The mammary gland is a branched epithelial organ comprised of myoepithelial, ductal and alveolar cells that are derived from resident stem and progenitor cells. The progression from mammary gland stem cell(s) to the differentiated mammary gland cell types is poorly understood. Here, I describe the identification and characterization of two luminal progenitor cell populations in the mouse mammary gland, and investigate the role of the transcription factor C/EBPβ in their development. In Chapter 2, I describe the isolation of two luminal progenitor cell populations (Sca1+ and Sca1- luminal cells) and show that they are differentially primed in their gene expression towards ductal and alveolar cell fates, respectively. Furthermore, I show that in vivo genetic priming affects the in vitro differentiation potential of Sca1+ and Sca1- luminal cells. In Chapter 3, I show that C/EBPβ is required for the appropriate specification of ductal and alveolar lineages, and in its absence, alveolar lineage priming is lost, and ductal lineage priming is up-regulated in both Sca1+ and Sca1- cells. Preliminary data also shows that in addition to severe proliferation defects, the changes in in vivo lineage priming in Cebpb-/- mice also affect the in vitro differentiation potential of Cebpb-/- Sca1+ and Sca1- luminal progenitors. Lastly, in Chapter 4, I describe the genome-wide binding characteristics of C/EBPβ in Sca1+, Sca1- and P16.5 alveolar cells. These experiments reveal that genome-wide C/EBPβ occupancy is correlated with alveolar cells fate, and that C/EBPβ target genes perform distinct cellular functions in alveolar cells (Sca1- cells and P16.5). Furthermore, I show that Elf5 is directly regulated by C/EBPβ, and posit that direct regulation of Elf5 by C/EBPβ may be one mechanism through which C/EBPβ exerts its alveolar cell fate programming.

Orientador: Wilter Ricardo Russiano Vicente / Banca: Maricy Apparicio Ferreira / Banca: Marcela Marcondes Pinto Rodrigues / Resumo: O estudo dos tumores mamários em cadelas revela-se como um excelente modelo para a investigação das neoplasias mamárias em mulheres e tanto no homem quanto nos animais a resposta imune pode interferir no desenvolvimento de neoplasias. Este trabalho teve como objetivo avaliar a eficácia e estimulação do sistema imune celular em tumores mamários de fêmeas caninas por meio da expressão de CD4, CD8, CD4+CD25+ (Foxp3), CD28, CD152 e CD56 (NK). A avaliação da expressão desses marcadores foi avaliada por imunoistoquimica, utilizando-se o método estreptoavidina-biotina-peroxidase. Para a realização desse estudo foram coletadas 20 amostras de tumores mamários de fêmeas caninas e essas divididas de acordo com a classificação histopatológica em 2 grupos: 10 carcinomas simples e 10 carcinomas complexos. Em relação aos resultados não se observou diferenças quanto à quantificação das células imunomarcadas quando comparadas segundo o tipo tumoral. No caso dos carcinomas simples evidenciou-se diferença entre a quantificação das células imunomarcadas (P< 0,05) pelo anticorpo CD28 (p=0,03) e Foxp3 (p=0,01) sendo menores que todas as demais marcações. Já para os carcinomas complexos houve diferença entre a quantificação das células imunomarcadas (P= 0,05) pelo anticorpo CD-152 que apresentou valor maior que as demais marcações com exceção do CD8. Os marcadores CD28, CD56 e Foxp3 foram menores que o CD8 (p=0,01) sendo o Foxp3 menor que todos os outros marcadores. No presente estudo observou-se que existe um controle negativo do sistema imune em ambos os casos, porém esse controle negativo foi mais evidente no carcinoma complexo / Abstract: The mammary tumor study in female dogs is revealed as an excellent model for the investigation of breast tumors in humans. On both species, the immune system can interfere on neoplasia progression. The aim of this study was to evaluate the immunolocalization and quantification of CD4, CD8, CD4+CD25+ (Foxp3), CD28, CD152 and NK in tumour lymphocyte infiltration, to evaluate the efficacy of stimulation and immune system defense in canine mammary carcinoma. The expression of these markers using immunohistochemistry was made by streptoavidin-biotin peroxidase method. The tissues were collected from twenty mammary carcinomas, subdivided in ten simple carcinomas and ten complex carcinomas of twenty