We applied single-molecule fluorescence (SMF) methods to probe the properties of individual fluorescent probes, and to characterize the proteins of interest to which these probes were attached. One remarkable advantage of SMF spectroscopy is the ability to investigate heterogeneous subpopulations of the ensemble, which are buried in ensemble averaging in other measurements. Other advantages include the ability to probe the entire dynamic sequences of a single molecule transitioning between different conformational states. For the purpose of having an extended observation of single molecules, while maintaining the native nanoscale surroundings, we developed an improved vesicle preparation method for encapsulating scarce biological samples. SMF investigations revealed that molecules trapped in vesicles exhibit nearly ideal single-emitter behavior, which therefore recommends the vesicle encapsulation for reproducible and reliable SMF studies. Hyperactive Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein contributes significantly to human cancers, such as leukemia and lymphoma. We have proposed a novel therapeutic strategy by designing a cholesterol-based protein membrane anchor (PMA), to tether STAT3 to the cell membrane and thus inhibit unwanted transcription at the cell nucleus. We designed in vitro proof-of-concept experiments by encapsulating STAT3 and PMAs in phospholipid vesicles. The efficiency and the stability of STAT3 anchoring in the lipid membrane were interrogated via quantitative fluorescence imaging and multiparameter SMF spectroscopy. Our in vitro data paved the way for the in vivo demonstration of STAT3 inhibition in live cells, thus demonstrating that PMA-induced protein localization is a conceptually viable therapeutic strategy. The recent discovery of intrinsically disordered proteins (IDPs) highlights important exceptions to the traditional structure-function paradigm. SMF methods are very suited for probing the properties of such highly heterogeneous systems. We studied in detail the effects of electrostatics on the conformational disorder of an IDP protein, Sic1 from yeast, and found that the electrostatic repulsion is a major factor controlling the dimensions of Sic1. Based on our data we also conclude that a rod-like shape seems a better candidate than a random Gaussian chain to describe and predict the behavior of Sic1.

We applied single-molecule fluorescence (SMF) methods to probe the properties of individual fluorescent probes, and to characterize the proteins of interest to which these probes were attached. One remarkable advantage of SMF spectroscopy is the ability to investigate heterogeneous subpopulations of the ensemble, which are buried in ensemble averaging in other measurements. Other advantages include the ability to probe the entire dynamic sequences of a single molecule transitioning between different conformational states. For the purpose of having an extended observation of single molecules, while maintaining the native nanoscale surroundings, we developed an improved vesicle preparation method for encapsulating scarce biological samples. SMF investigations revealed that molecules trapped in vesicles exhibit nearly ideal single-emitter behavior, which therefore recommends the vesicle encapsulation for reproducible and reliable SMF studies. Hyperactive Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein contributes significantly to human cancers, such as leukemia and lymphoma. We have proposed a novel therapeutic strategy by designing a cholesterol-based protein membrane anchor (PMA), to tether STAT3 to the cell membrane and thus inhibit unwanted transcription at the cell nucleus. We designed in vitro proof-of-concept experiments by encapsulating STAT3 and PMAs in phospholipid vesicles. The efficiency and the stability of STAT3 anchoring in the lipid membrane were interrogated via quantitative fluorescence imaging and multiparameter SMF spectroscopy. Our in vitro data paved the way for the in vivo demonstration of STAT3 inhibition in live cells, thus demonstrating that PMA-induced protein localization is a conceptually viable therapeutic strategy. The recent discovery of intrinsically disordered proteins (IDPs) highlights important exceptions to the traditional structure-function paradigm. SMF methods are very suited for probing the properties of such highly heterogeneous systems. We studied in detail the effects of electrostatics on the conformational disorder of an IDP protein, Sic1 from yeast, and found that the electrostatic repulsion is a major factor controlling the dimensions of Sic1. Based on our data we also conclude that a rod-like shape seems a better candidate than a random Gaussian chain to describe and predict the behavior of Sic1.

