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Content removed due to copyright restriction: Appendix 6 Cooper, C. V., Stelwagen, K., Singh, K., Farr, V. C., Prosser, C. G., and Davis, S. R. (2004): Expression of the tight junction protein zonula occludens-1 during mammary engorgement. Proceedings of the New Zealand Society of Animal Production 64,43-47. Singh, K., Dobson, J., Phyn, C. V. C., Davis, S. R., Farr, V. C., Molenaar, A. J., and Stelwagen, K. (2005): Milk accumulation decreases expression of genes involved in cell-extracellular matrix communication and is associated with induction of apoptosis in the bovine mammary gland. Livestock Production Science 98,67-78. Appendix 7 McMahon, C.D., Farr, V.C., Singh, K., Wheeler, T.T. and Davis, S.R. (2004). Decreased expression of ß1-integrin and focal adhesion kinase in epithelial cells may initiate involution of mammary glands. Journal of Cellular Physiology 200, 318-325 / Extended periods of milk accumulation result in loss of secretory activity, increased apoptosis and eventually, involution of mammary glands. This process is associated with increased permeability of the tight junction (TJ) complexes between adjacent mammary epithelial cells (MECs). The change in cell shape during mammary engorgement from a cuboidal to a flattened morphology may initiate changes in protein and gene expression (mechanotransduction) that trigger these processes. Therefore, this study examined the regulation of the major TJ protein components during mammary engorgement, and in particular the role of physical distension of the mammary epithelium in the regulatory process. Expression of the integral transmembrane TJ proteins, occludin and claudin-1, and the cytoplasmic TJ protein, ZO-1, were down-regulated in both bovine and rat mammary glands during the early stages of mammary apoptosis and involution following the abrupt cessation of milk removal. In the rat, these responses were locally regulated as they occurred only in teat-sealed glands in a hemi-suckled model. Furthermore, the down-regulation of TJ proteins is consistent with a loss of TJ integrity during mammary engorgement. Induced physical distension of rat mammary glands in vivo transiently up-regulated the expression levels of occludin protein and mRNA, and ZO-1 mRNA, followed by an accelerated decrease in expression compared with the effects of milk accumulation alone. This was associated with the initiation of apoptosis, the up-regulation of the pro-apoptotic factor pSTAT3, and the down-regulation of the cell-ECM survival factor βl-integrin. An in vitro model was also developed to stretch MECs, mimicking the flattening in cell shape during mammary engorgement in vivo. While stretching MECs in vitro did not conclusively alter TJ protein expression, the overall results of this project support further investigation into the role of the TJ complex in mechanotransduction pathways. In addition, the results point to crosstalk between cell-ECM survival signalling and STAT3 death signalling as a candidate for regulation by physical distension of the mammary epithelium. In conclusion, this study supports the hypothesis that physical distension during engorgement of the mammary glands with milk is a primary trigger initiating apoptosis of MECs through changes in the regulation of gene pathways controlling cell survival and death, and the disruption of TJ function.

<p>Sea urchins are fascinating marine creatures belonging to the phylum Echinodermata that serve as an essential ecological component and hold promise as a prospective source of therapeutics. However, sudden environmental changes, such as global warming and marine pollution, are placing significant stress on these organisms. To maintain natural resources and exploit sea urchins commercially, researchers are investigating aquaculture as a solution.</p> <p>This investigation discloses the physiological and immunological effects of physical and chemical stressors on one of the most common edible species of sea urchin, <em>Arbacia punctulata</em>. The study employed an elevated temperature as a physical stressor (1°C/day), lipopolysaccharides (LPS) inoculation as a chemical stressor (4µg/ml/day), and a combination of both LPS and elevated temperature as combined stressors. The results demonstrated a significant alteration in the total and differential coelomocyte count in the LPS-stressed group (p<0.05) and combined stressed group (p<0.05) followed by abnormal behavioral activity compared to those of control. Additionally, exposure to acute LPS exposure (at day 1 and day 3) and combined stressors led to an increase in phagocytic capacity (p<0.05) and lysozyme activity (p<0.05). Chronic exposure to LPS and combined stressors resulted in a decrease in gonadosomatic index (p<0.05, at day 10) and lysozyme activity (at day 7). A significant increase in coelomic fluid (CF) protein (p<0.05)was observed in the temperature-stressed group on days 5 and 10, while the combined stressed group had significantly more CF protein on days 1, 5, 7, and 10. An upregulation of Nf-kB gene expression was also observed (p>0.05) in temperature stressed group.  </p> <p>The study also revealed that sea urchins contain bioactive compounds that protect against external and internal injury, cell death, and body wall extract of sea urchin exhibited high antioxidant activity(p<0.05). Furthermore, it confirmed the antibacterial activity (p<0.05) of sea urchin (<em>Arbacia punctulata </em>and<em> Lytechinus variegatus</em>) body wall and coelomic fluid (cell-free plasma) extracts against ten pathogenic bacteria. The ethyl acetate body wall extract of both sea urchin species demonstrated higher inhibitory activity against the pathogenic bacteria tested. Overall sea urchin has potentials to meet the demand of food and medicine. </p>

<p> Down syndrome (DS) is a complex genetic disorder caused by the triplication of human chromosome 21 (Hsa21). The presence of an extra copy of an entire chromosome greatly disrupts the copy number and expression of over 350 protein coding genes. This gene dosage imbalance has far-reaching effects on normal development and aging, leading to cognitive and skeletal defects that emerge earlier in life than the general population.</p> <p> The present study begins by characterizing skeletal development in young male Ts65Dn mice to test the hypothesis that skeletal defects in male Ts65Dn mice are developmental in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical defects were present generally throughout development, but trabecular defects emerged at P30 and persisted until P42. </p> <p> The gene <i>Dual-specificity tyrosine-regulated kinase 1a </i>(<i>Dyrk1a</i>) is triplicated in both DS and in Ts65Dn mice and has been implicated as a putative cause of both cognitive and skeletal defects. To test the hypothesis that trisomic <i>Dyrk1a</i> is related to the emergence of trabecular defects at P30, expression of <i>Dyrk1a</i> in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown to fluctuate throughout development and overexpression generally aligned with the emergence of trabecular defects at P30.</p> <p> The growth rate in trabecular measures between male Ts65Dn and euploid littermates was similar between P30 and P42, suggesting a closer look into cellular mechanisms at P42. Assessment of proliferation of BMSCs, differentiation and activity of osteoblasts showed no significant differences between Ts65Dn and euploid cellular activity, suggesting that the cellular microenvironment has a greater influence on cellular activity than genetic background.</p> These data led to the hypothesis that reduction of <i>Dyrk1a</i> gene expression and pharmacological inhibition of DYRK1A could be executed during a critical period to prevent the emergence of trabecular defects at P30. To tests this hypothesis, doxycycline-induced cre-lox recombination to reduce <i>Dyrk1a</i> gene copy number or the DYRK1A inhibitor CX-4945 began at P21. The results of both genetic and pharmacological interventions suggest that trisomic <i>Dyrk1a</i> does not influence the emergence of trabecular defects up to P30. Instead, data suggest that the critical window for the rescue of trabecular defects lies between P30 and P42.

<p>Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear.</p> <p><br></p> <p>Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative <em>in vitro</em> model is critical to elucidate the signaling pathways orchestrating CSF production in the CP.</p> <p><br></p> <p>This research aims to characterize an <em>in vitro</em> culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.</p>