Recent advances in the field of bionanotechnology have enabled researchers to design a variety of tools to detect, image and monitor biological process in cells. Despite this progress, the limited understanding of nanomaterial-cellular interactions has hindered the widespread use of these nanomaterials in biological systems. In this thesis, we examined the potential effects of metallic nanoparticle geometry on important cellular processes such as membrane trafficking, intracellular transport and subcellular signalling. We found that the size of nanoparticles plays an important role on their ability to interact with the cell surface receptors thus dictating their subsequent ability to activate intracellular signalling cascades. Interestingly, trafficking of these nanoparticles was dependent on their size due to biochemical and thermodynamical constraints. These findings suggest that nanomaterials actively interact with biological systems, thus, directly modulating vital cellular processes. In addition, by utilizing various physical and chemical properties of nanomaterials, we developed a novel class of hybrid nanoscaled carrier systems capable of delivering semiconductor quantum dots (QDs) into live cells without inducing membrane damage. Using biodegradable polymeric nanoparticles, bioconjugated QDs were encapsulated and delivered into trafficking vesicles of live cells. The environmentally sensitive surface charge of the polymeric nanoparticles exhibited positive zeta potential inside acidic endo-lysosomes, thus enabling their escape from the vesicular sequestration into the cytosol. Hydrolytic-induced degradation then releases the bioconjugate QDs for active labelling of subcellular structures for real-time studies. Unlike previously described intracellular QD delivery methods, the proposed system offers an efficient way to non-invasively deliver bioconjugated QDs without inducing cell damage, enabling researchers to accurately monitor cellular processes in real-time. The understanding of both physical and chemical properties of nanomaterials is crucial to the design of biocompatible nanosystems to study fundamental processes in biological systems. Here, we demonstrated that both the size and surface chemistry of nanoparticles can be modified to obtain desired biological responses. Future experimental efforts to study other physical and chemical properties could allow the development of more sophisticated and effective platforms for biological applications.

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.

Survival of engineered tissues in vivo requires the presence of an internal vascular network and immediate connection to the host vasculature. Modular tissue engineering approaches the vascularization ‘design’ requirement through fabrication of submillimeter-sized collagen microtissues (‘modules’) with endothelial cells (EC) seeded on the surface of the modules and functional or vascular support cells inside the modules. Several modules are then packed together to build a larger tissue. In this work, we explored biomaterial-based strategies to build vascularized modular tissue engineered constructs. A photocrosslinkable poloxamine-polylysine acrylate biomaterial was first synthesized to improve the mechanical limitations of collagen modules under flow, while still supporting EC attachment. An extracellular matrix (ECM)-based strategy was then explored to enhance the vascularization of the modules in vivo. Manipulation of the ECM was accomplished through lentiviral transduction of EC to overexpress Developmental endothelial locus-1 (Del-1), a pro-angiogenic ECM molecule. Supporting the hypothesis that Del-1 overexpression ‘tilts’ the balance in EC from a quiescent to a pro-angiogenic phenotype, human umbilical vein endothelial cells transduced to overexpress Del-1 (Del-1 HUVEC) formed more sprouts and had a distinct expression profile of angiogenic genes in vitro, relative to control eGFP HUVEC. While very few blood vessels formed upon subcutaneous injection of empty collagen modules coated with Del-1 or eGFP HUVEC in a SCID/Bg mouse model, embedding adipose derived mesenchymal stem cells (adMSC) inside the modules increased blood vessel formation. Moreover, Del-1 HUVEC and adMSC modules consistently had more blood vessels (donor-derived and total number of vessels) compared to eGFP HUVEC and adMSC, over the 21 day duration of the study, with the greatest difference observed at day 7 post-transplantation. In addition, more α-smooth muscle actin (SMA+) staining was observed in Del-1 implants compared to eGFP, suggestive of increased vessel maturation through recruitment of SMA+ pericytes and smooth muscle cells. Perfusion studies showed that the implant vasculature was connected to the host vascular network as early as day 7, and throughout the 21 day duration of the study, for both Del-1 and eGFP implants. Nevertheless, further normalization of the vasculature is likely required to improve perfusion at early time points after transplantation.