Cervical cancer is the third most common cancer among women worldwide, and the fourth leading cause of cancer mortality. Despite significant declines in the incidence and mortality rates of cervical cancer in Canada, it remains the 4th most common cancer in women aged 20-29 years. In order to gain novel insights into cervical cancer tumourigenesis and clinical outcome, we investigated and characterized the alterations in microRNA (miRNA) expression in this disease. Firstly, we performed global miRNA expression profiling of cervical cancer cell lines (n=3), and patient specimens (n=79). From this analysis, we identified miR-196b to be significantly down-regulated in cervical cancer, and characterized its role in regulating the HOXB7~VEGF axis. The global miRNA expression data also led to the development of a candidate 9-miRNA signature that was prognostic for disease-free survival in patients with cervical cancer, although we were unable to validate this signature in an independent cohort. This report describes important considerations concerning the development and validation of microRNA signatures for cervical cancer. Our investigations also led us to a comparison of three methods for measuring miRNA abundance: the TaqMan Low Density Array, the NanoString nCounter assay, and single-well quantitative real-time PCR. Our findings demonstrated limited concordance between the TLDA and NanoString platforms, although each platform correlated well with PCR, which is considered the gold standard for nucleic acid quantification. Furthermore, we examined biases created by amplification protocols for microarray studies. Our analysis demonstrated that performing a correction using the LTR-method (linear transformation of replicates) could help mitigate, but not completely eliminate such biases. Overall, this report presents insights into the role of miRNAs in cervical cancer, as well as an evaluation of technical considerations concerning miRNA and mRNA expression profiling studies.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.

Bilateral implantation of a cochlear implant (CI) after a >2 year period of unilateral hearing with a second implant has been shown to result in altered latencies in brainstem responses in children with congenital deafness. In this thesis, a neural source localization method was developed to investigate the effects of unilateral CI use on cortical development after the implantation of a 2nd CI. The electroencephalography (EEG) source localization method is based on the linearly constrained minimum variance (LCMV) vector beamformer and utilizes null constraints to minimize the electrical artifact produced by the CI. The accuracy of the method was assessed and optimized through simulations and comparisons to beamforming with magnetoencephalography (MEG) data. After using cluster analyses to ensure that sources compared across subjects originate from the same neural generators, a study was done to examine the effects of unilateral CI hearing on hemispheric lateralization to monaural responses. It was found that a >2 year period of unilateral hearing results in expanded projections from the 1st implanted ear to the contralateral auditory area that is not reversed by implantation of a 2nd CI. A subsequent study was performed to examine the effects of unilateral CI hearing on the contributions of the 1st and 2nd implanted ears to the binaural response. It was found that in children with > 2 years of unilateral hearing, the binaural response is dominated by the 1st implanted ear. Together, these results suggest that the delay between the 1st and 2nd CI should be minimized in bilateral implantation to avoid dominance of auditory pathways from the 1st implanted ear. This dominance limits developmental competition from the 2nd CI and potentially contributes to poorer performance in speech detection in noise tasks.

Microbubble contrast agents for ultrasound are widely used in numerous medical applications, both diagnostic and therapeutic. Due to their size, similar to that of red blood cells, microbubbles are able to traverse the entire vascular bed, enabling their utilization for applications such as tumour diagnosis. Vaporizable submicron droplets of liquid perfluoro- carbon potentially represent a new generation of extravascular contrast agents for ultrasound. Droplets of a few hundred nanometers in diameter have the ability to extravasate selectively in regions of tumour growth while staying intravascular in healthy tissues. Upon extravasation, these droplets may be vaporized with ultrasound and converted into gas bubbles. In this thesis we argue that vaporized submicron perfluorocarbon droplets possess the necessary stability and acoustic characteristics to be potentially applicable as a new gener- ation of extravascular ultrasound contrast agents. We examine, separately, the ultrasound conditions necessary for vaporization of the droplets into microbubbles, the size and stability of these bubbles following vaporization, on timescales ranging from nanoseconds to minutes, and the bubbles’ acoustic response to incident diagnostic ultrasound. We show that submicron droplets may be vaporized into bubbles of a few microns in diameter using single ultrasound pulse within the diagnostic range. The efficiency of conversion is shown to be on the order of at least 10% of the exposed droplets converting into stable microbubbles. The bubbles are shown to be stabilized by the original coating material encapsulating the droplet precursors, and be stable for at least minutes following vaporization. Finally, vaporized droplets are shown to be echogenic, with acoustic characteristics comparable to these of the commercially available ultrasound contrast agents. The results presented here show that vaporized droplets possess the necessary stability properties and echogenicity required for successful application as contrast agents, suggesting potential for their future translation into clinical practice.

