The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

The post-translational modifications (PTMs) of histones regulate many cellular processes including transcription, replication, DNA repair, recombination, and chromosome segregation. A large number of combinations of PTMs are possible, with methylation being one of the most complex, since it is found in three states and is recognized in a sequence specific context. Methylation of histones at key lysine residues has been shown to work in concert with other modifications to provide a Histone Code that may determine heritable transcriptional conditions in normal and disease states. On the most basic level it is pivotal to understand how and by which proteins the numerous PTMs are recognized, as well as mechanisms for downstream signal propagation. To address this need we developed a high-throughput method that allows analysis of up to 600 PTMs in a single experiment. This approach was utilized to characterize macromolecules interacting with the specific modifications on histone tails and to screen for the marks that bound to Malignant Brain Tumor (MBT) proteins, important chromatin regulators implicated in cancer. All MBTs recognized either mono- or dimethyllysine histone marks, and using structure-based mutants we identified a triad of residues that were responsible for this discrimination. These results provide the foundation for the rational design of highly selective MBT inhibitors. Additionally, this thesis describes combinatorial recognition of histone modifications, as proposed in the original Histone Code hypothesis. We demonstrate that Tudor domains of UHRF1, a protein involved in epigenetic maintenance of DNA methylation, is able to read a dual modification state of histone H3 in which it is trimethylated at lysine 9 and unmodified at lysine 4. This study provides an elegant example of the combinatorial readout of histone modification states by a single domain. Together, our findings offer mechanistic insights into the recognition of methylated histone tails by MBT domains and Royal Family in general.

An all-optical micromanipulation technique is presented in the framework of precise cell selection within a cell culture and multiplexed transport capabilities for microfluidic single cell analysis applications. The technique was developed by combining an optical tweezer setup with a novel integrated waveguide cell propulsion method referred to as end-face waveguide propulsion (EFWP). The EFWP technique delivers optical forces to a particle generating thrust. The thesis is divided into two sections: simulation and experimental validation. In the first section a new simulation technique based on ray optics theory (ROT) and the beam propagation method (BPM) is used to predict particle velocity and trajectory along a microfluidic propagation channel. In this work, the ROT-BPM technique is used to analyse and optimize the waveguide geometry to maximize particle velocity. Analysis of the impact of common microchip manufacturing limitations on velocity is performed to determine acceptable fabrication process tolerances. The second section presents experimental results of polymer microspheres and acute myeloid leukemia (AML) cells as biological targets. The experimental results are compared with simulations performed in the first section. Correction factors are added to the simulations to reflect the experimental device parameters. Thermal e_ects due to photon absorption within the fluidic channels are also investigated and corrected for. The final analysis indicates that the ROT-BPM technique developed in this work can be used to adequately predict particle velocity and trajectory path. EFWP currently delivers the fastest particle velocities compared to other optical micromanipulation techniques currently available in microfluidic applications. While the technique is focused on addressing chemical cytometry precise particle selectivity and high throughput needs, EFWP can also be used in many other single cell applications.

Imaging techniques provide a method for non-invasive longitudinal monitoring of cancer therapies, but common metrics such as tumour size are late markers and do not indicate heterogeneity of response. Apoptotic cell death is an earlier marker of tumour response and produces molecular and cellular-level changes (macromolecular breakdown, membrane changes and cell shrinkage) that may be detectable by magnetic resonance imaging (MRI). Previous studies using conventional MRI methods have shown little sensitivity to apoptosis. In this thesis it is hypothesized that, using an extracellular contrast agent to affect the MRI property of relaxation for extracellular water preferentially, parameters related to water in the intracellular and extracellular environments and the exchange between them can be obtained and will be sensitive to apoptosis. It is also hypothesized that membrane changes and macromolecular breakdown are detectable by the technique of magnetization transfer. Measurements of relaxation in the presence of contrast agent in vitro demonstrated a decrease in extracellular water fraction and an increase in the rate of water exchange across the plasma membrane during apoptosis. In vivo, this method was complicated by the difficulty of delivering contrast agent to the tumour, but regions with good delivery showed correlation between high water exchange rates from MRI and apoptosis in histology. Magnetization transfer studies indicated only small changes in vitro during apoptosis and these were largely related to changes in the free water, so this method was not investigated further. Further work is required to determine the tumour lines where the water exchange methods may be applied reliably. Nevertheless, the method of measuring water exchange presented in this thesis can be performed in a clinically-feasible amount of time (~20 minutes). It therefore has potential in detecting apoptosis and predicting therapy response. It also emphasizes the role of water exchange in conventional MRI relaxation experiments.

