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    Effect of mechanical stress o the integrity, signalling mechanisms and function of bovine mammary epithelial cells : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Manawatū, Palmerston North, New Zealand
    (Massey University, 2014) Biet, Juliane
    Mammary gland engorgement due to milk accumulation in late lactation leads to changes in cell morphology and has been recognised as a potential key initiator of involution and remodelling of the mammary gland. The physical distension of mammary epithelial cells (MEC), due to udder filling, is likely to result in mechanical tension on cell-cell and cell-matrix interactions. Cell-cell and cellmatrix junctions provide tissue integrity, promote cell polarity, guarantee sufficient communication between cells to ensure synchronised milk secretion and support cell survival. Their disruption may be one of the early initiators of the mammary gland remodelling process. As a consequence, the primary goal of this study was to determine the potential effects of MEC stretch on changes in cell sensing within the mechanical micro-environment in the initiation of bovine MEC involution. During this investigation, particular emphasis was put on three potential mechanosensors: tight junctions (TJ), focal adhesions (FA) and primary cilia (PC), and their regulation in the early stages of involution using in vivo and in vitro experimental approaches. Static, biaxial in vitro cell stretch and acute physical distension in vivo resulted in changes in TJ protein expression levels implying a potential disruption of cell-cell communication as well as communication with the cell‟s cytoskeleton. Furthermore, down-regulation of Akt and pAkt following different periods of mechanical strain applied in vitro and decreased levels of pAkt following acute physical distension in vivo indicated a disruption of β1-integrin-FAK survival signalling through the PI3K-Akt pathway downstream of FA interactions. Increased numbers of ciliated MEC following extended periods of non-milking indicated a dedifferentiation of MEC. Furthermore, increased levels of STAT6 transcription (part of PC signalling following mechanical stimulation) factor indicates the initiation of macrophage accumulation and promotion of tissue remodelling of the bovine mammary gland. In conclusion, this study supports the hypothesis that local factors play an important role during bovine mammary gland involution and that mechanical stimulation may play a part in the initiation of this process.
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    Interactions between commensal obligate anaerobes and human intestinal cells : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Manawatu, New Zealand
    (Massey University, 2013) Ulluwishewa, Dulantha
    The human intestinal epithelium is formed by a single layer of epithelial cells which regulates intestinal barrier permeability. Increased permeability can result in the entry of potentially harmful compounds into the body, and is implicated in autoimmune, inflammatory and atopic diseases. The intestinal tract is inhabited by an estimated 1014 microbes and it is increasingly evident that they affect intestinal barrier function. However, over 90% of commensal intestinal bacteria are obligate anaerobes, making it difficult to co-culture them with oxygen-requiring mammalian cells in vitro. To investigate the interactions between obligate anaerobes and epithelial cells that regulate the intestinal barrier, an apical anaerobic model of the human intestinal epithelium, which utilises a dual-environment co-culture chamber, was developed and validated. The chamber allowed for polarised monolayers of the intestinal cell line Caco-2 to be grown such that the apical (luminal) side was exposed to an anaerobic environment, while maintaining an aerobic basal side. The cell viability and barrier function of Caco-2 monolayers was unaffected by culture in the apical anaerobic model for at least 12 hours. Global gene expression analysis predicted upregulation of cell survival and proliferation in Caco-2 cells cultured in the apical anaerobic model, compared to Caco-2 cells grown under conventional conditions, suggesting an adaptation of the Caco-2 cells to a lower supply of oxygen. The apical anaerobic model was used to co-culture the commensal obligate anaerobe Faecalibacterium prausnitzii with Caco-2 cells. The survival of F. prausnitzii was improved in the anaerobic apical environment compared to when cultured in an aerobic atmosphere. Live F. prausnitzii, but not non-viable (UV-killed) F. prausnitzii, were shown to increase permeability across Caco-2 monolayers. Furthermore, global gene expression analysis suggested that live F. prausnitzii cells have more profound effects on Caco-2 cells than non-viable F. prausnitzii, illustrating the importance of maintaining viability of obligate anaerobes in an in vitro co-culture system. The apical anaerobic model can be used to gain insights into the mechanisms of crosstalk between commensal obligate anaerobic bacteria and intestinal cells, and new knowledge generated using this model will assist in the development of strategies to improve intestinal barrier function.
