Contrast enhanced ultrasound offers a unique method to measure the blood flow, perfusion, vascular volume and morphology of microvascular networks. This is achieved by exploiting the ability of microbubble contrast agents to be disrupted and preferentially detected with contrast specific imaging techniques – using a method known as disruption-replenishment. In its current form, disruption-replenishment suffers from poor reproducibility and accuracy, largely due to the inappropriate application of a mono-exponential model of microbubble replenishment, and an incomplete understanding of the dependencies of the measurement. In this thesis, we hypothesize that disruption-replenishment measurements can be improved by applying a perfusion model that considers the physical elements of the measurement, including the haemodynamics and morphology of the vascular system, the ultrasound field distribution and microbubble properties. We present a flexible, theoretical framework to model microbubble replenishment within the microvasculature. The replenishment model is further developed by in vitro and in vivo validation, and clinical translation in a trial of anti-angiogenic therapy in patients, resulting in a proposed clinical protocol. The presented formalism was shown to be more robust and demonstrated better agreement of both fitting quality and estimates of flow velocity when compared to the established model (accuracy to within 3-9%). The reproducibility of repeated in vivo disruption-replenishment flow measurements was 11.9% using the proposed perfusion model compared to 24% using the established model. Variability of clinical perfusion measurements was also reduced with a method that discards the contribution of flow from larger arteries. Excluding the large vessel component in clinical measurements of tumour blood volume decreased the inter-plane variability by up to 20%. The proposed perfusion model can be used to generate parametric maps of vascularity through which additional quantitative parameters become available. These improvements will help translate the method of disruption-replenishment into routine clinical practice and clinical trials.

Heart failure is a complex clinical syndrome in which the heart cannot pump sufficient blood and nutrients to the organs in the body. Increasingly, alterations in cardiac energetics are being implicated as playing an important role in the pathogenesis of heart failure. An understanding of specific metabolic switches which occur during the development of heart failure in patients would be greatly beneficial as a new diagnostic method and for the development of new therapies for patients with failing hearts. This thesis deals with the non-invasive assessment of metabolism in the heart. New magnetic resonance imaging (MRI) methods for metabolic characterization of the heart using hyperpolarized carbon-13 MRI are presented. Spatially resolved images of hyperpolarized 13C substrates and their downstream products can provide insight into real-time metabolic processes occurring in vivo, within minutes of injection of a pre-polarized 13C-labeled substrate. Conventional 3D spectroscopic acquisitions require in excess of 100 excitations, making it challenging to acquire full cardiac and respiratory-gated, whole-heart metabolic volumes. Each of the developments described in this thesis is intended to advance cardiac hyperpolarized 13C metabolic imaging towards a routine, clinical exam which can be used for prognosis and treatment optimization in patients with cardiovascular disease. The major technical development is a new interleaved-frequency, time-resolved MRI pulse sequence that can provide robust and reliable measurements of cardiac metabolic signals. The technique was applied to several realistic pre-clinical models of cardiac disease and the work presented will hopefully lead towards significant improvement in the management of patients with heart failure.

Pulmonary arterial hypertension (PAH) alters pulmonary hemodynamics by changing the vascular wall mechanics. Currently, pulmonary vascular resistance is used clinically to assess the disease. However, the invasive nature of pressure measurements, needed for calculating the resistance, prevents longitudinal monitoring of patients during therapy. This work employs wave reflection as an alternative measure of the downstream stiffness and proposes a new method for wave reflection assessment using only non-invasive phase contrast magnetic resonance (PCMR) flow data. The feasibility of the proposed method was investigated in a numerical model of blood flow in the right pulmonary artery. Furthermore, it was validated experimentally using a flow phantom and compared with an existing invasive technique. Finally, the feasibility of the method was tested in a study of the right pulmonary artery of a volunteer. This approach may provide a non-invasive method to evaluate PAH and its response to therapy.

APLF (Aprataxin and Polynucleotide kinase-Like Factor), a novel protein with a forkhead-associated (FHA) domain and two poly(ADP-ribose)-binding zinc fingers (PBZ), interacts with core non-homologous end-joining (NHEJ) repair factors, Ku and XRCC4-DNA ligase IV, and facilitates NHEJ. However, how APLF functions in NHEJ is undefined. This thesis demonstrates that the Ku-binding domain on APLF is mapped to amino acid residues 180-200, where conserved amino acid residue W189 strongly contributes to the APLF-Ku interaction. Remarkably, the APLF-Ku interaction is involved in the nuclear localization of APLF. Furthermore, we demonstrate that the N-terminal region (amino acids 1-200), containing the XRCC4-Ligase IV and Ku binding domains, is required for APLF- dependent NHEJ. Collectively, these findings suggest that Ku contributes to APLF nuclear localization, and that once APLF is retained in the nucleus, the N-terminal portion of APLF, which facilitates interactions with the core NHEJ proteins Ku and XRCC4-DNA ligase IV, is required for efficient NHEJ.