female dogs. This study identified no significant differences on quantification of positive cellular staining in simple carcinoma compared with complex carcinoma. The samples of simple carcinoma resulted significant differences on positive staining between the antibodies CD28 (p=0,03) and Foxp3 (p=0,01) and these markers revealed less quantitative staining compared with the others markers (CD4, CD8, CD152 and NK). The samples of complex carcinoma resulted significant differences on cellular staining quantification by the antibody CD152 (p<0,05) compared with the others markers (CD4, Foxp3, CD28, and NK) with exception when compared with CD8. The immune quantification of positive cellular staining about the markers CD28, CD56 and Foxp3 were smaller than CD8 (p=0,01), and the Foxp3 was smallest than those others markers. It was concluded that exist a negative control of immune system to complex and simple mammary carcinoma, but, this negative control was more significant in female dogs with complex mammary carcinoma / Mestre

Ultrawideband (UWB) technology has received considerable attention in recent years as it is regarded to be able to revolutionise a wide range of applications. UWB imaging for breast cancer detection is particularly promising due to its appealing capabilities and advantages over existing techniques, which can serve as an early-stage screening tool, thereby saving millions of lives. Although a lot of progress has been made, several challenges still need to be overcome before it can be applied in practice. These challenges include accurate signal propagation modelling and breast phantom construction, artefact resistant imaging algorithms in realistic breast models, and low-complexity implementations. Under this context, novel solutions are proposed in this thesis to address these key bottlenecks. The thesis first proposes a versatile electromagnetic computational engine (VECE) for simulating the interaction between UWB signals and breast tissues. VECE provides the first implementation of its kind combining auxiliary differential equations (ADE) and convolutional perfectly matched layer (CPML) for describing Debye dispersive medium, and truncating computational domain, respectively. High accuracy and improved computational and memory storage efficiency are offered by VECE, which are validated via extensive analysis and simulations. VECE integrates the state-of-the-art realistic breast phantoms, enabling the modelling of signal propagation and evaluation of imaging algorithms. To mitigate the severe interference of artefacts in UWB breast cancer imaging, a robust and artefact resistant (RAR) algorithm based on neighbourhood pairwise correlation is proposed. RAR is fully investigated and evaluated in a variety of scenarios, and compared with four well-known algorithms. It has been shown to achieve improved tumour detection and robust artefact resistance over its counterparts in most cases, while maintaining high computational efficiency. Simulated tumours in both homogeneous and heterogeneous breast phantoms with mild to moderate densities, combined with an entropy-based artefact removal algorithm, are successfully identified and localised. To further improve the performance of algorithms, diverse and dynamic correlation weighting factors are investigated. Two new algorithms, local coherence exploration (LCE) and dynamic neighbourhood pairwise correlation (DNPC), are presented, which offer improved clutter suppression and image resolution. Moreover, a multiple spatial diversity (MSD) algorithm, which explores and exploits the richness of signals among different transmitter and receiver pairs, is proposed. It is shown to achieve enhanced tumour detection even in severely dense breasts. Finally, two accelerated image reconstruction mechanisms referred to as redundancy elimination (RE) and annulus predication (AP) are proposed. RE removes a huge number of repetitive operations, whereas AP employs a novel annulus prediction to calculate millions of time delays in a highly efficient batch mode. Their efficacy is demonstrated by extensive analysis and simulations. Compared with the non-accelerated method, RE increases the computation speed by two-fold without any performance loss, whereas AP can be 45 times faster with negligible performance degradation.

