Targeting angiogenic vasculature has been validated as a viable approach for cancer imaging and therapy. The tumour vasculature-specific ligand asparagine-glycine-arginine (NGR) peptide targets the isoform of aminopeptidase N (CD13) expressed on endothelial cells lining angiogenic vessels. CD13 has become widely recognized as a rational target for therapeutic development and several NGR-conjugated agents are now in pre-clinical and clinical development. In the current study, a CT image-based approach is used to evaluate the in vivo performance of several NGR-conjugated liposome formulations that vary in terms of NGR density and PEG spacer arm length. Indeed, for the first time it is demonstrated that CT imaging can be used for quantitative and longitudinal assessment of the pharmacokinetics and biodistribution of an actively targeted liposome formulation. In comparison to conventional methods, CT imaging enables visualization of the intratumoural distribution of liposomes and quantification of the fraction of tumour occupied by the vesicles over time.

The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.

Programmed Cell Death 4 (PDCD4) is a known tumour suppressor, lost in carcinomas of the breast, prostate, colon, lung and ovary. This study found significantly reduced levels of PDCD4 mRNA and protein in both primary patient oral squamous cell carcinomas (OSCCs) and OSCC cell lines. Moreover, lower PDCD4 mRNA levels were significantly correlated with nodal metastasis (P=0.019). To determine the functional significance of PDCD4 down-regulation in OSCC we asked whether PDCD4 played a role in invasion. In fact, over-expression of PDCD4 decreased invasion of OSCC lines. We then sought to determine a mechanism for PDCD4 down-regulation in OSCC. Previous studies in breast and colon carcinomas suggested that reduced PDCD4 expression was due to over-expression of miR-21. Interestingly, miR-21 was inversely correlated to PDCD4 mRNA (P=0.002) and PDCD4 protein (P<0.001) levels in OSCC patient samples. Moreover, we found that miR-21 directly regulated PDCD4 protein expression in OSCC cell lines. This is the first report in OSCC that demonstrates that PDCD4 is down-regulated by miR-21 and may play a role in OSCC invasion.

Optical methods can provide useful tissue characterization tools. For this project, two-photon microscopy and polarized light examinations (polarimetry) were used to assess the organizational state of myocardium in healthy, infarcted, and stem-cell regenerated states. Two-photon microscopy visualizes collagen through second-harmonic generation and myocytes through two-photon excitation autofluorescence, providing information on the composition and structure/organization of the tissue. Polarimetry measurements yield a value of linear retardance that can serve as an indicator of tissue anisotropy, and with a dual-projection method, information about the anisotropy axis orientation can also be extracted. Two-photon microscopy results reveal that stem-cell treated tissue retains more myocytes and structure than infarcted myocardium, while polarimetry findings suggest that the injury caused by temporary ligation of a coronary artery is less severe and more diffuse that than caused by a permanent ligation. Both these methods show potential for tissue characterization.

Distant metastases are a limiting factor in cancer patient survival as they are least accessible to conventional therapies. Effective therapy should treat primary tumours and metastatic disease. Use of image-guided radiation therapy (IGRx) enables high doses of radiation to be delivered for better tumour control while minimizing toxicity to healthy tissues. Systemic effects on distant non-irradiated tissues have been observed following IGRx. This phenomenon, termed the abscopal effect, is hypothesized to be mediated by the immune system. The inflammatory milieu generated following IGRx may activate immune cells to mount specific anti-tumour responses. The work described in this thesis aims to develop a model to study the abscopal effect, and evaluate the potential of combining IGRx and immunotherapy to enhance such distant tumour killing. Results from these studies may have clinical implications, where a combined IGRx and immunotherapy approach may prove useful in eliciting regression of local tumours and distant metastases.

Targeting angiogenic vasculature has been validated as a viable approach for cancer imaging and therapy. The tumour vasculature-specific ligand asparagine-glycine-arginine (NGR) peptide targets the isoform of aminopeptidase N (CD13) expressed on endothelial cells lining angiogenic vessels. CD13 has become widely recognized as a rational target for therapeutic development and several NGR-conjugated agents are now in pre-clinical and clinical development. In the current study, a CT image-based approach is used to evaluate the in vivo performance of several NGR-conjugated liposome formulations that vary in terms of NGR density and PEG spacer arm length. Indeed, for the first time it is demonstrated that CT imaging can be used for quantitative and longitudinal assessment of the pharmacokinetics and biodistribution of an actively targeted liposome formulation. In comparison to conventional methods, CT imaging enables visualization of the intratumoural distribution of liposomes and quantification of the fraction of tumour occupied by the vesicles over time.

