This thesis consisted of three major components: 1. A sample of randomized controlled trials of herbal medicines was collected and assessed with a recently developed extension of the CONSORT statement for herbal medicine trials. 2. A methodological review of proposed methods of assessing clinical heterogeneity in meta-analyses of randomized controlled trials, 3. The application of permutation based resampling in meta-regression of multiple covariates. An analysis of 406 RCTs of herbal medicine interventions revealed that these trials are regularly under reporting important aspects of the intervention. Next, the second project showed that there are many resources providing suggestions for investigating clinical heterogeneity in systematic reviews of controlled clinical trials and though there is minimal consensus some recommendations are common across sources. Finally, the third project found that permutation tests result in more conservative, larger, p-values potentially reducing the rate of false positive findings when exploring multiple covariates.

Lung transplantation is a life-saving therapy for patients suffering from end-stage lung disease; however, the majority of donor lungs are injured and attempts to transplant them results in a high risk of primary graft dysfunction in the recipient, a type of severe acute lung injury. Previously, a novel method of lung preservation known as ex vivo lung perfusion (EVLP) has been developed in which donor lungs are continuously perfused and ventilated at normothermia using a protective strategy. Donor lungs have been shown to tolerate at least 12 h of preservation in this manner without the accrual of injury. Hence, EVLP could act as a platform on which injured donor lungs could potentially be evaluated and repaired. To explore this concept, we utilized interleukin-10 (IL-10), an anti-inflammatory cytokine, as a prototypical drug for ex vivo delivery. Because IL-10 protein has a prolonged half-life during EVLP, we delivered recombinant IL-10 by the intravascular and intratracheal routes to clinically-rejected injured human lungs. Intratracheal delivery resulted in elevated levels of IL-10 in both tissue and perfusate whereas intravascular delivery resulted in elevated levels of IL-10 only in the perfusate over 12 h of EVLP. There was, however, no beneficial effect to either lung function or lung inflammation. This was thought to be a result of intratracheally delivered IL-10 leaking out into the perfusate where it may not be biologically active. Constant IL-10 production within the lung tissue could be achieved using a gene therapy approach. Thus, we subsequently explored the delivery of IL-10 by adenoviral gene therapy during EVLP. Ex vivo administered intratracheal adenoviral gene therapy could increase transgene protein levels within the lung. More importantly, it did so with less vector-associated inflammation when compared to in vivo delivery of adenoviral gene therapy. Having explored drug delivery, we sought to develop a large animal injury model on which to test ex vivo therapies. Given that the majority of organ donors are brain dead and therefore exposed to the injurious sequelae resulting from brain death, we developed a brain-death injury model in pig. Use of EVLP as a platform for repair necessitates an accurate recognition of both lung injury and lung improvement during EVLP. Thus, we utilized this injury model to explore the profile of physiological parameters when an injured lung is perfused during EVLP. Because of the alteration of the PO2 to oxygen content relationship of an acellular perfusate, we found that PaO2 changes are less dramatic than in the in vivo situation. However, as injured lungs begin to become edematous, the mechanical effects on the lung by the increased water content can be measured by corresponding falls in compliance and increases in airway pressure. Overall, use of EVLP demonstrates promise for reducing the organ shortage currently prevalent in clinical lung transplantation. Improved evaluation will instill confidence in transplant clinicians to transplant previously questionable organs. Lungs which prove to be injured during evaluation can potentially be repaired using IL-10 therapy as explored herein or with other therapies using the delivery methods described.

Large intergenic RNAs (lincRNA) are involved in numerous cellular processes, including many relevant to normal development and cancer progression. In my doctoral research, we hypothesized that lncRNAs are functionally important to human endothelial biology, more specifically, to the process of human blood vessel formation or angiogenesis. To detect lincRNAs that are functionally important to human angiogenesis, a custom microarray was used to profile long noncoding transcripts in human vascular endothelium in two-dimensional versus three-dimensional pro-angiogenic cultures, with or without VEGF-A165. We identified a VEGF-A-responsive lincRNA near the VEGFR1 gene, which we termed lincRNA-VEGFR1 (LIVE1). Unbiased mRNA microarrays defined a number of potential target genes when LIVE1 was functionally disrupted using RNA interference. Importantly, knockdown and over-expression studies indicated that LIVE1 exerts transcriptional control over VEGFR1 as well as other VEGF receptors and direct angiogenesis in vitro. Furthermore, we found that LIVE1 is highly expressed in glioblastoma, and is enriched in glioma stem cell (GSC) fractions and neoplastic endothelial progenitor populations. In vivo knockdown of LIVE1 in a glioblastoma xenograft model decreased microvascular density, vascular perfusion, pericyte coverage, tumor volume and slowed tumour progression. Our results establish LIVE1 as a key mediator of angiogenesis and demonstrate the potential of lincRNA-based therapeutics.