Obesity is an adverse factor in the development and severity of breast cancer. Obesity is often accompanied by an increase in circulating insulin, which is also associated with poor BC prognosis. Although not expressed in normal breast tissue, the insulin receptor (IR) is highly expressed in BC, therefore insulin signaling in BC cells may be responsible for the negative prognostic effects associated with hyperinsulinemia. This thesis describes the development of a cell-based system to study how insulin affects BC. My work shows that MCF10A, untransformed human breast epithelial cells that express the IR, require insulin for normal proliferation and morphology. Interestingly, I discovered hyperactivation of ERK1/2 in MCF10A cells in response to insulin withdrawal, resulting in a loss of epithelial phenotype. Unexpectedly, while losing epithelial phenotype, MCF10A cells depleted of insulin failed to migrate. In conclusion, breast cells that express IR require insulin for migration and maintenance of epithelial characteristics.

Monitoring treatment efficacy is a large area of cancer research as it can increase the effectiveness of therapy regimens. Diffusion weighted Magnetic Resonance imaging (DWI), allows assessment of tissue microstructure without exogenous contrast agents. In this thesis, two different DWI techniques were used to acquire data from acute myeloid leukemia cells undergoing apoptosis, and data was fitted to an analytical model of re- stricted diffusion. Results indicated a decrease in average restriction size from 6.4 to 2.7μm, and an increase in the restricted diffusion coefficient from 0.17 to 0.82μm^2/ms in untreated versus treated cells. The free diffusion coefficient was constant indicating changes in restrictions, rather than any intrinsic changes in the intra-cellular or extra- cellular fluid. This combination of techniques has the potential for use in preclinical and clinical settings as it demonstrates that apoptotic changes may be measured consistently.

Our cells are constantly dealing with DNA damage generated by endogenous cellular activity (e.g. DNA replication) and exogenous agents (e.g. ultraviolet and ionizing radiation (IR)). The cellular stress response to DNA damage requires strict co-ordination between cell cycle checkpoint control and DNA repair. In response to DNA double-strand breaks (DNA-dsbs), members of the phosphatidylinositol 3-kinase–related kinase family (e.g. ATM and DNA-PKcs kinases) have been shown to redundantly phosphorylate substrates including the DNA-dsb marker, gamma-H2AX, and the p53 tumour suppressor protein. The p53 protein is best known as the guardian of the genome through its transcriptional-dependent and -independent functions. Despite a clear link between ATM-dependent phosphorylation of p53 with cell cycle checkpoint control and various modes of DNA damage repair, the intracellular biology and sub-cellular localization of p53 and specifically its phosphoforms during DNA damage induction and repair remains poorly characterized. Using G0/G1 confluent primary human diploid fibroblast cultures, this thesis shows that endogenous p53, phosphorylated at serine 15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following the induction of DNA breaks. This biologically distinct sub-pool of p53Ser15 is ATM-dependent and resistant to 26S-proteasomal degradation. p53Ser15 co-localizes and co-immunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA-dsb rejoining. Sub-nuclear microbeam irradiation studies confirm that p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATMSer1981 and DNA-PKcsThr2609 in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX-association and altered DNA-dsb kinetics following DNA damage. We further hypothesized that the non-specific DNA binding activity of the p53 carboxy-terminus mediates chromatin anchoring at sites of DNA damage. YFP-p53 fusion constructs expressing carboxy-terminus deletion mutants of p53 were transfected into p53-null H1299 cells to determine the role of the carboxy-terminus in chromatin-binding pre- and post-IR, independent of transcriptional activity. Within this isogenic human cell system, we observed exogenous YFP-p53WT associated with ATMSer1981 and 53BP1 within cellular chromatin in a dynamic manner. We confirmed that these associations also occurred between endogenous WTp53 with ATMSer1981 and 53BP1 within the chromatin of primary human diploid fibroblasts. YFP-p53del1-299 fusion proteins, which lack transcriptional activity and the Ser15-residue, also associated within chromatin. Ser15-phosphorylation was found not to be essential for DNA damage-induced association of p53 with chromatin or with ATMSer1981 and 53BP1. These data suggest a unique biology for p53Ser15 phosphoforms in the initial steps of DNA damage signaling and implicates ATM-p53-53BP1 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair. And we propose a model whereby a pre-existing pool of p53 that constantly scans the genome, responds immediately to radiation-induced DNA damage by virtue of its association with chromatin through its carboxy-terminus. The consequences for these p53-ATMSer1981-53BP1 complexes following DNA damage remains to be investigated: could residual complexes be associated with decreased DNA-dsb rejoining or error-prone repair, or could these complexes signal for cell survival or cell death? Since altered p53 function and biology is an important factor in cellular carcinogenesis and response to cancer therapy, this study provides a step towards a greater understanding of WTp53 and MTp53 biology in tumour development and therapeutic resistance, in the hopes to contribute towards predicting therapeutic response and/or improving p53-targeted therapies.