Alzheimer’s disease is the most common form of dementia and is classified as a progressive neurodegenerative disease that impairs memory and cognition. Definitive diagnosis requires access to brain tissue and clinicians rely primarily on behavioural observation. Few specific, reliable, and well-characterized quantitative tools are in development. The accumulation of misfolded amyloid-beta protein in the brain is one of the hallmark pathological features of Alzheimer’s disease. Molecular imaging strategies have focused on measuring the amount of cerebral amyloid-beta. Antibody-mediated molecular imaging of amyloid-beta offers a promising strategy to measure specific types of amyloid-beta pathology in the central nervous system. This work characterizes the attempted translation of 4 anti-amyloid-beta antibodies from histological tools to live animal positron emission tomography imaging contrast agents. Several mass transfer properties of the classical anti-amyloid-beta antibody 6E10 were measured as a function of age in the TgCRND8 mouse model of Alzheimer’s disease. 6E10 was used to extensively label amyloid-beta plaques after direct injection into the cortex of TgCRND8 mice. 6E10 was subsequently covalently modified with poly(ethylene glycol) (PEG) in order to increase the blood concentration and promote higher brain uptake of the compound. PEG-modification of 6E10 enabled differentiation of TgCRND8 mice from wild type control mice using live animal imaging. Three additional antibodies were screened in a similar fashion; two of these antibodies targeted parenchymal amyloid-beta plaques and one targeted vascular amyloid-beta deposits. One of the antibodies that targeted parenchymal amyloid-beta plaques and the antibody that targeted vascular amyloid-beta were used to differentiate between TgCRND8 and wild type control mice using live animal imaging. This work demonstrates the successful use of 3 anti-amyloid-beta antibodies to detect amyloid-beta pathology using non-invasive imaging techniques and presents a credible framework for translating promising antibodies into contrast agents.

Split-protein systems are invaluable tools used for the discovery and investigations of the complexities of protein functions and interactions. Split-protein systems rely on the non-covalent interactions of two fragments of a split protein to restore protein function. Because of this, they have the ability to restore protein functions post-translationally, thus allowing for quick and efficient responses to a milieu of cellular mechanisms. Despite this, split-protein systems have been largely limited as a reporting tool for protein-protein interactions. The recent discovery of inteins has the potential of broadening the scope of split-protein systems. Inteins are protein elements that possess the unique ability of post-translationally ligating protein fragments together with a native peptide bond, a process termed protein splicing. This allows split-proteins to reassemble in a more natural state. Exploiting this property and utilizing protein engineering techniques and methodologies, several approaches are described here for restoring and controlling split-protein functions using inteins. First, the protein splicing behaviour was demonstrated with the development of a simple in vitro visual fluorescence assay that relies on examining the subcellular localization of different fluorescent proteins. Inteins were then used to reassemble and restore function to artificially split genetically encoded Ca2+ indicators. Second, inteins were shown to be able to simultaneously restore protein function to two target proteins. The first target protein was restored through the normal protein splicing pathway while the second was restored through non-covalent interactions of the split-protein fragments. This is a previous unknown property of inteins. Lastly, an intein was engineered to respond to an external light-stimulus that triggered protein splicing to restore split-protein function. The photoactivatable intein, coupled with the versatility of light, allows exquisite control in both space and time for the restoration of protein function within cells. The modularity of the photoactivatable intein can be simply attached to a variety of split-proteins. This was demonstrated with the restoration of various split-protein functions.

