This thesis describes a novel imaging methodology for visualization and quantitative analysis of the vascular topology of different cortical regions of the mouse brain in 3D. The brain is perfused with a fluorescent contrast agent, and rendered fully transparent via optical clearing. This procedure enables images through the whole cortical depth to be obtained with a 2-photon microscope without sectioning. The Allen Reference Atlas (ARA) (Lein et al., 2007) is registered to the 2-photon data for delineation of the cortical regions. Quantitative metrics are then extracted from the different regions using an automatic vessel segmentation algorithm. These metrics are compared with those obtained by other investigators to validate this technique, and are found to be in agreement. Since this methodology possesses the resolution to visualize vessels of all sizes, and provides reasonable estimates of quantitative parameters, it shows strong potential for quantitative analysis of normal and abnormal cortical vascular architecture.

This thesis describes a novel imaging methodology for visualization and quantitative analysis of the vascular topology of different cortical regions of the mouse brain in 3D. The brain is perfused with a fluorescent contrast agent, and rendered fully transparent via optical clearing. This procedure enables images through the whole cortical depth to be obtained with a 2-photon microscope without sectioning. The Allen Reference Atlas (ARA) (Lein et al., 2007) is registered to the 2-photon data for delineation of the cortical regions. Quantitative metrics are then extracted from the different regions using an automatic vessel segmentation algorithm. These metrics are compared with those obtained by other investigators to validate this technique, and are found to be in agreement. Since this methodology possesses the resolution to visualize vessels of all sizes, and provides reasonable estimates of quantitative parameters, it shows strong potential for quantitative analysis of normal and abnormal cortical vascular architecture.

TNF is a proinflammatory cytokine whose pleiotropic biological properties are signaled through the receptor TNFR1. Activation of this signaling pathway has been implicated in a broad range of biological functions, including host defense, inflammation, apoptosis, autoimmunity, and cancer. TRADD is an adaptor protein that is recruited to TNFR1 upon receptor engagement. Using a Tradd-deficient murine model, we demonstrated that TRADD is essential for both TNF-mediated apoptosis and inflammatory responses. In addition to refining the role of TRADD in TNFR1 signaling, we have also identified a novel function of TRADD in TLR3 and TLR4 pathways, which are key drivers of the innate immune response. We showed that TRADD is involved in NF-κB activation upon TLR3 and TLR4 stimulation, and Tradd-deficient macrophages showed impaired inflammatory cytokine production in response to TLR ligands in vitro. These data reveal the multifaceted functions of TRADD in immune signaling pathways. Beyond its role in the immune response, TNF has also been shown to play a crucial, cell-non-autonomous role in driving tumor growth in various models of cancer. We initially sought to determine whether TRADD is essential for this aspect of TNF function by employing the use of a chemical induced skin carcinogenesis model in which the tumor-promoting role of TNF is very well established. In this model, H-Ras is the major driving oncogene. We found that Tradd deficiency accelerated tumor formation in mouse skin, in strong contrast to what was observed in Tnfr1-deficient mice. Further in vitro analyses revealed that upon expression of oncogenic H-Ras, Tradd-deficient murine fibroblasts displayed both reduced cell cycle arrest and repression of Ras induced cellular senescence. Importantly, the level of p19Arf induced by H-Ras expression was reduced in Tradd-deficient fibroblasts in a post-translational manner. Our biochemical evidence suggests that TRADD can shuttle dynamically between the cytoplasm and the nucleus; in doing so, nuclear TRADD interacts with ULF, a newly identified E3 ubiquitin ligase for p19Arf. Interaction between nuclear TRADD and ULF sequesters ULF away from p19Arf, leading to p19Arf stabilization and tumor suppression. Together, these data demonstrate the functional diversity of TRADD in different compartments of the cell.

Improving stroke recovery is a topic of considerable interest in the developed world. Motor deficits following stroke can significantly impact quality of life, and motor rehabilitation strategies are urgently needed to promote brain recovery. One technique that has had therapeutic success in other domains is neurofeedback (NF), a strategy whereby a person is trained to gain volitional control of their neurological activity. The NF technique has proven efficacious in disorders ranging from epilepsy to attention deficit disorders based on feedback of electroencephalography (EEG) signals, and the potential exists for NF applications in the recovery of motor function following stroke. Using functional magnetic resonance imaging (fMRI), NF can be applied to precisely defined regions of interest (ROIs), facilitating the targeted treatment of affected functional areas. The combination of fMRI and NF is still relatively new, and much work remains in the characterization and optimization of fMRI NF strategies. In a two-pronged approach, this thesis focuses on the application of ROI-based NF to the motor system using traditional fMRI measurements and also the development and analysis of acquisition strategies intended for use in ROI-based NF. First, a study of the mechanisms governing the successful application of NF in primary motor cortex ROIs is presented, using kinaesthetic motor imagery (imagining the sensation and execution of movement) to engage the motor system. Second, an investigation of fMRI signal contrast enhancement properties using multi-echo fMRI acquisition methods in dense sampling regimes is considered. Third, a novel acquisition method is introduced, designed using parallel MRI principles to provide fast and detailed sampling of fMRI signals in selected ROIs, called Constrained Source Space Imaging (CSSI). To conclude, the potential future directions for fMRI NF research and the CSSI technique are discussed, including thoughts toward the continued development of NF as a potential motor therapy for stroke patients.

