Osteoarthritis is arguably the most common ailment in both dogs and people in the developed World. Treatment for osteoarthritis is currently symptomatic. Development of therapies designed at stopping the progression of osteoarthritis, require a method of evaluating efficacy. Objective analysis by measuring joint metabolism via clinical trials of the cross-over design is currently not possible with the methods utilized. Extracellular matrix degradation is a hallmark of osteoarthritis. The matrix metalloproteinases are major degradative enzymes. There are currently no commercially available assays to measure canine matrix metalloproteinases. Human and canine matrix metalloproteinases are highly homologous. The use of antibodies and assays designed to detect human matrix metalloproteinases may also detect canine molecules. A commercially available human enzyme-linked immunosorbent assay (ELISA) was tested against canine samples without success. Casein zymography detected the presence of canine enzymes weighing the same as human matrix metalloproteinase 3 (MMP-3) and matrix metalloproteinase 13 (MMP-13). Inhibition of the casein degradation was achieved by the addition of ethylenediamine tetra-acetic acid (EDTA). The presence of a protein of the correct molecular weight with the ability to digest casein that is inhibited by EDTA is only circumstantial and does not definitively identify the enzymes. Enzyme-assisted immunoelectroblotting (Western blotting) utilizing human polyclonal antibodies was unsuccessful at positively identifying the canine molecule.

Adult adipose derived mesenchymal stromal cells (ASCs) have been characterized in various species. Many factors may affect ASC fate and ASCs from different species may have different response to these factors. The first study was to identify the differences of the canine ASCs isolated from subcutaneous and infrapatellar adipose tissues, and evaluate the impact of cryopreservation on the cells. Based on paired comparisons of fresh and cryopreserved ASCs, cryopreserved ASCs had lower CD29 and CD44 protein expression and lower proliferation rates. The cryopreserved ASCs had relative lower mitochondria in the cytoplasm compared to the fresh ASCs regardless of tissue sources. The second study was to apply human ASCs for bone regeneration. The spinner flask bioreactor system was employed to load human ASCs onto three commercial scaffolds and the cell-scaffold constructs were cultured in stromal, osteogenic, or osteogenic for 48 hours followed by stromal medium for up to 28 days. The distinct scaffold upregulated different osteogenic signaling pathways, suggesting distinct osteogenic cell signaling pathways were selectively upregulated by scaffold composition. The third study was designed to quantify in vivo equine multipotent stromal cell (MSC) osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Addition of mineral to polymer scaffolds enhanced equine MSC osteogenesis over polymer alone, and contributions by both exo- and endogenous MSCs were confirmed. The fourth study was designed to evaluate the effects of collagenase digestion and cryopreservation on equine ASCs. Higher collagenase concentration yielded more nucleated cells, and the percentages of MHCII-, CD44+, CD105+ cells in freshly isolated and cryopreserved cells were similar. The embryonic gene expression was enhanced and the essential gene expression decreased after cryopreservation. The fifth study was to demonstrate the endodermal transdifferentiation capability in feline ASCs. xvi Feline-specific pancreatic β cell induction medium was developed in the study, and islet-like cell clusters that secrete insulin in response to glucose stimulation were created. Overall, the investigations in this dissertation provide critical information for canine, feline, equine and human MSC-based tissue engineering therapies and may contribute to better efficiency and efficacy of cell

