The apoptotic function of p53 is specifically regulated by iASPP and ASPP2 and their additional modulation of p63, a key regulator of squamous epithelial homeostasis, has been demonstrated in mice. In this study the role of iASPP and ASPP2 in human carcinomas was explored with a particular focus on the development of squamous cell carcinomas. The predominant expression of cytoplasmic ASPP2 is seen in the superficial, terminally differentiated cell layers of normal squamous epithelia and its loss is documented in areas of dysplasia and in squamous cell carcinomas. In addition the absence of nuclear ASPP2 in association with human papillomavirus infection suggests it could be a novel viral target. Furthermore in a novel mouse model of lung tumourigenesis, the somatic deletion of ASPP2 is shown to promote the over expression of markers associated with specific lung progenitor cells and to be associated with the development of non small cell lung carcinomas. Conversely nuclear iASPP expression is seen in p63 expressing, replication competent, basal and parabasal squamous epithelial cells and is increased in areas of dysplasia and in squamous cell carcinomas. Moreover the combination of nuclear iASPP, p63 and loss of ASPP2 expression is seen to be associated with cell survival and characterises the invasive edges of lingual squamous cell carcinomas. The novel interaction of iASPP with ERα is also demonstrated together with the negative impact of nuclear iASPP on ER positive breast cancer survival, potentially identifying iASPP as a novel player in estrogen signalling and an important factor in breast tumourigenesis. Together the data presented here provide significant corroborative evidence implicating ASPP2 and iASPP in tumourigenesis. Specifically ASPP2 is shown to promote cellular differentiation and inhibit the expansion of proliferative populations whereas nuclear iASPP promotes the survival of potentially neoplastic clones. It is likely that the balance of these proteins is a key factor in determining individual cell fate decisions.

Semen cryopreservation has allowed the establishment of genome banks and the large scale propagation of species. The development of simple techniques to cryopreserve semen or alternatives to efficiently use cryopreserved semen from males of valuable genetics that have become infertile will permit continuous propagation of the genetics from these males and may serve as a model for preservation and propagation of endangered species. Sperm cryopreservation without cryoprotectants is a simple process, and offspring have been produced following intracytoplasmic sperm injection (ICSI); however the ability of frozen-thawed sperm refrozen without the addition of cryoprotectants to activate oocytes following ICSI was unknown. In the series of experiments performed, bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants was used to activate intra- and interspecies oocyte following ICSI. Additionally, equine cumulus-oocyte complexes (COCs) glucose metabolism during in vitro maturation was evaluated. The first experiment demonstrated that bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants had their plasma membrane damaged; however the DNA was unaffected. The second experiment demonstrated that bull and stallion frozen-thawed sperm refrozen without the addition of cryoprotectants had the ability to activate bovine oocytes following intracytoplasmic sperm injection; although at a lower rate compared to frozen-thawed sperm. The third experiment demonstrated that frozen-thawed stallion sperm refrozen without the addition of cryoprotectants was unable to activate equine oocytes. The exact reason for this failure could not be explained from the experiment; however COC metabolism during in vitro maturation impacts embryo activation/development and required further investigation. The fourth experiment demonstrated that equine COCs consume and metabolize glucose through glycolysis during in vitro maturation; however, results from this experiment were unable to explain the failure of refrozen stallion sperm to activate equine oocytes. To our knowledge, this is the first report of the use of bull or stallion frozen-thawed sperm refrozen without the addition of cryoprotectants to activate oocytes following ICSI. Furthermore, this is also the first report of equine COCs glucose metabolism during in vitro maturation.

Histone H3K79 methylation has been shown to play roles in different DNA repair pathways. Histone H4 residues serine 64 to threonine 80 surround histone H3K79 residue. We have analyzed the effect of the mutations of the residues on UV sensitivity, H3K79 methylation, nucleotide excision repair, chromatin state and homologous recombination. We found that mutation of the residues 64 to 72 cause resistance to killing by UV whereas mutation of the residues 73 to 80 cause sensitivity to killing by UV compared to wild type. In general, we found that the mutations make nucleotide excision repair more proficient at the constitutively active RPB2 loci. We found global genomic repair is faster in most of the mutants except H75E. Transcription coupled repair is normal in most of the mutants except mutant Y72T. In mutant H75E, Rad26 independent transcription coupled repair is also defective. The mutations T73D, T73F and T73Y affect mono, di and tri methylation of H3K79 but they have faster or normal nucleotide excision repair. We have also found that these histone mutations make chromatin more accessible to micrococcal nuclease. The UV sensitive histone mutants have normal or faster nucleotide excision repair. Methyl methane sulfonate (MMS) sensitivity test, Rad14 and Rad52 epistasis analysis suggests that the UV sensitive histone H4 mutants could play role in homologous recombination repair pathway. Taken together, the results imply that the histone mutations remodel the chromatin that helps to recruit nucleotide excision repair factors for efficient repair.