Effective early detection and monitoring of patient response to cancer therapy is important for improved patient outcomes, avoiding unnecessary procedures and their associated toxicities, as well as the development of new therapies. Dynamic contrast-enhanced magnetic resonance imaging shows promise as a way to evaluate tumour vasculature and assess the efficacy of new anti-angiogenic drugs. However, unavoidable patient motion can decrease the accuracy of subsequent analyses rendering the data unusable. Motion correction algorithms are challenging to develop for contrast-enhanced data since intensity changes due to contrast-enhancement and patient motion must somehow be differentiated from one another. A novel method is presented that employs a motion-informed intensity correction in order to facilitate the registration of contrast enhanced data. The intensity correction simulates the presence or absence of contrast agent in the image volumes to be registered in an attempt to emulate the level of contrast-enhancement present in a single reference image volume.

Therapeutic efficacy of drugs depends on their ability to reach the treatment target. Drugs that exert their effect within cells are constrained by an inability to cross the cell membrane. Methods are being developed to overcome this barrier including biochemical and biophysical strategies. The application of ultrasound with microbubbles increases the permeability of cell membranes allowing molecules, which otherwise would be excluded, to enter the intracellular space of cells; a phenomenon known as sonoporation. This thesis describes studies aimed at improving our understanding of the mechanism underpinning sonoporation and of the exposure parameters affecting sonoporation efficiency. Cancer cells (KHT-C) in suspension were exposed to ultrasound and microbubbles – total of 97 exposure conditions. The effects on cells were assessed through uptake of cell-impermeable molecules (10 kDa to 2 MDa FITC-dextran), cell viability and microscopic observations of the plasma membrane using flow cytometry, colony assay and electron microscopy techniques. Sonoporation was a result of the interaction of ultrasound and microbubbles with the cell membrane. Disruptions (30-100 nm) were generated on the cell membrane allowing cell impermeable molecules to cross the membrane. Molecules up to 2 MDa in size were delivered at high efficiency (~70% permeabilisation). Sonoporation was short lived; cells re-established their barrier function within one minute, which allowed compounds to remain inside the cell. Following uptake, cells remained viable; ~50% of sonoporated cells proliferated. Sonoporation efficiency depended on ultrasound and microbubble exposure conditions. Microbubble disruption was a necessary but insufficient indicator of ultrasound-induced permeabilisation. The exposure conditions can be tailored to achieve a desired effect; cell permeability of ~70% with ~25% cell death versus permeability of ~35% with ~2% cell death. In addition, sonoporation depended on position in the cell cycle. Cells in later stages were more prone to being permeabilised and killed by ultrasound and microbubbles. This study indicated that sonoporation can be controlled through exposure parameters and that molecular size may not be a limiting factor. However, the transient nature may necessitate that the drug be in close vicinity to target cells in sonoporation-mediated therapies. Future work will extend the investigation into in vivo models.

Contrast enhanced ultrasound offers a unique method to measure the blood flow, perfusion, vascular volume and morphology of microvascular networks. This is achieved by exploiting the ability of microbubble contrast agents to be disrupted and preferentially detected with contrast specific imaging techniques – using a method known as disruption-replenishment. In its current form, disruption-replenishment suffers from poor reproducibility and accuracy, largely due to the inappropriate application of a mono-exponential model of microbubble replenishment, and an incomplete understanding of the dependencies of the measurement. In this thesis, we hypothesize that disruption-replenishment measurements can be improved by applying a perfusion model that considers the physical elements of the measurement, including the haemodynamics and morphology of the vascular system, the ultrasound field distribution and microbubble properties. We present a flexible, theoretical framework to model microbubble replenishment within the microvasculature. The replenishment model is further developed by in vitro and in vivo validation, and clinical translation in a trial of anti-angiogenic therapy in patients, resulting in a proposed clinical protocol. The presented formalism was shown to be more robust and demonstrated better agreement of both fitting quality and estimates of flow velocity when compared to the established model (accuracy to within 3-9%). The reproducibility of repeated in vivo disruption-replenishment flow measurements was 11.9% using the proposed perfusion model compared to 24% using the established model. Variability of clinical perfusion measurements was also reduced with a method that discards the contribution of flow from larger arteries. Excluding the large vessel component in clinical measurements of tumour blood volume decreased the inter-plane variability by up to 20%. The proposed perfusion model can be used to generate parametric maps of vascularity through which additional quantitative parameters become available. These improvements will help translate the method of disruption-replenishment into routine clinical practice and clinical trials.