A sensitive and simple cell counting method is necessary in many pathologies including HIV [1] and cancer [2]. Cell counting sensors are used in the clinic for diagnosis of leukemia [3] or HIV [4]. Furthermore, genetic analysis of these cells is crucial for better prognosis and diagnosis [5]. However, a simple method for cell counting that allows further analysis is still lacking. This study aims to design a sensor that counts cells in the complex matrix of cell media or in the presence of non-target cells. The chip designed uses the anti-EpCAM antibody to selectively count cells via differential pulse voltammetry. The device can selectively count prostate cancer cells in both complex media with serum and a mixed cell population with a sensitivity of 125 cells per sensor. A simple and sensitive cell cytosensor was designed that can be used to count cancer cells effectively.

The study of synthetic gene regulatory networks allows the isolation and investigation of components and motifs in natural regulatory networks. Many synthetic gene networks are regulated at the transcriptional level. In this work, two methods of regulating gene expression at the transcriptional level were studied with the objective of gaining finer control over network behaviour. The first approach focuses on activation and repression of promoters by transcription factors. A synthetic repressor-activator network was engineered using the cI and cro genes and the PRM promoter from bacteriophage λ. The cI and cro genes activated and repressed PRM, respectively, and the monomeric red fluorescent protein (mrfp) gene reported PRM activity. Experimental testing showed an increase in mrfp expression in response to CI, a decrease in mrfp expression in response to Cro, and a differential output that reflected the relative concentrations of CI and Cro when both inputs were applied together. A positive feedback network was then created by placing a cI gene downstream of PRM. The network showed increased expression in response to CI and decreased expression in response to Cro. A negative feedback network was created by placing a cro gene downstream of PRM. Experimental testing showed decreased mrfp expression in response to both inputs. The second approach employed two methods for tuning expression levels without modifying the genes or promoters. First, using a series of networks with tandem mrfp genes under the control of the PLtet0-1 promoter, it was demonstrated that magnitude and range of expression levels could be tuned by adjusting the number of genes in the operon. A network was tuned using this principle by placing luxR genes in tandem to increase the activity of the luxPR promoter. It was then demonstrated that the level of gene expression could be varied through the placement of the gene within an operon. Operons that were three, five and seven genes and contained one green fluorescent protein gene in the first, middle, or end position were created. By comparing green fluorescence levels in induced and uninduced networks, it was found that the gene closest to the promoter was the most inducible.

Signalling states of cells are heterogeneous even within clonally derived populations due to cell cycle status and their local microenvironment. As a result multiplexed single-cell signal transduction measurements represent a powerful tool which could potentiate a much greater understanding of subcellular processes. However, multiplexed single-cell analysis remains challenging due to several factors, most notably the low copy number of analytes present, difficulties in cellular manipulation and the availability of well characterized and stable probes for use in intact cells. In order to address these issues, a capillary electrophoresis system with laser induced fluorescence (CE-LIF) suitable for screening methods to facilitate single cell analysis was designed and assembled. This system has the requisite sensitivity for single-cell analysis, with the capability of detecting down to 10000 molecules of fluorescein, and has been designed to reduce the time required for analyte separations compared to similar systems by integrating a compact detection module which allows for shorter electrophoretic separation distances. This system has been employed to develop a method to determine the sampling efficiency of laser-based cell lysis of single cells allowing more accurate quantitative measurements of fluorescent peptides from single cells. Furthermore, a fluorescent probe based on amyloid precursor protein (β-APP peptide) has been designed and conditions were found which allowed resolution of enzyme-modified versions within single cells. To identify the enzymatic conversion products produced, a novel method was developed employing bulk cell samples in conjunction with LC-MS. As a testament to the resolution afforded by this technique, the peptide fragments identified from single cells represented peptides which differed by single uncharged amino acids. Together, the methods here developed are able to provide higher quality quantitative data and more informative analysis of fluorescent signal transduction reporters in single cells and represents progress towards being able to obtain highly multiplexed data needed for accurate cellular models.