The purpose of this research was to determine whether adipocytes express ACE2 and its role in obesity-associated hypertension with diet-induced obesity. To determine if ACE2 was expressed in adipose tissue and its regulation in the setting of diet-induced obesity, we fed male mice either a low fat (LF) or high fat (HF) diet acutely (1 week) or chronically ( 4 months). We demonstrated that ACE2 was regulated specifically in adipose tissue with consumption of a HF diet. However, with chronic HF feeding adipose ACE2 was dysregulated resulting in activation of the systemic RAS and increased blood pressure. To determine the role of ACE2 in obesity-associated hypertension, we used ACE2 deficient male and female mice. Wild type and ACE2 deficient mice were chronically fed either a LF or HF diet. Metabolic parameters were measured during the entire course of the study and blood pressure was measured by telemetry at the end of the study. Results from these studies demonstrate that HF diet promotes obesity-associated hypertension in male mice which is further augmented with ACE2 deficiency. Further, ACE2 deficiency resulted in marked glucose intolerance suggesting that stimulation of ACE2 may blunt the progression of obesity-associated diabetes. In contrast to the males, females are protected against obesity-associated hypertension. However, this protection in the females is lost with ovariectomy and ACE2 deficiency. These results suggest that female sex hormones protect the females against obesity-associated hypertension by regulating ACE2. To define mechanisms for HF diet-induced regulation of ACE2 in adipose tissue we examined various fatty acids for their ability to regulate ACE2 mRNA abundance in 3T3-L1 adipocytes. We revealed that omega-3 fatty acids, known regulators of PPARγ, increased ACE2 mRNA abundance in adipocytes. Therefore, we examined in vitro and in vivo regulation of ACE2 in 3T3-L1 cells and adipose tissue by PPARγ receptor ligands (TZDs). Results demonstrate regulatory effects of PPARγ to promote ACE2 gene transcription. These effects were associated with changes in glucose tolerance. Taken together, these results demonstrate that adipose ACE2 plays a protective role against obesity-associated hypertension in male and female mice and is regulated by natural and synthetic ligands of PPARγ.

The objectives of this study were to investigate concentration dependent effects of canine prostatic fluid (PF) on in vitro seminal parameters of cooled canine semen. Sperm motility parameters, plasma membrane integrity and stability, acrosome integrity and DNA fragmentation were measured after the addition of 0%, 10%, 25%, or 50% PF to extended semen of fertile dogs. Assessments were made at 0 h pre-cooling, at 24, and 48 h of cooled storage (4 °C), and after freezing and thawing followed by incubation (37 °C) at 0, 4 and 24 h. Our hypothesis was that lower dilutions of canine semen with PF in an egg yolk-Tris extender would improve plasma membrane stability and acrosome integrity, and preserve sperm kinetics and reduce DNA fragmentation in comparison with higher concentrations of PF in fertile dogs during cooling. Sperm motility parameters were assessed by computer assisted sperm analysis, and plasma membrane integrity by the hypo-osmotic swelling test. Flow cytometry was used after staining with YO-PRO-1/Ethidium Homodimer 1 (EthD-1) to evaluate membrane stability, fluorescent isothiocynate-PNA (Arachis Hypogaea)/propidium iodide to assess acrosome integrity, and sperm chromatin structure assay to assess DNA fragmentation. The data was analyzed using a mixed linear model (ANOVA) and in case of significant effects of time, treatment, or treatment*time interaction (P < 0.05), least square means were used for pairwise comparisons. Acrosome integrity and DNA fragmentation were not affected by treatment with PF. During the cooling period motility parameters were not influenced by PF treatment. A lower proportion of early apoptotic and higher proportion of early necrotic cells was seen during cooling with 50% PF (YO-PRO-1/EthD-1). Although lower concentrations of PF did not improve the evaluated spermatozoal parameters, they did not seem to compromise sperm motility and plasma membrane stability. The presence of 50% PF prior to cryopreservation decreased post thaw motility and produced a shift towards early necrotic cells after thawing. Therefore admixture with more than 10% PF should be avoided prior to cryopreservation of canine semen.

