The helper-dependent adenoviral (HD-Ad) vector is the latest generation of Ad vectors. It ameliorates the vector-associated immunogenic problems with increased capacity for carrying DNA because all viral coding genes are removed. I hypothesize that HD-Ad vectors can be effective vehicles for retinal gene delivery. The objectives of this study are to determine if HD-Ad vectors can deliver reporter genes, GFP or lacZ, driven by a CMV or a MOPS promoter, into specific retinal layers. Subretinal injections were performed and eyes removed at time points from 1 week to 3 months, processed for fluorescent microscopy, X-gal staining, and H&E staining. Transgene expression was detected for at least 3 months. A dose dependent relationship was revealed between the level of transgene expression and viral vector dose. Distinctively, the MOPS promoter drove photoreceptor cell specific expression. Notably, no sign of inflammation or tissue toxicity was detected, demonstrating the benefits of the HD-Ad vector.

Cervix cancer affects women of all ages and causes significant morbidity and mortality. Locally advanced disease is curable with radiotherapy (RT) in about 50% of patients, although often at the expense of serious side effects. In order to improve the therapeutic ratio of tumour control versus normal tissue toxicity, conformal intensity-modulated radiotherapy (IMRT) is being investigated. However, inter- and intra-fractional motion of cervix cancer can contribute to both geographical miss of the target and overdosing of surrounding normal tissues, particularly in the setting of conformal IMRT with steep dose gradients. Defining the target volume accurately and understanding the dose consequence of these complex intra-pelvic organ dynamics during external beam radiotherapy forms the essential foundations for future treatment optimization and adaptation. This in turn will lead to improvements in tumour control and disease-free survival while minimising treatment toxicity.

Objective: To evaluate the anti-tumor and histone deacetylase (HDAC) inhibitory activity of the dietary isothiocyanate, sulforaphane (SFN) in the paediatric cancer,neuroblastoma (NB). Materials and Methods: NB cell line (NUB-7), fibroblasts (FLF; negative control) and MCF-7 (positive control), were treated with SFN for up to 7 days and effects on growth, cytotoxicity, differentiation and tumorigenicity assessed. HDAC inhibition was determined by histone (H3/ H4) acetylation. Results: 10 μM SFN significantly decreased in vitro growth and survival of NUB-7 to 10.22 ± 0.71% (p < 0.001) with no significant effect on FLF. SFN induced G1, G2 and S phase cell cycle arrests and stimulated H3/H4 histone acetylation. SFN markedly decreased NUB-7 clonogenicity and tumorigenicity in vivo. Conclusion: Results suggest that low dose SFN reduces proliferation, survival and tumorigenicity of NB NUB-7. As a dietary factor of negligible intrinsic toxicity SFN is a promising therapeutic agent for the treatment of NB.

There have been many advances in surgical residency education and similar interest exists in fellowship education. This study evaluated perceptions among spine surgeons about the specific competencies required for successful spine surgical fellowship training, and then compared these perceptions to clinical practice. Firstly, a questionnaire was administered to spine fellow trainees and academic spine surgeons across Canada in order to identify the cognitive and technical skills required for successful spine fellowship training. Fellowship trainees and supervisors had similar perceptions on the relative importance of specific cognitive and technical competencies. Differences in perceptions were found when comparing surgeons based on background residency specialty training (orthopaedic surgical or neurosurgical). Secondly, using administrative data, a retrospective study assessed volumes, surgeon characteristics, and outcomes for surgery of the degenerative lumbar spine in Ontario between 1995 and 2001. Neurosurgeons were found to perform more decompressions, and more total procedures, than orthopaedic surgeons with similar outcomes.