The human gut is host to a vast consortium of microorganisms, collectively referred to as the microbiota or microflora, which play important roles in health and disease. Current applications focus only on a single type of bacteria, which are not the most dominant numerically, and without detailed knowledge of the specific functions of these bacteria. A good indicator of the function of a bacterial species involves detailed analysis of its enzymes. Bacteroides thetaiotaomicron is one of the predominant bacterial species with a great representation of the carbohydrate processing enzymes, glycoside hydrolases in its proteome. This thesis reports the production and purification of one such enzyme, BT3299, suitable for kinetic and structural studies. The enzyme displayed a broad substrate specificity with a slight preference for 1-->3 and 1-->6 glycosidic linkages and longer chain saccharides. Future work will focus on structural analysis as an aid to the understanding of the enzyme function.

Revascularization of Chronic Total Occlusions (CTO) by percutaneous coronary interventions is limited by low success rates, primarily due to difficulty in guidewire crossing. There are a number of contributing factors that make guidewire crossing challenging. Two of the most significant impediments are: a) inability to adequately visualize the CTO to appropriately plan a pathway to the distal lumen, and b) difficulty in physically crossing the rigid endcap at the proximal end of CTO without using stiff wires. Moreover, there is a significant knowledge gap in the composition of CTOs, and the consequent impact of that composition on crossability. This thesis presents tools and techniques to help mitigate the current shortcomings, while shedding new light on CTO composition and maturation. The tools and techniques presented herein are based upon ultrasound approaches with the intent of eventually developing these strategies into catheter based solutions. Recent studies have suggested that the presence of microvessels in CTO may provide a preferred pathway for guidewire crossing. However, due to limited resolution and a lack of soft tissue contrast in angiography, microvessels within CTO cannot generally be detected by in-vivo angiographic techniques, and when they are visualized, it is unknown whether or not they are intraluminal. In this thesis, high frequency ultrasound with Power Doppler overlays is shown to be capable of detecting and tracking transluminal recanalization channels using an in vivo porcine model of CTO. It is also shown that ultrasound is a more sensitive technique to detect and map these channels than MRI. Furthermore, features of microvasculature in CTOs that had not previously been seen are presented. A technique was then developed to facilitate guidewire crossing through the proximal endcap, also known as the proximal fibrous cap (PFC). In order to assess the ease with which a probe is able to iv perforate the PFC, a system was designed and to measure the force required for PFC puncture. This system was validated by examining the required puncture forces for CTOs of different ages. It was shown that CTOs less than 6 weeks in age are significantly easier to puncture than those greater than 12 weeks. This coincides with differences in composition, with the presence of softer materials at the earlier time point, such as thrombus and proteoglycans compared to stiffer fibrotic materials which predominate at late timepoints. After development and validation of a reliable technique to measure ease of PFC puncture, the efficacy of therapies designed to modify PFC compliance could be assessed. The use of ultrasound mediated microbubble (UMM) disruption to act as an adjuvant to accelerate collagenase therapy in CTO was examined. A significant reduction in puncture force and an increase in the amount of collagen degraded was achieved using a combined UMM + collagenase treatment compared with collagenase therapy alone and UMM treatment alone.