A single application of cyclic compression to bioengineered cartilage improves tissue formation through cell shape changes that are mediated by α5β1 integrin and membrane-type metalloprotease (MT1-MMP). To determine if this response is controlled by calcium, we investigated how calcium regulated cell shape changes, MT1-MMP and integrin activity in response to stimulation. Stimulation-induced changes in cell shape and MT1-MMP expression were abolished with chelation of extracellular calcium, and reinstated with its re-introduction. Spreading and retraction were inhibited by blocking the stretch-activated and L-Type voltage-gated channels, respectively; channel blocking also inhibited MT1-MMP upregulation. Channels’ role was confirmed through treatment with calcium A23187 ionophore, which alleviated the effects of channel blocking. Calcium regulated the integrin-mediated signalling pathway, which was facilitated through the kinase Src. Both calcium- and integrin-mediated pathways converged on activating ERK in response to stimulation. Understanding the molecular mechanisms regulating chondrocyte mechanotransduction may lead to the development of improved bioengineered cartilage.

Glucocorticoids (GCs) are commonly used in the clinic as a treatment for Chronic Lymphocytic Leukemia (CLL). The exact mechanism of GC action remains unclear and patients eventually develop resistance to this group of agents. Our findings show that GC-cytotoxicity in circulating CLL cells is caused by bioenergetic restriction resulting from the down-regulation of a key glycolytic enzyme, pyruvate kinase, muscle isozyme 2 (PKM2). Conversely, GCs were shown to promote fatty acid oxidation instead by up-regulating the expression of peroxisome proliferator activated receptor α (PPARα). These findings establish PPARα and fatty acid oxidation as novel mediators of GC resistance in CLL. Our findings also demonstrate that GCs enhance the cytotoxic effects of membrane-damaging agents such as ionophores and complement-mediated cytotoxicity. A clinically relevant agent known to intercalate in the cell membrane, Danazol was also found to have activity against CLL and can be combined safely with GCs for enhanced treatment efficacy.

In clinical oncology and experimental therapeutics, changes in tumour growth rate or volume have been traditionally the first indication of treatment response. These changes typically occur late in the course of therapy. Currently, no routinely available imaging modality is capable of assessing tumour response to cancer treatment within hours or days after delivery of radiation treatment. Therefore, the goal of this thesis is to develop the use of ultrasound imaging and ultrasound characterization methods with frequencies of 10 to 30 MHz to assess non-invasively tumour response to radiotherapy, early, within hours to days after delivery of radiotherapy. Responses to radiotherapy were characterized initially in vitro in a well-controlled environment using cell samples. It was demonstrated experimentally that the changes in ultrasound integrated backscatter and spectral slopes were the direct consequences of cell and nuclear morphological changes associated with cell death. The research in vitro provided a basis for the in vivo research that characterized responses to radiotherapy in cancer mouse models. The results from mouse tumour models indicated that quantitative ultrasound could detect the regions in a tumour that corresponded in histology to areas of cell death. In order to understand the cellular morphological changes responsible for ultrasound scattering at these frequencies and assist in the interpretation of experimental data, numerical simulations of ultrasound scattering from four different cell lines exposed to radiotherapy were conducted and compared to experimental results. It was concluded that the increases measured in ultrasound backscatter could in part be explained by the increase in the randomization of cell nuclei resulting from the increase in the variance of cell sizes following cell death. In this thesis, it is demonstrated that ultrasound imaging and quantitative ultrasound methods were able to detect non-invasively early responses to radiotherapy in vitro and in vivo. The mechanism behind this detection was linked to changes in the acoustic properties of nuclei and changes in the spatial organization of cells and nuclei following cell death. This provides the groundwork for future investigations regarding the use of ultrasound in cancer patients to individualize treatments non-invasively based on their responses to specific interventions.