Large intergenic RNAs (lincRNA) are involved in numerous cellular processes, including many relevant to normal development and cancer progression. In my doctoral research, we hypothesized that lncRNAs are functionally important to human endothelial biology, more specifically, to the process of human blood vessel formation or angiogenesis. To detect lincRNAs that are functionally important to human angiogenesis, a custom microarray was used to profile long noncoding transcripts in human vascular endothelium in two-dimensional versus three-dimensional pro-angiogenic cultures, with or without VEGF-A165. We identified a VEGF-A-responsive lincRNA near the VEGFR1 gene, which we termed lincRNA-VEGFR1 (LIVE1). Unbiased mRNA microarrays defined a number of potential target genes when LIVE1 was functionally disrupted using RNA interference. Importantly, knockdown and over-expression studies indicated that LIVE1 exerts transcriptional control over VEGFR1 as well as other VEGF receptors and direct angiogenesis in vitro. Furthermore, we found that LIVE1 is highly expressed in glioblastoma, and is enriched in glioma stem cell (GSC) fractions and neoplastic endothelial progenitor populations. In vivo knockdown of LIVE1 in a glioblastoma xenograft model decreased microvascular density, vascular perfusion, pericyte coverage, tumor volume and slowed tumour progression. Our results establish LIVE1 as a key mediator of angiogenesis and demonstrate the potential of lincRNA-based therapeutics.

Background: Researchers and research ethics boards (REBs) in Toronto were unprepared for the SARS outbreak. There is a paucity of literature about how to review emergency-related protocols during a public emergency and so REBs had no guidance about how to review SARS-related protocols. Research questions: The thesis presents four related research inquiries based on the following four objectives: 1) to conduct a comprehensive evaluation of the one-year outcomes in SARS survivors; 2) to explore the ethical issues that emerged during the conduct of the SARS outcomes study; 3) to understand the impact of the SARS outbreak on research ethics review (RER) of SARS-related protocols; and 4) to propose a new framework of RER for use during public emergencies. Methods: Included in this thesis are an observational study, an analytic reflection, a grounded theory study, and a translation of the knowledge gained in the first three parts of the thesis into a framework of RER that is meaningful and actionable. Results: Part I describes the recovery made by SARS survivors from their acute illness. In part II, I explore ethical issues that arose during the conduct of the study including: social and scientific value and scientific validity of emergency research, and respect for privacy and confidentially. Part III presents a theory about how researchers, REBs and public health interacted during the outbreak and in part IV I propose ‘emergency review’ a framework for RER for use during a publicly declared emergency. Conclusions: The natural experiment that was the SARS outbreak in Toronto revealed the vulnerabilities in the structure of REBs. I highlight three conclusions which are the highest priority to provide further development in this field. These are: 1) when REBs, researchers and public health are not effectively communicating during a public emergency, the work of each group is disrupted; 2) institutional conflict of interest occurred during the research ethics review of SARS-related protocols and may be amplified during a public emergency and 3) there is a need for a multi-site review structure that could be activated on short notice to review protocols related to the emergency situation.

Bone morphogenetic protein-2 (BMP-2) plays a critical role in bone formation. The aim of this study was to compare the activity of the mammalian cell BMP-2 to the E-coli cell BMP-2. In vitro, the potency of mammalian and E-coli BMP-2 was compared by adding BMP-2 to C2C12 cells and measuring the level of alkaline phosphatase activity. In vivo, the activity was evaluated by placing the bioimplants in the thigh muscle of mice, and measuring the amount of bone induced. The in vitro assay clearly showed that mammalian BMP was significantly more potent than E-coli BMP. In vivo, on the calcium phosphate carrier, mammalian BMP produced more bone than E-coli BMP, but E-coli BMP produced higher density tissue than mammalian BMP. On both mammalian and E-coli BMP, the calcium phosphate carrier had a significant effect on the density but not the quantity of bone produced versus the absorbable collagen sponge carrier.