O câncer de mama permanece como o segundo tipo de câncer mais freqüente no mundo e o primeiro entre as mulheres (INCA, 2007). Porém, os mecanismos envolvidos no processo de gênese dos tumores mamários mesmo sendo intensamente estudados nos últimos 30 anos, ainda não são bem definidos. Vários estudos apontam que a susceptibilidade em função da genética é uma causa relevante ao surgimento do tumor, porém não a principal. Outros fatores tais quais o ambiente e dieta tendem a ser mais significantes nesse processo. Para a indução dos tumores em animais, a maioria dos modelos utiliza carcinógenos pertencentes à família dos hidrocarbonetos aromáticos policíclicos, dentre eles o DMBA (7,12-dimetil bezantraceno). O DMBA foi utilizado neste estudo com o objetivo de induzir tumor mamário, estabelecendo-se assim um modelo para estudos futuros, quantificando e classificando as lesões nas diferentes concentrações do carcinógeno, avaliando também a proliferação celular através do método de imunohistoquímica PCNA nos diferentes tumores encontrados. Neste estudo, em todos os grupos houve o desenvolvimento de tumores mamários, sendo estes mais freqüentes nos grupos de 3, 6 e 9 mg. O tipo de tumor mais freqüente foi o Adenocarcinoma A, seguido de Adenoacantoma e Adenocarcinoma Misto em menor freqüência. Sendo assim, concluiu-se através deste trabalho que o DMBA produz um modelo de carcinogênese mamária em camundongos. / The breast cancer remains the second most common cancer type in the world and first among women (INCA, 2008). However, the mechanisms involved in the origin even being intensively studied in the past 30 years, are still not well defined. Several studies suggest that genetic susceptibility is a relevant issue to the tumor development, however others factors can favor tumor growing. Among such factors the environment and diet are considered more significant. For mice tumor induction, most significant carcinogens used belonging to the polycyclic aromatic hydrocarbons family, where DMBA (7,12-dimethyl bezantraceno) take place. The DMBA was used in this study in order to induce mammary tumors, establishing the real conditions for future studies in our mice colony, classifying and quantifying the lesions. Cell proliferation was also evaluated though the immunohistochemistry against PCNA in different tumors classified. In this study, all concentration resulted in breast tumor development, which was more frequently observed in groups of 3, 6 and 9 mg. The most common type tumor regarded was Adenocarcinoma A, followed by Adenoacantoma and Mixed A/B in lower frequency. In conclusion, DMBA was able to produces a model of mammary carcinogenesis in mice.

Breast cancer arises from a series of acquired mutations that disrupt normal mammary epithelial homeostasis and create multi-potent cancer stem cells that can differentiate into clinically distinct breast cancer subtypes. Despite improved therapies and advances in early detection, breast cancer remains the leading diagnosed cancer in women. A predominant mechanism initiating invasion and migration for a variety of cancers including breast, is epithelial-to-mesenchymal transition (EMT). EMT— a trans-differentiation process through which mammary epithelial cells acquire a more aggressive mesenchymal phenotype—is a regulated process during early mammary gland development and involves many transcription factors involved in cell lineage commitment, proliferation, and growth. Despite accumulating evidence for a broad understanding of EMT regulation, the mechanism(s) by which mammary epithelial cells maintain their phenotype is unknown. Mitotic gene bookmarking, i.e., transcription factor binding to target genes during mitosis for post mitotic regulation, is a key epigenetic mechanism to convey regulatory information for cell proliferation, growth, and identity through successive cell divisions. Many phenotypic transcription factors, including the hematopoietic Runt Related Transcription Factor 1 (RUNX1/AML1), bookmark target genes during mitosis. Despite growing evidence, a role for mitotic gene bookmarking in maintaining mammary epithelial phenotype has not been investigated. RUNX1 has been recently identified to play key roles in breast cancer development and progression. Importantly, RUNX1 stabilizes the normal breast epithelial phenotype and prevents EMT through repression of EMT-initiating pathways. Findings reported in this thesis demonstrate that RUNX1 mitotically bookmarks both RNA Pol I and II transcribed genes involved in proliferation, growth, and mammary epithelial phenotype maintenance. Inhibition of RUNX1 DNA binding by a specific small molecule inhibitor led to phenotypic changes, apoptosis, differences in global protein synthesis, and differential expression of ribosomal RNA as well as protein coding genes and long non-coding RNA genes involved in cellular phenotype. Together these findings reveal a novel epigenetic regulatory role of RUNX1 in normal-like breast epithelial cells and strongly suggest that mitotic bookmarking of target genes by RUNX1 is required to maintain breast epithelial phenotype. Disruption of RUNX1 bookmarking results in initiation of epithelial to mesenchymal transition, an essential first step in the onset of breast cancer.