Stem cells exist in various tissues, including dental follicles and dental pulps. Adult stem cells (ASC) can be isolated from patients for autologous transplantation, which eliminates the risk of immune rejection with low or no tumorigenesis. However, one of the challenges is that ASC progressively lose their differentiation ability when cultured in vitro. This prevents expansion of large quantities of high-potential stem cells for therapeutics, especially for stem cells with limited tissue source, such as dental pulp stem cells (DPSC). The goal of this study is to define possible molecular regulation causing loss of differentiation. To achieve this goal, we determined that DPSC at passages 3 and 5 (early passage) possessed strong differentiation capability, and such differentiation capability is completely lost at passage 11 (late passage). Using whole-genome microarray to compare the transcriptomes, we found that the expression of 34 genes were decreased for more than 10-fold in p11 DPSC when compared to p3. After confirming gene expression with RT-PCR, heat shock protein B8 (HspB8) and the GIPC PDZ domain-containing family (Gipc2) were selected for siRNA knockdown study. Knockdown of HspB8 in early-passage DPSC resulted in the cells losing differentiation, but knockdown of Gipc2 had no effect, suggesting that HspB8 plays an important role in maintaining DPSC differentiation. To further study HspB8, we constructed 2 vectors, one containing the coding sequence (CDS) and 3 untranslated region (3UTR) and another containing only the CDS. Transfection of the vectors into early passage DPSC dramatically increased both HspB8 mRNA and protein. However, transfection of the vectors into the late passage DPSC resulted in overexpression of HspB8 mRNA, but increase of HspB8 protein was seen only in CDS transfection. Given that 3UTR of mRNA is the major target region for microRNAs (miRNAs), the results indicate that miRNAs are responsible for down-regulation of HspB8 in long-term culture of DPSCs. We conclude that high-level HspB8 expression is essential for differentiation of DPSC, and down-regulation of HspB8 in cultured DPSC is likely due to increased expression of miRNAs. These are novel findings regarding HspB8 and miRNAs on the regulation of stem cell fate.

Utilization of patient-specific stem cells in regenerative medicine provides a novel treatment approach for diseases and disorders. Embryonic stem cells and induced pluripotent stem cells can differentiate into any cells found within the body; however, ethical, technical and safety concerns have to be overcome before they can be used in clinics. Patient-specific stem cells can be isolated from adult tissues with no ethical, fewer technical, and safety concerns. Obtaining tissues for stem cell isolation usually requires invasive procedures, but impacted teeth are often extracted in the clinics and can be used for isolation of dental follicle stem cells (DFSCs). The overall goal of this dissertation is to characterize the osteogenic potential of DFSCs and to explore the possibility of using DFSCs for the treatment of craniofacial defects. In this regard, we first showed that DFSCs can be induced to differentiate primarily toward the osteoblast lineage. Our experiments showed that DFSCs at passages 3 to 5 have a strong osteogenic capability that is reduced during in vitro expansion. Comparing DFSCs with non-stem cell dental follicle cells (DFCs), we determined that dentin matrix protein 1 (DMP1) is highly expressed in DFSCs. Further study suggests that DMP1 is likely necessary to maintain the osteogenic differentiation capability of DFSCs via regulating expression of osteogenic genes. Given that adult stem cells exist in a quiescent state under normal physiological conditions, we attempted to activate DFSCs with heat-stress. Culturing DFSCs under mild heat-stress (39ºC-40ºC) could effectively promote their proliferation and osteogenic differentiation. In the final part of this project, in vivo transplantation experiments were conducted to evaluate the osteogenic potential of DFSCs for treatment of calvarial critical-size defects using a rat model. Bone regeneration was assessed by micro-computed tomography (micro-CT) and histological analysis at 4 and 8 weeks post-transplantation. The results showed that transplantation of DFSCs seeded into PCL scaffold significantly improved bone regeneration. An average of 50% bone recovery was observed with treatment of PCL-DFSC transplantation at 8 weeks. In conclusion, this study found that DFSCs are valuable tissue stem cells possessing strong osteogenic potential that can be used for repairing craniofacial defects.