In 1962, Gross and Lapiere described collagenolytic activity in the degradation of tadpole tails during amphibian metamorphosis. This activity was later attributed to a collagenase enzyme belonging to the matrix metalloproteinase family. Over the past 49 years, steady growth in the field of metalloproteinase biology has uncovered that degradation of extracellular matrix components represents only a fraction of the functions performed by these enzymes. The regulatory roles of these enzymes in numerous aspects of mammalian biology remains poorly understood. This thesis investigates the metalloproteinase ADAM17 and its natural inhibitor TIMP3 in acute and chronic inflammation. My work describes the generation of new murine experimental systems of compartmentalized ADAM17 or TIMP3 deficiency and their applications in acute liver inflammation (i.e. fulminant hepatitis and T-cell mediated autoimmune hepatitis) and atopic dermatitis. Loss of Timp3 protected mice against fulminant hepatic failure caused by activation of the death receptor Fas. We determined that TIMP3 simultaneously promotes pro-apoptotic signaling through TNFR1 while suppressing anti-apoptotic EGFR activation in the liver. Mechanistically, we identified that ADAM17 is critical in shedding TNFR1 and EGFR ligands (e.g. Amphiregulin, HB-EGF, TGF) and extended this finding to clinically relevant drug-induced hepatitis. Adult TIMP3 deficient mice also exhibited spontaneous accumulation of CD4+ T cells in the liver. Consequently, polyclonal T cell activation with the lectin Concanavalin A (con A) in a model of autoimmune hepatitis resulted in accelerated liver injury. We identified that this immunopathology relied on TNF bioavailability as mice lacking both Timp3 and Tnf were resistant to con A. Using bone marrow chimeras we established that non-hematopoietic tissues were the physiologically relevant source of TIMP3 in vivo, thereby highlighting an immunosuppressive role for this stromal metalloproteinase inhibitor in cellular immunity. Finally, we investigated epithelial:immune crosstalk in the epidermis by generating tissue-specific ADAM17 deficiency in basal keratinocytes. These mice developed spontaneous inflammatory skin disease that was physiologically consistent with atopic dermatitis. Focused investigation of keratinocyte-specific signaling deregulated by ADAM17 deficiency revealed its requirement for tonic Notch activation, which in turn antagonized transcriptional activity of AP-1 transcription factors on the promoters of epithelial cytokines TSLP and G-CSF. In summary, these works identify cellular mechanisms governing cytokine-mediated communication between epithelial and immune cells to modulate inflammation. The findings that TIMP3 and ADAM17 act as regulators of key inflammatory, proliferative and developmental pathways provide impetus to expand our understanding of this important family of enzymes in mammalian signal transduction.

Revascularization of Chronic Total Occlusions (CTO) by percutaneous coronary interventions is limited by low success rates, primarily due to difficulty in guidewire crossing. There are a number of contributing factors that make guidewire crossing challenging. Two of the most significant impediments are: a) inability to adequately visualize the CTO to appropriately plan a pathway to the distal lumen, and b) difficulty in physically crossing the rigid endcap at the proximal end of CTO without using stiff wires. Moreover, there is a significant knowledge gap in the composition of CTOs, and the consequent impact of that composition on crossability. This thesis presents tools and techniques to help mitigate the current shortcomings, while shedding new light on CTO composition and maturation. The tools and techniques presented herein are based upon ultrasound approaches with the intent of eventually developing these strategies into catheter based solutions. Recent studies have suggested that the presence of microvessels in CTO may provide a preferred pathway for guidewire crossing. However, due to limited resolution and a lack of soft tissue contrast in angiography, microvessels within CTO cannot generally be detected by in-vivo angiographic techniques, and when they are visualized, it is unknown whether or not they are intraluminal. In this thesis, high frequency ultrasound with Power Doppler overlays is shown to be capable of detecting and tracking transluminal recanalization channels using an in vivo porcine model of CTO. It is also shown that ultrasound is a more sensitive technique to detect and map these channels than MRI. Furthermore, features of microvasculature in CTOs that had not previously been seen are presented. A technique was then developed to facilitate guidewire crossing through the proximal endcap, also known as the proximal fibrous cap (PFC). In order to assess the ease with which a probe is able to iv perforate the PFC, a system was designed and to measure the force required for PFC puncture. This system was validated by examining the required puncture forces for CTOs of different ages. It was shown that CTOs less than 6 weeks in age are significantly easier to puncture than those greater than 12 weeks. This coincides with differences in composition, with the presence of softer materials at the earlier time point, such as thrombus and proteoglycans compared to stiffer fibrotic materials which predominate at late timepoints. After development and validation of a reliable technique to measure ease of PFC puncture, the efficacy of therapies designed to modify PFC compliance could be assessed. The use of ultrasound mediated microbubble (UMM) disruption to act as an adjuvant to accelerate collagenase therapy in CTO was examined. A significant reduction in puncture force and an increase in the amount of collagen degraded was achieved using a combined UMM + collagenase treatment compared with collagenase therapy alone and UMM treatment alone.