Acute and chronic hypoxia exists within the microenvironment of solid tumours and drives therapy resistance, genetic instability and metastasis. Despite its importance in solid tumour progression, very little is known regarding the functional consequences of hypoxia-mediated changes in the expression of DNA repair proteins. I studied the relationship between hypoxia and DNA repair using a prolonged chronic hypoxic gas treatment model in a variety of human tumour cell lines to mimic the dynamic state of proliferation and DNA repair in cells distant from the tumour blood vasculature. I observed decreased expression of homologous recombination (HR) and base excision repair (BER) proteins due to a novel mechanism involving decreased protein synthesis. Error-free HR was suppressed 3-fold under 0.2% O2 as measured by the DR-GFP reporter system and functional BER was impaired as assessed with a functional glycosylase assay. This decrease in protein expression and function resulted in increased sensitivity to the DNA damaging agents MMC, cisplatin, H2O2 and MMS. Additionally, chronically hypoxic cells were relatively radiosensitive (OER = 1.37) when compared to acutely hypoxic or anoxic cells (OER = 1.96 - 2.61). As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, I evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. I observed increased clonogenic killing in HR-deficient hypoxic cells following inhibition or depletion of PARP1. PARP-inhibited hypoxic cells accumulated γH2AX foci consistent with an accumulation of collapsed replication forks. Additionally, tumour xenografts exposed to PARP1 inhibition showed increased γH2AX and cleaved caspase-3 expression in hypoxic subregions with suppressed RAD51 protein expression and decreased ex vivo clonogenic survival. I conclude that persistent down-regulation of DNA repair components by the microenvironment could result in faulty DNA repair with significant implications for therapeutic response and genetic instability in human cancers. Specifically, hypoxic cells may be sensitized to PARP inhibitors and other agents targeting repair pathways down-regulated by hypoxia as a consequence of microenvironment-mediated “contextual synthetic lethality”.

TNF is a proinflammatory cytokine whose pleiotropic biological properties are signaled through the receptor TNFR1. Activation of this signaling pathway has been implicated in a broad range of biological functions, including host defense, inflammation, apoptosis, autoimmunity, and cancer. TRADD is an adaptor protein that is recruited to TNFR1 upon receptor engagement. Using a Tradd-deficient murine model, we demonstrated that TRADD is essential for both TNF-mediated apoptosis and inflammatory responses. In addition to refining the role of TRADD in TNFR1 signaling, we have also identified a novel function of TRADD in TLR3 and TLR4 pathways, which are key drivers of the innate immune response. We showed that TRADD is involved in NF-κB activation upon TLR3 and TLR4 stimulation, and Tradd-deficient macrophages showed impaired inflammatory cytokine production in response to TLR ligands in vitro. These data reveal the multifaceted functions of TRADD in immune signaling pathways. Beyond its role in the immune response, TNF has also been shown to play a crucial, cell-non-autonomous role in driving tumor growth in various models of cancer. We initially sought to determine whether TRADD is essential for this aspect of TNF function by employing the use of a chemical induced skin carcinogenesis model in which the tumor-promoting role of TNF is very well established. In this model, H-Ras is the major driving oncogene. We found that Tradd deficiency accelerated tumor formation in mouse skin, in strong contrast to what was observed in Tnfr1-deficient mice. Further in vitro analyses revealed that upon expression of oncogenic H-Ras, Tradd-deficient murine fibroblasts displayed both reduced cell cycle arrest and repression of Ras induced cellular senescence. Importantly, the level of p19Arf induced by H-Ras expression was reduced in Tradd-deficient fibroblasts in a post-translational manner. Our biochemical evidence suggests that TRADD can shuttle dynamically between the cytoplasm and the nucleus; in doing so, nuclear TRADD interacts with ULF, a newly identified E3 ubiquitin ligase for p19Arf. Interaction between nuclear TRADD and ULF sequesters ULF away from p19Arf, leading to p19Arf stabilization and tumor suppression. Together, these data demonstrate the functional diversity of TRADD in different compartments of the cell.

Improving stroke recovery is a topic of considerable interest in the developed world. Motor deficits following stroke can significantly impact quality of life, and motor rehabilitation strategies are urgently needed to promote brain recovery. One technique that has had therapeutic success in other domains is neurofeedback (NF), a strategy whereby a person is trained to gain volitional control of their neurological activity. The NF technique has proven efficacious in disorders ranging from epilepsy to attention deficit disorders based on feedback of electroencephalography (EEG) signals, and the potential exists for NF applications in the recovery of motor function following stroke. Using functional magnetic resonance imaging (fMRI), NF can be applied to precisely defined regions of interest (ROIs), facilitating the targeted treatment of affected functional areas. The combination of fMRI and NF is still relatively new, and much work remains in the characterization and optimization of fMRI NF strategies. In a two-pronged approach, this thesis focuses on the application of ROI-based NF to the motor system using traditional fMRI measurements and also the development and analysis of acquisition strategies intended for use in ROI-based NF. First, a study of the mechanisms governing the successful application of NF in primary motor cortex ROIs is presented, using kinaesthetic motor imagery (imagining the sensation and execution of movement) to engage the motor system. Second, an investigation of fMRI signal contrast enhancement properties using multi-echo fMRI acquisition methods in dense sampling regimes is considered. Third, a novel acquisition method is introduced, designed using parallel MRI principles to provide fast and detailed sampling of fMRI signals in selected ROIs, called Constrained Source Space Imaging (CSSI). To conclude, the potential future directions for fMRI NF research and the CSSI technique are discussed, including thoughts toward the continued development of NF as a potential motor therapy for stroke patients.