The adoption of Minimally Invasive Surgery (MIS) and Robot-Assisted MIS has resulted in the distortion of haptic cues surgeons rely on. The application of excessive force during port creation has lead to increased surgical access trauma. This study aims to quantify the forces experienced during port creation with a blunt-ended Threaded Visual Cannula (TVC) in an effort to ameliorate patient safety, provide a quantitative platform for surgeon training, and offer a gateway for the eventual automation of this problematic aspect of MIS. A method of determining the torque encountered during port creation was established. It was found that the magnitude of torque required to cannulate different materials was unique and was dictated by the friction observed at the tool/tissue interface. Furthermore, the ability to detect instantaneous changes in torque arising from the transition between two different media was not found to be possible with the current design of the TVC.

There is mounting evidence to support combined therapy with radiation (RT) and antiangiogenic agents (AA) for the treatment of brain tumors. However, the therapeutic benefit of this combined treatment hinges on the specific dose, schedule, and duration of each treatment. Early biomarkers that reflect tumor physiological responses provide key information that could guide these aspects of treatment. Pre-clinical tumor models are invaluable tools for identifying potential biomarkers, their optimal timing for measurement and their ability to guide therapy in clinical translation. This thesis demonstrates the feasibility and potential of serial MRI to guide the design, delivery and measure of early response to combined AA and RT in a murine intracranial glioma model. We identified promising biomarker changes reflecting early treatment response that may ultimately facilitate individualized spatio-temporal delivery of radiotherapy (RT) and anti-angiogenic agents (AA) for brain tumors.

Introduction: The objective of this study is to investigate the role of osteoblasts in the pathophysiology of premature suture fusion in infants. Methods: Bone and periosteal tissue from fused and patent cranial sutures and adjacent bone were harvested from infants undergoing surgery for craniosynostosis and used to develop primary osteoblast cell cultures. Dural tissue was obtained from neurosurgical procedures in order to generate an osteoblast-dural co-culture. Osteoblast proliferation, differentiation, mineralization, protein expression (Noggin, BMP3 and Runx2) and response to exogenous FGF2 stimulation were assessed. Results: Cell cultures demonstrated significant (p<0.05) regional variations in osteoblast proliferation, differentiation markers and in vitro bone nodule formation. The expression of anti-osteogenic molecules (Noggin and BMP3) was decreased in osteoblasts from fused suture regions. Conclusion: The creation of a pro-osteogenic environment through the decreased expression of anti-osteogenic signalling molecules and increased expression of osteogenic factors may be responsible for premature suture fusion in infants.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease (CHD). The understanding of the genetics and inheritance of TOF is limited. Although about 15% of cases are associated with a 22q11.2 deletion, the majority have no known aetiology. Even in 22q11.2 Deletion Syndrome (22q11DS), factors that increase the likelihood of CHD expression are poorly understood. We aimed to determine the prevalence and phenotypes of CHD in relatives of adults with TOF. We also investigated the prevalence of CHD in relatives without a 22q11.2 deletion of individuals with 22q11DS. Offspring of patients with TOF had the greatest prevalence of CHD. Diverse cardiac phenotypes, including left heart obstructive lesions, were found in families. We also found that unaffected relatives of individuals with 22q11DS had a greater prevalence of complex CHD over population expectations, suggesting that modifier genetic factors may be involved in expression of CHD in 22q11DS.

Subarachnoid hemorrhage (SAH) comprises 7% of all stroke cases, and is associated with a disproportionately high morbidity and mortality with few therapeutic options available. The goal of this project was to understand the mechanism of neurological deterioration after experimental SAH, with a focus on cerebral vasospasm and brain injury after SAH. We tested the hypothesis that endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled after SA, resulting in exacerbated neurological injury in a mouse model of SAH. The project entailed the investigation of eNOS-dimer uncoupling, its association with oxidative and nitrosative stress in the brain parenchyma and finally its association with secondary complications after SAH. In our studies we demonstrated the crucial role eNOS plays in anti-microthromboembolism, anti-apoptosis and maintenance of physiological superoxide (O2-)/NO balance. This study suggests that SAH up-regulates and disrupts eNOS, producing peroxynitrite (OONO-) and other radicals that further exacerbate the oxidative insult and neurological injury.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.