The tumour suppressor p53-binding protein 1 (53BP1) is phosphorylated following DNA double strand breaks (DSBs); however, little is understood about the upstream signaling pathways that control this phosphorylation. Additionally, it is not known how these processes combine with 53BP1 to control the survival of cells following DNA damage such as that imparted by ionizing radiation (IR), which is the basis of radiotherapy. In this thesis, I have shown that 53BP1 is phosphorylated specifically in S-phase cells, but not relocalized to intranuclear foci, in response to severe oxygen stress. This occurs with only partial dependence on the ATM kinase (Chapter 2). Following IR, I find that both ATM and DNA-PKcs contribute to intranuclear phosphorylated 53BP1 foci, but that this phosphorylation is independent of proximal signaling molecules that control the localization of 53BP1 to initial DSBs (Chapter 3). Furthermore, I show that 53BP1 loss confers sensitivity to IR and this can be further augmented by inhibition of ATM and DNA-PKcs kinases suggesting that there are both 53BP1-dependent and -independent pathways of survival from IR (Chapter 4). These findings may have important implications for molecular pathology and personalized medicine as 53BP1 has recently been found to be activated or lost in subsets of human tumours. I have collaborated to initiate the development of a novel system to interrogate the implications of 53BP1 loss as traditional siRNA approaches in human cancer cells were not feasible (Chapter 5 and Appendix 2). This system can be used in vivo as tumour xenografts to further understand how 53BP1 and the tumour microenvironment interact endogenously and in response to IR. I also present the possibility and proof of concept for the use of 53BP1 as a biomarker in primary human prostate cancer tissue where little is known about 53BP1 biology (Chapter 5).

The goal of this thesis was to produce an animal model that develops atherosclerotic plaque featuring plaque neovascularization leading to intraplaque hemorrhage and is suitable for noninvasive imaging studies. Several strategies were tested for their effectiveness in producing such plaques in the rabbit aorta, including: a high cholesterol diet, vascular endothelial growth factor injections, therapeutic contrast ultrasound, and balloon catheter injury. It was found that a combination of the high cholesterol diet and balloon injury was able to achieve plaque neovascularization in a manner dependent on circulating plasma cholesterol levels. In addition, a contrast-enhanced magnetic resonance imaging technique implemented in the animal model was able to detect plaque neovascularization and monitor its change over time in a single group of animals. In conclusion, an animal model was created where plaque neovascularization occurs in a predictable fashion and can be studied with non-invasive magnetic resonance imaging.

Radiotherapy plays a significant role in cancer treatment, and is thought to be curative by mainly killing tumor cells through damage to their genetic material. However, recent findings indicate that the tumor’s vascular blood supply is also a major determinant of radiation response. The goals of this thesis are to: (1) develop an experimental platform for small animals to deliver ionizing radiation and perform high-resolution optical imaging to treatment targets, and (2) use this toolkit to longitudinally monitor the response of tumors and the associated vasculature. The thesis has achieved: (1) customization of a novel micro-irradiator for mice, (2) technical development of an improved optical coherence tomography imaging system, (3) comprehensive experimental protocol and imaging optimization for optical microscopy in a specialized animal model, and (4) completion of a feasibility study to demonstrate the capabilities of the experimental platform in monitoring the response of tumor and vasculature to radiotherapy.

A primary objective of modern biology is to understand the molecular mechanisms which underlie cellular functions and a crucial part of this task is the ability to manipulate and analyze individual cells. As a result of interdisciplinary research, microfluidics may become the forefront of analytical methods used by biologists. This technology can be used to gain unprecedented opportunities for cell handling, lysis and investigation on a single cell basis. This thesis presents the development of a microfluidic device capable of selecting individual cells and performing selective electrical lysis of the plasma membrane, while verifying intactness of the nuclear membrane. The device is fabricated by an improved photolithography method and integrates molten solder as electrodes for lysis by a DC electric field. Quantification of lysis is accomplished by video and image analysis, and measurement of the rate of ion diffusion from the cell.

Dynamic nuclear polarization and dissolution of 13C-labeled metabolite allows dynamic imaging of metabolism in-vivo. However, the spatial and temporal resolutions of magnetic resonance spectroscopic imaging are limited by the duration of free-induction decay acquisitions and the T1-based, non-recoverable polarization decay. This thesis describes the implementation of a spectral-spatial radiofrequency excitation pulse with a `flyback' echo-planar readout trajectory to dynamically image [1-13C]-pyruvate and [1-13C]-lactate in an interleaved manner. This technique excites a single resonance of either [1-13C]-pyruvate or [1-13C]-lactate and generates dynamic images with 5mm in-plane resolution. Metabolite dynamics extracted from the images and the corresponding non-localized spectroscopic data reveal similar kinetic rates upon fitting to a kinetic model. This demonstrates the feasibility of probing metabolism in heterogeneous tissues in-vivo with dynamic interleaved 13C MR imaging.

In vivo real-time data of ultrasound and microbubble assisted blood-brain barrier disruption is centrally based on low-resolution magnetic resonance images. Additional information can be gained using online microscopic monitoring. This study presents the first ever in vivo two-photon microscopy, four-dimensional data sets of ultrasound and microbubble assisted blood-brain barrier disruption. It characterized the threshold pressures and mechanical index needed to disrupt the vasculature with 800 kHz ultrasound, and found three different leakage constants from the compromised vasculature. Furthermore, using numerical models, an ultrasound array was designed and optimized to perform specifically with our two-photon microscope. It was fabricated, fully characterized, and its performance met both the required pressure field profile and the pressure values needed for our in vivo two-photon microscopy experiments. This array is an important step in microscopically characterizing ultrasound and microbubble assisted blood-brain barrier disruption.