The orphan nuclear receptor EAR-2 (NR2F6) is a gene that I previously found to be expressed at a higher level in clonogenic leukemia single cells than in leukemia cells that can not divide. For this thesis I undertook to perform the first investigations of the roles EAR-2 may play in normal haematopoiesis and in the pathogenesis of acute myelogenous leukaemia. Here, I show that EAR-2 is overexpressed in the bone marrow of patients with MDS, AML and CMML compared to healthy controls and that EAR-2 is a gatekeeper to hematopoietic differentiation. Over-expression of EAR-2 prevents the differentiation of cell lines, while knock down induces their spontaneous differentiation. In vitro, primary bone marrow cells that over-express EAR-2 do not differentiate into granulocytes in suspension culture, but have greatly extended replating capacity in colony assays. In vivo, overexpression of EAR-2 in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome characterised by hypercellular bone marrow, an increase in blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice. Furthermore, animals that are transplanted with grafts of sorted bone marrow develop a rapidly fatal leukemia that is characterized by pancytopenia, enlargement of the spleen, infiltration of blasts into the spleen, liver and peripheral blood. Interestingly, development of leukemia is preceded by expansion of the stem cell compartment. Overexpression of EAR-2 increases the maintenance of KSL primitive bone marrow cells in ex vivo suspension culture, while knockdown of EAR-2 induces rapid differentiation of KSL cells into granulocytes. These data establish that EAR-2 is a novel oncogene that regulates hematopoietic cell differentiation. Furthermore, I show that EAR-2 is also a novel negative regulator of T-cell lymphopoiesis, and demonstrate that down-regulation of EAR-2 is important for the survival, proliferation and differentiation of T-cell progenitors. Overall, this work establishes that expression of EAR-2 is an important determinant of cell fate decisions in the hematopoietic system.

Heart failure is a complex clinical syndrome in which the heart cannot pump sufficient blood and nutrients to the organs in the body. Increasingly, alterations in cardiac energetics are being implicated as playing an important role in the pathogenesis of heart failure. An understanding of specific metabolic switches which occur during the development of heart failure in patients would be greatly beneficial as a new diagnostic method and for the development of new therapies for patients with failing hearts. This thesis deals with the non-invasive assessment of metabolism in the heart. New magnetic resonance imaging (MRI) methods for metabolic characterization of the heart using hyperpolarized carbon-13 MRI are presented. Spatially resolved images of hyperpolarized 13C substrates and their downstream products can provide insight into real-time metabolic processes occurring in vivo, within minutes of injection of a pre-polarized 13C-labeled substrate. Conventional 3D spectroscopic acquisitions require in excess of 100 excitations, making it challenging to acquire full cardiac and respiratory-gated, whole-heart metabolic volumes. Each of the developments described in this thesis is intended to advance cardiac hyperpolarized 13C metabolic imaging towards a routine, clinical exam which can be used for prognosis and treatment optimization in patients with cardiovascular disease. The major technical development is a new interleaved-frequency, time-resolved MRI pulse sequence that can provide robust and reliable measurements of cardiac metabolic signals. The technique was applied to several realistic pre-clinical models of cardiac disease and the work presented will hopefully lead towards significant improvement in the management of patients with heart failure.

Pulmonary arterial hypertension (PAH) alters pulmonary hemodynamics by changing the vascular wall mechanics. Currently, pulmonary vascular resistance is used clinically to assess the disease. However, the invasive nature of pressure measurements, needed for calculating the resistance, prevents longitudinal monitoring of patients during therapy. This work employs wave reflection as an alternative measure of the downstream stiffness and proposes a new method for wave reflection assessment using only non-invasive phase contrast magnetic resonance (PCMR) flow data. The feasibility of the proposed method was investigated in a numerical model of blood flow in the right pulmonary artery. Furthermore, it was validated experimentally using a flow phantom and compared with an existing invasive technique. Finally, the feasibility of the method was tested in a study of the right pulmonary artery of a volunteer. This approach may provide a non-invasive method to evaluate PAH and its response to therapy.