The pituitary hormone prolactin (Prl) is essential for alveolar morphogenesis and plays a role in breast carcinogenesis, however the mechanism that underlies these actions remains to be defined. Alterations in serum Prl provide the primary endocrine signal regulating developmental events in the mammary gland in sexually mature mammals. Prl production and post-translational phosphorylation by the pituitary is regulated by the neuropeptide Galanin (Gal) in response to hypothalamic signals integrating neuronal and endocrine inputs. Prl exerts its effects on the mammary epithelium in two ways, indirectly by modulation of the systemic hormonal environment, for example the release of progesterone from the corpus luteum, and directly by binding to Prl receptors (Prlr) within the mammary epithelium. Prl binding to Prlr initiates signalling predominantly via activation of the Jak2/Stat5 pathway, leading to altered patterns of gene transcription. One of these target genes is the ets transcription factor Elf5, which is required by the epithelium for alveolar morphogenesis. This thesis aims to further our understanding of the mechanisms by which prolactin exerts its influence on the mammary gland during alveolar morphogenesis and carcinogenesis. Transcript profiling revealed a lactation signature of 35 genes in Prlr+/- mice, Gal-/- mice and mice treated with a Prl mutant (S179D) that mimics phosphorylated Prl. We discovered that the majority of changes in gene expression were produced by prolactin rather than by Gal. The action of Gal was predominantly via modulation of Prl phosphorylation and release, as its effects were very similar to that of S179D. Knockout of Elf5 phenocopied knockout of Prlr, resulting in failure of alveolar morphogenesis and reduced expression of milk and lipid synthesis genes. Forced Elf5 expression at puberty resulted in aberrant differentiation of the terminal end buds and milk protein synthesis during ductal morphogenesis. Re-expression of Elf5 in Prlr-/- mammary epithelial cells completely rescued alveolar morphogenesis. These observations indicate that Elf5 is a master regulator of alveolar morphogenesis downstream of the Prlr. Loss of mammary epithelial Prlr resulted in reduced proliferation of low-grade neoplastic lesions resulting in increased tumour latency in the C3(1)/SV40T model of mammary carcinogenesis. There was no change in the growth rate, proliferation nor the morphology of tumours in Prlr-/-/C3(1)/SV40T transplants, thus Prl acts early in carcinogenesis to drive the proliferation of pre-invasive lesions resulting in faster progression to cancer.

Bovine mastitis is one of the most costly dairy-based diseases worldwide. Streptococcus uberis is a prevalent causative organism of mastitis and resides naturally in the environment of the dairy cow making prevention of the disease difficult. New strategies need to be developed to control this pathogen. However, a fundamental understanding of the complex relationships that exist between the cow, the pathogen and the environment are required in order to advance the development of prevention strategies. Microarray technology was used to evaluate the complex transcriptional changes which occur in the bovine mammary gland following the onset of clinical S. uberis mastitis. A 22,000 bovine cDNA microarray indicated that S. uberis mastitis led to the up-regulation of 1,283 genes and the down-regulation of 1,237 genes by greater than 1.5 fold. Gene ontology analysis demonstrated that S. uberis mastitis was typically associated with the up-regulation of genes that are involved in the immune response and homeostasis and a down-regulation of genes involved in lipid metabolism. Quantitative real-time analyses for a selection of genes associated with the immune response validated the microarray data. Mammary epithelial cell cultures did not show an increase in the expression of any of these immune factors in response to the same S. uberis strain used to induce clinical mastitis. This indicates that the expression of immune-related genes by mammary epithelial cells may be initiated by host factors and not S. uberis. The application of bacteriocins, proteinaceous antimicrobials produced by bacteria which typically inhibit the same or closely-related species to that of the producer organism, has been suggested as one possible approach in the control of mastitis. S. uberis have been previously found to commonly produce bacteriocin-like inhibitory substances (BLIS). The BLIS activities of a set of fifteen S. uberis and S. bovis strains were assessed. The