Background: The metabolic syndrome is associated with an increased risk of diabetes and vascular disease. In order to understand the pathophysiological processes underlying such risk, it is necessary to develop a better understanding of normal fat metabolism and abnormalities associated with the syndrome. The hypothesis tested in this thesis is that specific abnormalities in adipose tissue and muscle fat metabolism characterise the metabolic syndrome. Methods: Fasting biochemical parameters were measured in a cohort of overweight men with and without the metabolic syndrome. Stable-isotope labeling and arterio-venous difference measurements were conducted in 18 men to elucidate pathways of exogenous and endogenous fat metabolism under fasting and postprandial conditions in adipose tissue and skeletal muscle. In addition, a pilot study of the effects of heat and electrical stimulation on adipose tissue metabolism was undertaken. Results: Cohort study - The prevalence of the metabolic syndrome depended on the definition used. Total cholesterol and apoB were greater in those with the metabolic syndrome than in those without. There was no difference in fasting NEFAs. Metabolic investigation - There was significant postprandial uptake of NEFA from the circulating NEFA pool by adipose tissue. Chylomicrons were confirmed as the preferred substrate of LPL. There was preferential uptake of FAs derived from chylomicron hydrolysis. There was release of NEFA across muscle. In the metabolic syndrome, adipose tissue NEFA output is lower during fasting and falls less following a meal than in the healthy obese. Clearance of dietary-derived TG is lower across both adipose tissue and muscle in the metabolic syndrome. Pilot study – Heat increased measures of lipolysis whereas electrical stimulation had no effect. Conclusions: Fat metabolism in individuals with the metabolic syndrome is characterised by metabolic inflexibility but not insulin resistance.

The objective of the survey study was to measure the level of knowledge and attitudes of the study groups with respect to the field of veterinary prosthetics. The objective of the implant study was to validate the biomechanical feasibility of two basic prosthetic implants for horses. The objective of the foot study was to virtually design a simple, inexpensive and accessible prosthetic foot for amputee horses, as well as biomechanically investigate it using finite element analysis (FEA) to define the optimal materials and dimensions. The survey study showed that there is not enough information available about veterinary prosthetics and student populations are more amicable to the field. The implant study showed that both implant designs were adequate in their abilities to withstand equine specific loads in axial compression, cycling and 4-point bending; the implant with and abutment performed statistically significantly better for axial loads and the interlocking screw implant was practically significantly better for bending loads. Using FEA, the most ideal dimensions, out of 0.5, 0.75 or 1 inch heel fillets and toe lengths, for an average-sized equine prosthetic foot were a 0.5 inch diameter heel fillet and 0.75 inch toe length at breakover for both midstance and heel strike. The most ideal material was found to be acrylonitrile butadiene styrene polycarbonate (ABSPC). The survey study indicates that collaborative efforts of different professionals is required to successfully provide veterinary prosthetics and produce more relevant information for advancement of the field. The implant study indicated that the implants, as they are, would best biomechanically resist the forces of small horses or ponies and that more research is required to assess the biomechanical potential of both implant designs together for average or large-sized horses. The prosthetic foot would likely perform better with a rubber heel and rubber lining the sole to absorb shock. It is entirely customizable to any amputee horses specific needs, 3D printable or machinable, inexpensive and accessible as an open source design with free permission to build on and modify as necessary.

Possession of the Major Histocompatibility Complex (MHC) allele, HLA-B27 (B27), strongly predisposes to the development of spondyloarthritis. Furthermore, B27 exists as polymorphic variants, with some subtypes (such as B*2705) being more strongly associated with disease than others (B*2709). The immunological function of MHC molecules is to present peptides in a heterotrimeric complex with beta-2-microglobulin (β2m); however, B27 has also been observed to form non-classical (β₂m –free) homodimers at the cell surface. It has been suggested that there may be a pathogenic role for cell surface B27 homodimer interactions with Natural Killer (NK) cell receptors, such as Leukocyte Immunoglobulin like Receptors (LILRs). In this thesis I characterise these interactions and investigate molecular differences between two B27 subtypes. Here I show that the B*2705 subtype forms homodimers more readily than the B*2709 subtype, but once formed, B27 homodimers of the 2 different subtypes exhibit comparable binding specificities and affinities to the NK receptors. On the other hand, I show significant differences in the binding specificities and affinities of these receptors to B27 homodimers and heterotrimers. LILRB1 does not bind B27 homodimers, but does bind B27 heterotrimers. LILRB2 binds B27 heterotrimers with a KD of 22μM, whereas LILRB2 binds B27 homodimers more strongly with a KD of 2.5μM. In addition to these main findings, I have characterised the specificity and affinity of candidate B27 homodimer-specific antibodies. I have performed epitope-mapping experiments and developed a model for binding to the B27 homodimer. Finally, I have identified crystallisation conditions for the B27 homodimer in complex with a Fab, allowing for X-ray crystallography studies. In this thesis, I have characterised for the first time the molecular interactions of the B27 homodimer with NK cell ligands and show that they are different from those with the B27 heterotrimer. This work supports a hypothesis of B27 homodimer induced pathology involving NK receptors.