The pathophysiology of psychiatric disorders such as schizophrenia is not fully understood due, in part, to the shortcomings of available neurophysiological techniques. Previous studies have shown that patients with schizophrenia have deficits in dorsolateral prefrontal cortex (DLPFC). In this regard, two major deficits were observed: impairments in gamma-aminobutyric-acid (GABA) neurotransmission and cortical gamma (30-50Hz) oscillations. Previous in vitro and animal studies have linked the modulation of gamma oscillations with GABAB receptor mediated inhibition. Objectives: The first objective was to examine the effect of GABAB receptor mediated inhibition on cortical oscillations in the motor cortex and DLPFC in healthy subjects by using the novel technique of transcranial magnetic stimulation (TMS) combined with electroencephalography (EEG) and through the TMS paradigm long interval cortical inhibition (LICI), which has been associated with GABAB receptor mediated inhibition. Second, to evaluate the psychometric properties of this neurophysiological paradigm, the validity and reliability of EEG indices of LICI were examined. Finally, the effect of LICI on cortical oscillations was examined in the DLPFC and motor cortex of patients with schizophrenia compared to healthy subjects and patients with bipolar disorder. Hypothesis: It was predicted that EEG measures of LICI would show validity and reliability, and it was hypothesized that patients with schizophrenia would show deficits in inhibition of gamma oscillations in DLPFC compared to healthy subjects and patients with bipolar disorder. Results: The first experiment showed that in healthy subjects LICI inhibited gamma oscillations in the DLPFC but not in the motor cortex. The second experiment demonstrated the validity and reliability of EEG indices of LICI were confirmed in healthy subjects. Finally, patients with schizophrenia had a selective deficit in inhibition of gamma oscillations in the DLPFC which appeared to be independent of illness duration or antipsychotic medication, and it was not observed in bipolar disorder. Conclusions: TMS combined with EEG allows for measuring modulatory effect of LICI on cortical oscillations. Inhibition of gamma oscillations in the DLPFC may be an essential neurophysiological process that may be impaired in schizophrenia. Future studies should ascertain the potential of gamma inhibition deficit as a biological marker for this illness.

In spite of burgeoning of new technologies in the field of maxillofacial surgery, such as novel methods for osteosynthesis, bone substitution and bone regeneration, the reconstruction of the craniofacial skeleton (CFS) remains a challenge. Complications and failure in existing technologies and treatments for the CFS may be attributed in part to an incomplete understanding of the biomechanical environment in which these technologies are expected to perform. Characterizing the morphology and biomechanical behaviour of this complex and unique structure is important to understanding its global response to mechanical demands. This thesis aims to characterize the biomechanical behaviour of thin bone regions and sutures in the CFS. We investigated the impact of image degradation in CT scans on the ability to develop accurate specimen-specific FE models. Image degradation resulted in large increases in cortical thickness and decreases in scan intensity, which corresponded to significant changes in maximum principal strains in the FE models. A new semi-automated connectivity technique was developed to quantify the degree of fusion in sutures and revealed varying degrees of connectivity and interdigitation depending on the suture location. Morphological features characterized using this technique were incorporated into idealized suture FE models and analysed under multiple loading directions. The idealized FE models revealed that the impact of the number of interdigitations on the strain energy absorption in the suture/bone complex is dependent on the loading direction (inversely related under pressure and directly related under perpendicular and pressure loading); similar behaviour was seen in a μCT based specimen-specific FE model. Three-point bending tests on bone samples containing sutures revealed a positive correlation between the number of interdigitations and bending strength. Finally, experimental testing of full cadaveric heads demonstrated inter-specimen consistency in strain magnitude and direction under muscle loading in spite of morphological differences. Overall, these findings provide new insight into the complex morphology of the CFS, limitations of current clinical imaging and the biomechanical behaviour of thin bone structures and their articulations. This work forms a solid foundation for future development of image analysis, modeling and experimental investigations focused on characterizing the global behaviour of the CFS.