Disease-specific (e.g. urinary) and generic problems (e.g. fatigue) impact health-related quality of life (HRQOL) of patients with bladder cancer (BC). A questionnaire addressing both sets of problems and generating utilities, a measure of overall HRQOL, is needed for this population. In consultation with 47 BC patients and 12 BC experts we created a novel HRQOL questionnaire for BC in a stepwise, iterative fashion with conceptual framework development, item generation, item reduction, question design and pilot testing. Patients and experts identified urinary problems, bowel problems, sexual problems, body image, pain, and decreased vigor as domains, which impact daily activities and impair HRQOL. Support from patients’ family and friends and their medical team were also paramount to HRQOL. The Bladder Utility Symptom Scale is a comprehensible instrument that was created to facilitate patient-oriented care and guide clinical policy by grounding guidelines, health resource allocation, and policy decisions in the expressed preferences of BC patients.

Background: Researchers and research ethics boards (REBs) in Toronto were unprepared for the SARS outbreak. There is a paucity of literature about how to review emergency-related protocols during a public emergency and so REBs had no guidance about how to review SARS-related protocols. Research questions: The thesis presents four related research inquiries based on the following four objectives: 1) to conduct a comprehensive evaluation of the one-year outcomes in SARS survivors; 2) to explore the ethical issues that emerged during the conduct of the SARS outcomes study; 3) to understand the impact of the SARS outbreak on research ethics review (RER) of SARS-related protocols; and 4) to propose a new framework of RER for use during public emergencies. Methods: Included in this thesis are an observational study, an analytic reflection, a grounded theory study, and a translation of the knowledge gained in the first three parts of the thesis into a framework of RER that is meaningful and actionable. Results: Part I describes the recovery made by SARS survivors from their acute illness. In part II, I explore ethical issues that arose during the conduct of the study including: social and scientific value and scientific validity of emergency research, and respect for privacy and confidentially. Part III presents a theory about how researchers, REBs and public health interacted during the outbreak and in part IV I propose ‘emergency review’ a framework for RER for use during a publicly declared emergency. Conclusions: The natural experiment that was the SARS outbreak in Toronto revealed the vulnerabilities in the structure of REBs. I highlight three conclusions which are the highest priority to provide further development in this field. These are: 1) when REBs, researchers and public health are not effectively communicating during a public emergency, the work of each group is disrupted; 2) institutional conflict of interest occurred during the research ethics review of SARS-related protocols and may be amplified during a public emergency and 3) there is a need for a multi-site review structure that could be activated on short notice to review protocols related to the emergency situation.

Bone morphogenetic protein-2 (BMP-2) plays a critical role in bone formation. The aim of this study was to compare the activity of the mammalian cell BMP-2 to the E-coli cell BMP-2. In vitro, the potency of mammalian and E-coli BMP-2 was compared by adding BMP-2 to C2C12 cells and measuring the level of alkaline phosphatase activity. In vivo, the activity was evaluated by placing the bioimplants in the thigh muscle of mice, and measuring the amount of bone induced. The in vitro assay clearly showed that mammalian BMP was significantly more potent than E-coli BMP. In vivo, on the calcium phosphate carrier, mammalian BMP produced more bone than E-coli BMP, but E-coli BMP produced higher density tissue than mammalian BMP. On both mammalian and E-coli BMP, the calcium phosphate carrier had a significant effect on the density but not the quantity of bone produced versus the absorbable collagen sponge carrier.

Disease-specific (e.g. urinary) and generic problems (e.g. fatigue) impact health-related quality of life (HRQOL) of patients with bladder cancer (BC). A questionnaire addressing both sets of problems and generating utilities, a measure of overall HRQOL, is needed for this population. In consultation with 47 BC patients and 12 BC experts we created a novel HRQOL questionnaire for BC in a stepwise, iterative fashion with conceptual framework development, item generation, item reduction, question design and pilot testing. Patients and experts identified urinary problems, bowel problems, sexual problems, body image, pain, and decreased vigor as domains, which impact daily activities and impair HRQOL. Support from patients’ family and friends and their medical team were also paramount to HRQOL. The Bladder Utility Symptom Scale is a comprehensible instrument that was created to facilitate patient-oriented care and guide clinical policy by grounding guidelines, health resource allocation, and policy decisions in the expressed preferences of BC patients.