Hypertension and type two diabetes mellitus (Type 2 DM) are serious chronic illnesses that impact on the lives of millions of people around the world. Various epidemiological studies have shown a relationship between early life events such as intrauterine growth retardation (IUGR) resulting in low birth weight and the development of these chronic illnesses in adult life. To explain the link between these two events, it has been suggested that an ‘insult’ at a critical time point of development can ‘program’ alterations in gene expression, organ size, and cell number. This has been termed “the early life origins of disease’. There is also evidence that these programmed effects are not limited to the first generation but can also be passed to subsequent generations. With changes in lifestyle in modern society, the prevalence of obesity is increasing, in association with problems such as type 2 DM, hypertension, fatty liver, atherosclerosis and the metabolic syndrome. Obesity during pregnancy is linked to problems such as gestational diabetes, hypertension and early miscarriage as well as a higher risk of congenital malformations. Maternal obesity has also been recognised as one of the factors capable of ‘programming’ the offspring, increasing the risk of childhood and adult disorders such as obesity and hypertension. In this thesis I have used two animal models to explore the underlying mechanisms of programming and its intergenerational effects: i) a rat model of prenatal glucocorticoid over-exposure (the dexamethasone-programmed rat) and ii) a mouse model of obesity during pregnancy. Using the dexamethasone-programmed rat, I have shown that prenatal glucocorticoid overexposure reduces fetal and placental weight in the first generation (F1) offspring, in association with alterations in gene expression in placenta and liver. In addition, I have shown effects on fetal and placental weights and gene expression in the second generation (F2) offspring. The observed changes in gene expression in the F2 offspring differ from those in the first generation. Thus, although effects on fetal growth are seen in both generations, the underlying mechanisms appear to be different. We also observed marked parent of origin effects on fetal and placental growth and gene expression in the second generation. In the mouse model of maternal obesity, birth weight was decreased in the F1 offspring. At weaning, the offspring of obese mothers were heavier than controls, however this difference in weight was not persistent. At three months of age, F1 female offspring of obese mothers showed altered expression of hepatic genes important in lipid regulation and metabolism. More striking changes were seen in the F2 generation in which there was an effect of paternal exposure to maternal obesity to decrease birth weight. There were also parent of origin effects on organ weights and insulin levels at six months of age. These results provide evidence for the transmission of programming effects to a second generation in two different programming models and suggest that the mechanisms leading to these effects differ between generations.

Cell-to-cell variation in expression of pluripotency- and lineage-determining factors has been proposed to be integral to the process of cell fate commitment in pluripotent cells both in vitro and in vivo. Understanding the sources of this heterogeneity in pluripotent stem cells promises greater insight into the mechanisms underlying cell fate choice. I identify mitochondrial membrane potential as an axis of heterogeneity in mouse embryonic stem cell populations, and show that high mitochondrial membrane potential marks cells that are in a stable self-renewing state. Partial overlap with previously described metastable subpopulations is demonstrated through gene expression analysis. I present evidence that similarly to previous findings in HeLa, heterogeneity in mitochondrial membrane potential is associated with variation in global transcription rate in mESCs. The direct impact of global transcription rate on differentiation propensity is demonstrated through manipulation of RNA Pol II transcription elongation rate. Mitochondrial variability is therefore likely a functionally relevant source of extrinsic gene expression variability in mouse embryonic stem cells.

Battlefield retinal laser injury is an infrequent but potentially devastating cause of irreversible blindness. Resultant laser-induced photoreceptor death may occur by necrosis or apoptosis, the latter which is a form of programmed cell death that may be physiological or pathological. Though necrosis cannot be prevented, apoptosis may be inhibited under certain conditions. Therefore, following retinal laser injury, specific treatment aims to target apoptotic photoreceptors and may take the form of neuroprotection or cell replacement. The primary aim of this thesis was to construct an in vivo model in which to observe the effects of retinal laser exposure on cone photoreceptor apoptosis. Current methodology to determine the effects involves histological techniques and is therefore limited to being cross-sectional. An in vivo model would permit longitudinal study to observe the cone response to injury using clinically relevant applications, including fundus autofluorescence imaging. Such a construct would enable more sensitive evaluation of new therapies which would be of direct translational relevance. The secondary aim was to investigate potential therapeutic options for retinal laser injury by pharmacological means in the form of CNTF or cell transplantation. To identify the possible molecular signals involved in neurotrophic factor-induced photoreceptor cell survival, apoptotic gene expression was investigated focusing on those genes modulated by the CNTF pathway.

NKX3.1, a prostate specific homeobox gene is a known marker of prostate epithelium during embryogenesis and is also expressed subsequently through different stages of prostate differentiation. However, all studies on NKX3.1 are focused on its regulation by androgen receptor (AR). The aim of this project is to establish the role of NKX3.1 in differentiation in prostate cancer, independent of AR regulation. In this thesis, I characterize the cell lines in terms of their differentiation capabilities in 3D, expression levels of NKX3.1 and the mismatch repair status. The genes potentially involved in differentiation and regulators of NKX3.1 are also identified using microarray data of the cell lines (<b>Chapter 3</b>). Although NKX3.1 plays a key role in prostate development no studies have been conducted on the effect of NKX3.1 expression on differentiation capabilities of prostate cell lines. In <b>Chapter 4</b>, this was investigated by siRNA mediated knockdown of NKX3.1 in 22Rv1 cell line and overexpression of NKX3.1 in PC3 (designated PC3-Nkx3.1) and PNT1a cells followed by growth in 3D. These functional studies show that the expression of NKX3.1 is vital for lumen formation in 3D, which is used as a measure of differentiation. The microarray data and overexpression of NKX3.1 studies suggest that this gene may also be involved in inhibiting epithelial to mesenchymal transition (EMT). Homeobox B13 (HOXB13) was identified as one of the downstream targets of NKX3.1. NKX3.1 and HOXB13 expression levels are positively correlated not only in the panel of prostate cell lines but also in the NKX3.1 overexpression and knockdown studies (<b>Chapter 5</b>). The results of the work presented in this thesis demonstrate that there is a striking parallel between the function of NKX3.1 in prostate and Caudal-type homeobox 1 (CDX1) in the colon and rectum. In conclusion, NKX3.1 plays a key role as a tumour suppressor in prostate cancer by controlling differentiation of prostate cancer cells.