results confirmed the prolific and varied nature of BLIS production by S. uberis and S. bovis and also indicated that these strains may commonly produce more than one inhibitory agent. This survey of BLIS production led to the detection and characterisation of a novel circular bacteriocin, uberolysin, produced by S. uberis strains 233 and 42. The structural gene of uberolysin was subsequently identified in nine (64%) of the fifteen test strains. Multiplex PCR analysis showed that 93% of 158 New Zealand S. uberis isolates contained the structural genes of at least one of the four known S. uberis bacteriocins (uberolysin, nisin U, ubericin A and ubericin 63). However, no apparent direct association was identified between any one of these bacteriocin-related loci and apparent ability to cause mastitis on New Zealand dairy farms. The uberolysin structural gene was detected in 91% of the isolates and this widespread distribution prompted the advancement and evaluation of a potential role for uberolysin in immunomodulation within the bovine mammary gland. Two different preparations of uberolysin were found to have different stimulatory effects on monocytes, neutrophils and epithelial cells. The less highly purified preparation appeared to diminish the production of TNF-α by monocytes in the presence of a bacterial stimulus and to decrease neutrophil phagocytosis. By contrast, the relatively more highly purified preparation of uberolysin itself induced a significant immune response by monocytes. Consistent with this, the purer preparation of uberolysin induced an increase in C3, IL-1β, IL-6, IL-8, the β-defensin LAP, the acute-phase protein MSAA, the calcium-binding protein S100A12 and TLR2 by quantitative real-time analysis. Although currently only two S. uberis bacteriocins (uberolysin and nisin U) have been fully characterised, the present study has shown that this species may be an important source of novel antimicrobials. Furthermore, bacteriocin production by S. uberis may have an immunomodulation role within the mammary gland. A better understanding of the complex immune response initiated at the onset of clinical S. uberis mastitis and of the role that bacteriocins have in S. uberis pathogenesis may lead to development of improved strategies to combat this disease.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 89-103).

The role that each of the Notch receptors play in controlling alveolar development and cell fate determination in the mouse mammary gland has remained unclear. By utilizing a cre-conditional constitutively active intracellular Notch1 knock-in I define, in vivo, that ectopic Notch1 activation is sufficient to inhibit ductal outgrowth, cause the formation of alveolar-like cell accumulations, and promote Elf5+/ER- cell fate, at the expense of ER+ cell fate, in the mammary gland of pubescent mice. Furthermore, ectopic Notch1 in the pregnant mammary gland is sufficient to promote the formation of pregnancy/lactation-dependent lactating adenomas. These lactating adenomas consist of differentiated secretory cells and normally regress during involution but progress into non-regressing tumours after multiple pregnancies. These lactating adenomas exhibit decapitation secretions characteristic of apocrine differentiation. Together these results suggest that Notch1 may function to promote Elf5+/ER- cell fate and may be misregulated in pregnancy-associated masses and apocrine-carcinoma of the breast in humans.

The role that each of the Notch receptors play in controlling alveolar development and cell fate determination in the mouse mammary gland has remained unclear. By utilizing a cre-conditional constitutively active intracellular Notch1 knock-in I define, in vivo, that ectopic Notch1 activation is sufficient to inhibit ductal outgrowth, cause the formation of alveolar-like cell accumulations, and promote Elf5+/ER- cell fate, at the expense of ER+ cell fate, in the mammary gland of pubescent mice. Furthermore, ectopic Notch1 in the pregnant mammary gland is sufficient to promote the formation of pregnancy/lactation-dependent lactating adenomas. These lactating adenomas consist of differentiated secretory cells and normally regress during involution but progress into non-regressing tumours after multiple pregnancies. These lactating adenomas exhibit decapitation secretions characteristic of apocrine differentiation. Together these results suggest that Notch1 may function to promote Elf5+/ER- cell fate and may be misregulated in pregnancy-associated masses and apocrine-carcinoma of the breast in humans.