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    The role of the mammary fat pad during mammogenesis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, New Zealand
    (Massey University, 1996) Hovey, Russell Charles; Hovey, Russell Charles
    Development of the female mammary gland involves the proliferation and morphogenesis of epithelial cells within a matrix of adipose and connective tissue which constitutes the mammary fat pad. The objective of this research was to investigate the mechanisms by which this stromal environment locally regulates postnatal mammogenesis. Initial experiments showed that the mouse mammary fat pad liberates a diffusible activity in vitro which stimulates the growth of mouse mammary epithelial cells and enhances their proliferative response to insulin-like growth factor-I, epidermal growth factor and insulin. This effect was specific to these mitogens, and of a variety of cell lines tested was most pronounced for mouse mammary epithelial cells. Subsequent investigations indicated that these responses were likely induced by unsaturated fatty acids, particularly linoleic acid, from mammary adipocytes. Such responses may be effected by increased intracellular signalling via the actions of protein kinase C. The mitogenic capacity of the mouse mammary fat pad was also evaluated across several physiological states. Mammary fat pad-stimulated proliferation during the estrus cycle was increased at estrus concomitant with a phase of ductal elongation in vivo. In certain medium treatments there was evidence for epithelial upregulation of the mitogenic effect of the mammary fat pad, where intact mammary tissue was more stimulatory than mammary fat pad cleared of endogenous epithelium. Further experiments demonstrated that while the mitogenic effect of the mammary fat pad was unaltered by ovariectomy, ovarian function was required for this effect to be increased by exogenous progesterone. The effect of estrogen was independent of ovarian function but was altered by the local epithelial-stromal interaction, where it increased the mitogenic effect of epithelium-free mammary fat pad and decreased that of intact mammary tissue. Mitogenic stimulation by mammary tissues also declined during virginal development to be least in mature virgin and mid-pregnant states. Stimulation by intact mammary tissue increased during lactation, while that from epithelium-free mammary fat pad remained constant in the presence of steroid hormones and increased in the presence of growth factors. Further experiments investigated the stromal regulation of epithelial growth within the ruminant mammary gland. Differences between the ruminant and rodent mammary fat pad were emphasised in vitro where ovine mammary fat pad stimulated the growth of mouse mammary epithelial cells but did not markedly potentiate their growth factor-responsiveness. A subsequent study examined the expression of stroma-derived growth factors within the ruminant mammary gland during postnatal development, and their regulation by several physiological influences. The level of insulin-like growth factor (IGF)-I mRNA in the ovine mammary fat pad was elevated prior to puberty and during late gestation, while IGF-II mRNA was upregulated in mammary parenchyma of virgin ewes in a transcript-specific manner. Abundance of IGF-I mRNA in mammary tissues of prepubertal ewe lambs tended to be increased by exogenous estrogen whereas IGF-II mRNA levels were reduced. Messenger RNA for keratinocyte growth factor (KGF) was detected within the ovine mammary fat pad throughout development as 2.4 and 1.5 kb mRNA transcripts which were expressed by stromal adipocytes and fibroblasts, respectively. The level of KGF mRNA in mammary tissues of prepubertal lambs was increased by ovariectomy and decreased by estrogen, while KGF mRNA expression in cultures of mammary fibroblasts was suppressed by dexamethasone. Messenger RNA for hepatocyte growth factor, a paracrine mitogen and morphogen for mammary epithelial cells, was expressed in the ovine mammary fat pad and by cultured mammary fibroblasts. The abundance of basic fibroblast growth factor (bFGF) mRNA was highest within the ovine mammary fat pad, while in vitro results suggest bFGF may be a paracrine/autocrine mitogen for multiple cell types within the mammary gland. Basic FGF gene expression in mammary tissues of prepubertal ewes was reduced by estrogen treatment. For each of these growth factors there was evidence suggesting that their expression within the mammary fat pad was upregulated by the adjacent mammary epithelium. In conclusion, these findings indicate that the mammary fat pad may stimulate the proliferation of mammary epithelial cells during postnatal mammogenesis by a variety of influences. Such mechanisms may involve the direct stimulation of epithelial growth or the modulation of epithelial responsiveness to other mitogens. These effects may function to mediate the actions of certain mammogenic hormones. Furthermore, strong evidence indicates that mammary growth may be locally regulated by the interaction between epithelial and stromal cells.