This poststructuralist qualitative study explored the discourses constructing women’s choice for a caesarean section in the absence of clinical indications, in the talk and texts of women, midwives, an obstetrician, professional journals and the media publications. The study affirms inscriptions surrounding choice in childbirth are shaped discursively through a multiplicity of discourses underpinned by social and institutional practices. With advances in technology, childbearing women have a greater variety of options from which to choose. Controversial, is the option of a caesarean section, regardless of clinical need. The issue is depicted in both professional and popular discourse as contentious, complex and contradictory. Its momentum into the 21st century, as a new object of obstetric discourse, has been played out on a number of platforms. In this thesis I draw from the theoretical ideas of French philosopher Michel Foucault, to examine this complex debate. I argue there is a volatile moment in the history of childbirth in which an explosion of discourses have sculptured choice for a caesarean, in the absence of clinical indications, out of a repartee of autonomy, convenience, desire, fear and risk. In this precarious moment, new meanings joust with the old on a shifting terrain awash with rhetoric that co-opts, competes, and contradicts to bring about a caché of mutable ‘truths’. Whether caesarean, as an optional extra, can be explained in terms of a libertarian imperative, an embodiment of lifestyle, the satiation of desire, the attenuation of fear or the avoidance of risk, the democratisation of this choice has exposed a pathologising paradox, whereupon the normal emerges as the abnormal, and the abnormal emerges as the normal. The deconstruction of choice through a poststructuralist lens has enabled insight into how contradiction and contest befall the ‘order of things ’ and in so doing, provides new openings for contemplating the discursive positioning of women through the competing discourses of childbirth.

The hypothesis underlying this research is that hand activity produces changes in the tissues of the hand which are reflected in the various functions of those tissues. Understanding the effect of hand activity upon hand function would allow occupational therapists to assess the efficacy of therapeutic interventions upon a clients ability to perform hand activity without damage to the tissues of the hand. Such information could assist in the design of safe and sustainable work tasks. The first step towards understanding how activity affects the hand is to measure its effects. The aim of this research is to determine which instruments can measure the effects of activity upon the hand.

Testicular function is stimulated from the pituitary gland by the gonadotrophic hormones FSH and LH. The testis in turn regulates gonadotrophin secretion by negative feedback. The agents of feedback are steroids, and in addition, probably a protein hormone, which has been given the name inhibin. A method capable of detecting inhibin-like activity in bull seminal plasma (bSP) was developed. Administration of bSP at the time of castration to five-week old male rats inhibited the post-castration rise of serum concentrations of FSH and LH otherwise seen 24 h later. The degree of inhibition depended on the dose; 0.5 ml bSP or the equivalent amount of bSP-extract always suppressed FSH and LH to levels typical of intact rats. The rats' sensitivity to 0.2 ml bSP varied with the time of year, possibly reflecting seasonal changes in the onset of puberty. The time-course of action of bSP-extract was studied in intact rats. Serum FSH was suppressed 12, 24 and 48 h after a single injection, but not at 3 or 6 h. LH was suppressed at 6, 12, 24 and possibly 48 h.

The hypothesis underlying this research is that hand activity produces changes in the tissues of the hand which are reflected in the various functions of those tissues. Understanding the effect of hand activity upon hand function would allow occupational therapists to assess the efficacy of therapeutic interventions upon a clients ability to perform hand activity without damage to the tissues of the hand. Such information could assist in the design of safe and sustainable work tasks. The first step towards understanding how activity affects the hand is to measure its effects. The aim of this research is to determine which instruments can measure the effects of activity upon the hand.