like breast cancers in humans. The murine mammary gland phenotypes seen in response to elevated Edar signalling, including the squamous metaplasia within Edar-induced tumours, are very similar to those observed when Wnt signalling is increased. We provide data to support a positive correlation between activation of the EDAR and Wnt pathways in murine mammary tumourigenesis and provide data to support a comparable interaction in the aetiology of basal-like breast cancer showing squamous differentiation in humans. In summary, this thesis identifies the EDAR pathway as a novel potential therapeutic target in the treatment of a subset of basal-like breast cancers, and provides evidence that signalling through the RBP-Jĸ-dependent Notch pathway is sufficient to induce mammary tumourigenesis, most likely through the suppression of apoptosis.

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Mammary gland development is controlled by hormones such as progesterone and prolactin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. This thesis investigates two molecules in detail, Elf5 and KIBRA, which were identified as potential prolactin targets in a transcript profiling screen for key members in this genetic program of mammary morphogenesis. We examined the effect of expression of Elf5, a transcription factor critical in alveolar differentiation, in a 3D culture model of non-transformed mammary epithelial MCF-10A cells. We discovered that Elf5 expression was selectively repressed over time in these cells when cultured on a basement membrane, and that Elf5 overexpression disrupted the architecture of acini resulting in luminal filling. This occurred due to an increase in the expression of epidermal growth factor receptor (EGFR) with repressed the induction of the pro-apoptotic molecule, Bim. We also observed that Elf5 is up-regulated with progesterone treatment, and that suppression of Elf5 expression in T47D breast cancer cells inhibits proliferation. Data obtained from the suppression of Elf5 expression in the presence of progesterone suggested that the role played by Elf5 in the Pg signalling pathway in T47D cells is relatively minor, and that rather than being a major downstream factor, the induction of Elf5 expression is utilised more to influence and potentiate other signalling pathways, such as the Prl pathway. We characterised expression of KIBRA in the mammary gland and breast cancer cell lines, and observed that KIBRA was also up-regulated with progesterone treatment. Using a bioinformatic approach, we identified the tyrosine kinase receptor DDR1 as a binding partner of KIBRA. We have demonstrated that the WW domains of KIBRA bind to a PPxY motif in DDR1, and that these molecules dissociate upon treatment with the DDR1 ligand, collagen. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodelling events of mammary gland development. Overall, the data presented in this thesis contributes to our growing knowledge of the genetic program responsible for mammary development and carcinogenesis.

<p>This study was carried out to determine the phenotype of special dog mammary gland tumours that were grown in nude mice. 26 tumours were examined by the immunohistochemical ABC-Elite protocol. The tumour tissues were labelled with following anti-human antibodies:</p><p>- AE1/AE3 (pankeratin antibody) labelled epithelial and myoepithelial cells</p><p>- CD 31 labelled endothelial cells</p><p>- desmin labelled cross-striated and smooth muscle cells</p><p>- myosin labelled cross striated muscle cells</p><p>- neurofilament (NF) labelled nerve cells</p><p>- osteopontin labelled preosteoblasts, osteoblasts and osteocytes</p><p>- p63 labelled nuclei of the myoepithelial cells</p><p>- smooth muscle actin (SMA) labelled the cytoplasm of myoepithelial cells</p><p>- type I collagen labelled the extracellular matrix in connective tissue and bone</p><p>- type II collagen labelled the extracellular matrix in cartilage</p><p>- vimentin labelled fibroblasts, fibrocytes, lipocytes, smooth muscle cells, endothelial cells, nerve cells, macrophages and myoepithelial cells</p><p>The tumours were also submitted to a double immunolabelling study using p63 and SMA.</p><p>The study could not give a final conclusion about the origin the tumours. There was still need for more research to answer that question. However, the immunohistochemical technique was analysed in detail, in order to obtain perfect labelings.</p><p>Initially, all the antibodies were tested on normal dog tissue, to acquire the best working dilutions with the lowest background problems. In the tumours, good results were obtained with these dilutions for the antibodies p63, SMA, vimentin, desmin, NF, AE1/AE3 and CD 31. Except for type I collagen, type II collagen and osteopontin that gave too much unspecific labelling of the mouse connective tissue. Even, when using the Vector® M.O.M. blocking kit, the results were still very difficult to interpretate.</p><p>The antigen retrieval methods were evaluated for all the antibodies. The antibodies p63, SMA, vimentin, desmin, AE1/AE3, myosin, neurofilament and CD 31 needed the antigen retrieval treatment. The antibodies type I collagen and type II collagen needed the treatment with the enzyme pepsin, while osteopontin did not need any pretreatment at all.</p><p>The double immunolabelling with p63 and SMA gave excellent results. Different combinations were tried out with different substrates, namely Vector® Nova RED, Vector® DAB and Vector® SG. Vector® methyl green was used as counterstaining, but it interfered with the other substrates, and better results were obtained without this counterstaining.</p>

Sim2s is a basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor. In Drosophila, the Sim2 homolog, sim, is necessary for cell fate determination during central nervous system (CNS) development. In mammals, both Sim2 isoforms are involved in development of various tissues, including muscle, cartilage, and mammary gland. Loss-of-function studies revealed a role for Sim2s in specifying epithelial cell fate during mammary development and inhibiting growth and invasion of aggressive breast cancer cells. This study determined the role of Sim2s in mammary epithelial cell differentiation. Our hypothesis is that Sim2s is sufficient to promote lactogenic differentiation in vivo, characterized by expression of lactation-specific genes. Two models were used to test this hypothesis: (1) a transgenic mouse, expressing Sim2s under control of the MMTV-LTR, and (2) the mouse mammary epithelial cell line HC11. Together, these models allow analysis of the effect of Sim2s on global mammary gland differentiation and the mechanism through which it accomplishes this in a relatively homogenous population of cells. We determined that precocious expression of Sim2s in vivo is associated with upregulation of a subset of milk protein genes in nulliparous females. During early pregnancy, Sim2s regulation of lactogenic differentiation extended to a larger group of genes. Following pup removal, Sim2s appears to promote survival of alveolar epithelial cells. In vitro, Sim2s expression is necessary for maximal Csn2 expression, as determined by loss-of-function studies. Overexpression of Sim2s is sufficient to enhance prolactin-mediated Csn2 expression. Chromatin immunoprecipitation assays performed in HC11 cells revealed enhanced recruitment of Stat5a and RNA Polymerase II (RNAPII) to the regulatory region of Csn2 in the presence of Sim2s. In addition, Sim2s and RNAPII were found in a complex that was localized to both the promoter and coding region of the Csn2 gene. These studies support the idea that Sim2s is upregulated in a developmental stage-specific manner in the mouse mammary gland to promote the survival and differentiation of alveolar epithelial cells expressing high levels of milk protein genes. Further, Sim2s may regulate the function of a specific subset of alveolar cells by targeting the RNAPII holoenzyme complex to genes expressed during lactogenic differentiation.