Science Collected Works - Theses
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ItemIdentifying the function of milk protein genes and factors regulating their expression by exploiting the lactation strategy of the Tammar Wallaby (Macropus eugenii)(University of Melbourne, 2010)Milk is a crucial element for the development of the young, as well as mammary gland function. Unlike eutherian species, marsupials have a unique reproduction strategy, consisting of a short gestation, followed by a birth of an altricial young and a relatively long lactation. Additionally, marsupials progressively change the composition of their milk during lactation, exhibiting a phase-specific expression of whey milk proteins. This project exploited the lactation strategy of the tammar wallaby (Macropus eugenii) to analyse the function of whey acidic protein (WAP) in relation to mammary gland development. Furthermore, cross species promoter analysis together with microarray assay was employed to discover factors involved in the regulation of WAP, late lactation protein-A (LLP-A) and late lactation protein-B (LLP-B) gene expression. Structural and expression differences between marsupial and eutherian WAPs have presented challenges to identifying physiological functions of the WAP protein. This study characterized the genomic structure of tammar WAP (tWAP) gene and investigated the potential function of the protein. The in vitro studies demonstrated that tWAP and domain III (Dill) of the protein alone stimulate proliferation of a mouse mammary epithelial cell line (HC11) and primary cultures of tammar mammary epithelial cells (Wall-MEC), whereas deletion of Dill from tWAP abolishes this proliferative effect of the protein. Furthermore, the ability of Wall-MEC cells to form mammospheres was increased in the presence of tWAP. However, tWAP does not stimulate proliferation of human embryonic kidney (HEK293) or human gastric cancer (AGS) cells. DNA synthesis and expression of cyclin D1 and cyclin dependent kinase-4 (CDK-4) genes were significantly up-regulated when Wall-MEC and HC11 cells were grown in the presence of either tWAP or DHL These data suggest that Dill is the functional domain of the tWAP protein and that evolutionary pressure has led to the loss of this domain in eutherians, most likely as a consequence of adopting a reproductive strategy that relies on greater investment in development of the newborn during pregnancy. The temporal expression of marsupial milk protein genes such as WAP, LLP-A and LLP- 13 in marsupials, suggests that these genes are likely to be regulated by different molecular mechanisms from those milk protein genes expressed in eutherians. The in silico characterization of tammar wallaby, stripe-faced dunnart and short-tailed opossum WAP gene promoters identified putative recognition sequences for transcription factors such as STAT5, GR, NF-1, C/EBP, YY1 and SPI, which were conserved between the three marsupial species. Furthermore, WAP promoters demonstrated a prolactin- dependent transcriptional activity in vitro. However, although the analysis of LLP-A and LLP-B gene promoters from tammar wallaby, brush-tailed possum and short-tailed opossum revealed number of recognition sequences for transcription factors mentioned above, no transcriptional activity was observed regardless of the hormone combination used in the assay. This suggests that tammar wallaby WAP, LLP-A and LLP-B genes are regulated by different mechanisms during lactation. This thesis also for the first time describes the cloning, characterization and expression of marsupial specific LLP-A and LLP-B genes from fat-tailed dunnart and short-tailed opossum. Furthermore, microarray-based analysis of changes in gene expression between phase 2B and phase 3 of tammar wallaby lactation detected number of up-regulated genes during later stages of tammar wallaby lactation, which could be playing a role in the regulation of LLP-A and LLP-B gene expression. Overall, this project has highlighted the benefit of studying animals with extreme adaptation to lactation, such as the tammar wallaby, as a model to unravel the function of milk proteins in relation to mammary gland development and identify factors regulating their expression.
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ItemMolecular mechanisms that regulate apoptosis and involution in the mammary gland(University of Melbourne, 2008)This thesis investigates the hypothesis that milk components play a role in regulating mammary gland function during involution, and explores an in vitro model system to examine the response of mammary tissue in the absence of significant milk accumulation. It was determined that a population of mammary epithelial cells have an intrinsic survival mechanism and that a milk protein can regulate cell fate, which is most likely central to the process of involution in the lactation cycle. Previous studies using eutherian species concluded that mammary explants require addition of exogenous macromolecules to remain hormone responsive in culture. In the absence of added hormones and growth factors, the alveoli and small terminal ducts of mouse explants showed degeneration (Elias, 1957). Furthermore, the withdrawal of hormones from bovine mammary gland explants resulted in DNA laddering (Accorsi et al., 2002). Therefore, the addition of growth factors to the culture media appeared to prevent the onset of apoptosis in mammary explants, which perpetuated the dogma that permissive growth factors are required for mammary epithelial cell survival in vitro. The research presented herein challenges this dogma. Investigation of mammary explants in chemically defined media and in the absence of hormones and growth factors demonstrated definitive evidence for mammary epithelial cell survival without any exogenous hormones. Furthermore, this phenomenon is well conserved, and occurs in the tammar wallaby (Macropus eugenii), the cow (Bos taurus) and the mouse (Mus musculus): three evolutionarily distinct species with markedly different lactation strategies. The specific mechanism that permits a population of mammary epithelial cells to survive in the absence of either hormones or growth factors in mammary explants was not confirmed, however two possibilities were investigated. Although milk proteins are synthesised and secreted by mammary explants incubated with lactogenic hormones, milk constituents do not accumulate to elevated levels such as those at weaning (Nagamatsu and Oka, 1980; Nicholas and Tyndale-Biscoe, 1985). Therefore, milk accumulation that provides the trigger for involution upon milk stasis (Green and Streuli, 2004; Quarrie et al., 1996) is absent in this culture model, which may allow a population of mammary epithelial cells to survive. Alternatively, microarray analysis of wallaby and cow mammary explants showed that a number of antioxidant cytoprotective genes involved in stress-induced cellular defence mechanisms, not yet defined in the mammary gland, are upregulated. The induction of these genes is most likely due to the stress of culture on the mammary tissue, which may be responsible for maintaining a population of mammary epithelial cells in explants. Although it was unclear whether the absence of an accumulated milk factor facilitated mammary epithelial cell survival in mammary explants from the wallaby, cow and mouse, a possible trigger of involution due to milk accumulation was investigated. It is well established that chemically induced forms of the milk protein ?-lactalbumin (LALBA) can cause apoptosis of transformed and immature cells, while sparing healthy cells (Hakansson et al., 1995). More recently, it was demonstrated that some commercial preparations of bovine LALBA (bLALBA) had direct apoptotic effects on an intestinal cell line (Xu et al., 2005) and primary fur seal (Arctocephalus pusillus) mammary epithelial cells (Sharp et al., 2007). Similarly, the results presented in this thesis show that bLALBA had a potent apoptotic effect on a number of mammary cell types, and the effect appears to be a function of a 28kD variant. The naturally occurring 28kD form of bLALBA caused apoptosis of primary mammary epithelial cells, immortal HC11, AGS and Hek293 cell lines, and a number of human breast cancer cell lines. The cells became irreversibly apoptotic within 1-3 hours of exposure to the 28kD bLALBA form, and an equal and potent apoptotic effect occurred when cells were exposed to either the naturally occurring 28kD form of bLALBA or bLALBA treated with EDTA to remove bound calcium. Furthermore, incubation of cells with calcium-depleted bLALBA in calcium free media did not alter the apoptotic effects, which suggests the protein is a stable complex. In contrast to the chemically induced LALBA variants, multimeric LALBA (MAL) and human LALBA made lethal to tumour cells (HAMLET), cell death also occurred after fatty acids were sequestered from bLALBA by pre-incubation with BSA. It therefore appears the apoptotic bLALBA form is a naturally occurring stable structure present in bovine whey with strong and irreversible apoptotic potential. Microarray analysis of HC11 cells exposed to bLALBA showed that important genes involved in the apoptosis pathway were upregulated, and genes previously identified in serum starved apoptotic HC11 cells (Seol et al., 2005) were also differentially expressed. Analysis of the abundance of 28kD bLALBA during lactation showed this form was highest in whey from cows during the drying off period, when cells are undergoing apoptosis due to involution of the mammary gland. We propose that the apoptotic effects of 28kD LALBA may be involved in mammary epithelial cell death during involution, a process initiated by the accumulation of milk. Although the trigger of involution is associated with milk accumulation, a myriad of downstream signaling pathways are required to conduct the apoptosis stimuli (Watson, 2006; Green and Streuli, 2004). This study shows that the conditional deletion of STAT6 delays involution in the mouse mammary gland by 72 h, which highlights that apoptosis regulation in vivo is multifactorial and depends on the critical juxtaposition of several signaling pathways. Using microarray analysis it was demonstrated that the expression of important involution genes involved in apoptosis and immune-responses have been altered in STAT6-/- mice. Involution requires immuno-modulatory signals to suppress the potentially damaging effects of cellular inflammatory responses, which may correlate with a Th2 cytokine environment that operates through the actions of STAT6 within the immune system. The precise mechanisms that regulate these actions remains unclear, however, as the specific roles of the immune system in mammary gland involution have not been clearly defined these results emphasize the importance of considering immune response signaling events in any future studies of involution.
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ItemThe function and evolutionary significance of genomic imprinting in the marsupial placenta(University of Melbourne, 2006)