Physiological interpersonal synchrony, the spontaneous alignment of indicators of physiological activity, is highly associated with the level of empathy between people in emotionally meaningful relationships. However, synchrony has not been studied with nonverbal, severely-disabled individuals, many of whom have a limited means to communicate. In this study, dyadic physiological synchrony in client-parent-therapist triads was quantified through simultaneously recording electrodermal activity, heart rate, cortical brain activity (client-parent only), and cortisol level. For the majority of the trials, a greater level of synchrony was observed in the client-parent dyads compared to the client-therapist dyads; however, the client-therapist dyads demonstrated an increased level of synchrony in the later trials. Electroencephalography analysis revealed widespread interbrain synchrony involving empathy-related regions. Our results suggest that even in the presence of disabilities, synchrony due to empathy exists and that the measurement of this synchrony may serve as a clinical adjunct to the self-report of interpersonal relationships.

Despite the importance of inter-cellular (between cell) communication networks in regulating homeostasis in multicellular organisms, very little is known about their topology, dynamics, or functional significance. Inter-cellular communication networks are particularly relevant in stem cell biology, as stem cell fate decisions (self-renewal, proliferation, lineage specification) are tightly regulated based on physiological demand. Using human blood stem cell cultures as an experimental paradigm, we present an integrated experimental and computational approach to interrogate a hierarchically organized tissue network. We have developed a novel mathematical model of blood stem cell development incorporating cell-level kinetic parameters as functions of secreted molecule-mediated inter-cellular networks. By relation to quantitative cellular assays, our model is capable of predictively simulating many disparate features of both normal and malignant hematopoiesis, relating internal parameters and microenvironmental variables to measurable cell fate outcomes. Through integrated in silico and experimental analyses we show blood stem and progenitor cell fate is regulated by cell-cell feedback, and can be controlled non-cell autonomously by dynamically perturbing inter-cellular signalling. Furthermore, we have compiled genome-scale molecular profiles (transcriptome and secretome), publicly available databases, and literature mining to reconstruct soluble factor-mediated inter-cellular signalling networks regulating cell fate decisions. We find that dynamic interactions between positive and negative regulators, in the context of tuneable cell culture parameters, tip the balance between stem cell supportive vs. non-supportive conditions. The cell-cytokine interactions can be summarized as an antagonistic positive-negative feedback circuit wherein stem cell self-renewal is regulated by a balance of megakaryocyte-derived stimulatory factors vs. monocyte-derived inhibitory factors. To understand how the experimentally identified positive and negative regulatory signals are integrated at the intra-cellular level, we define a literature-derived blood stem cell self-renewal network wherein these extracellular signals converge for coherent processing into cell fate decisions. In summary, this work demonstrates the utility of integrating experimental and computational methods to explore complex cellular systems, and represents the first attempt to comprehensively elucidate non-autonomous signals balancing stem cell homeostasis and regeneration.

Physiological interpersonal synchrony, the spontaneous alignment of indicators of physiological activity, is highly associated with the level of empathy between people in emotionally meaningful relationships. However, synchrony has not been studied with nonverbal, severely-disabled individuals, many of whom have a limited means to communicate. In this study, dyadic physiological synchrony in client-parent-therapist triads was quantified through simultaneously recording electrodermal activity, heart rate, cortical brain activity (client-parent only), and cortisol level. For the majority of the trials, a greater level of synchrony was observed in the client-parent dyads compared to the client-therapist dyads; however, the client-therapist dyads demonstrated an increased level of synchrony in the later trials. Electroencephalography analysis revealed widespread interbrain synchrony involving empathy-related regions. Our results suggest that even in the presence of disabilities, synchrony due to empathy exists and that the measurement of this synchrony may serve as a clinical adjunct to the self-report of interpersonal relationships.

Communication disorders affect one in ten Canadians and the incidence is particularly high among those with Cerebral Palsy. A vocalization-activated switch is often explored as an alternative means to communication. However, most commercial speech recognition tools to date have limited capability to accommodate dysarthric speech and thus are often prematurely abandoned. We developed and evaluated a novel vocalization-based access technology as a writing tool for a pediatric participant with cerebral palsy. It consists of a high quality condenser headmic, a custom classifier based on Gaussian Mixture Modeling (GMM) and Mel-frequency Cepstral Coefficients (MFCC) as features. The system was designed to discriminate among five vowel sounds while interfaced to an on-screen keyboard. We used response efficiency theory to assess this technology in terms of goal attainment and satisfaction. The participant’s primary goal to reduce switch activation time was achieved with increased satisfaction and lower physical effort when compared to her previous pathway.

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.

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.

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.

Recent evidence suggests that “mesenchymal stem cells” (MSCs) are resident in the perivascular compartment of connective tissues. However, since the definition of a stem cell assumes that these progenitors have clonal self-renewal and multi-lineage differentiation potential, the term “MSC” has been criticised, as it has been impossible to isolate definitive clonally derived “MSCs.” To test for this most basic definition of a stem cell, here it is shown that human umbilical cord perivascular cells (HUCPVCs) are capable of multilineage differentiation in vitro and, more importantly, in vivo, displaying the ability to differentiate into functionally synthetic cells that direct and contribute to rapid connective tissue healing by producing bone, cartilage and fibrous stroma in a mouse injury model. Uniquely, these cells can be enriched to >1:3 clonogenic frequency in early passage culture, making it possible to isolate clones and daughter sub-clones from mixed gender suspensions, determined to be definitively single-cell-derived by Y-chromosome fluorescent in situ hybridization (FISH) analysis. Each clone was assayed for multi-lineage differentiation capacity into the five mesenchymal lineages: myogenic, adipogenic, chondrogenic, osteogenic and fibroblastic (stroma). The observation that daughter sub-clones possess equal or lesser differentiative potential to their respective parent clones demonstrated the two intrinsic properties of stem cells in vitro: clonal self-renewal and multi-lineage differentiation. This evidence provides a new hierarchical structure of robust MSCs self-renewing to produce more restricted progenitors that gradually lose differentiation potential until a state of complete restriction to the fibroblast is reached. The methods described herein combined with recognition of this lineage hierarchy provides a significant advance to the understanding of MSC biology, and will enable interrogation of the properties of robust self-renewal and differentiation of MSCs in serially transplanted living recipients.

The emergence of bionanotechnology has allowed the design of novel technological tools for a variety of biomedical applications. Despite the incompatibility of native semiconductor quantum dots with biological environment, this class of nanocrystals was among the first nanomaterials to demonstrate their use in biological labeling and imaging applications. In this thesis, new surface modification chemistry and synthetic strategies were developed to produce high quality biocompatible and bioconjugatable near-infrared emitting quantum dots for deep tissue in vivo imaging and detection applications. By carefully selecting a specific mixture of semiconductor elements to obtain a desired bandgap energy and optimizing the procedure for surface coating, successful synthesis of high quality, watersoluble, near-infrared emitting quantum dots were demonstrated. These developments allows for the use of quantum dots as alternative contrast agents for sophisticated biological imaging applications that are currently unachievable using conventional uorophores. In addition, using metallic nanoparticles, it was found that cells possess the ability to differentiate nanoparticles of various sizes upon their binding with specific membrane receptors. These receptors undergo rapid cellular internalization which altered their trafficking dynamics and down-stream signaling processes. The amplitude of such alteration was highly dependent on the size of the nanoparticle with most efficient internalization occurring at 40 nm - 50 nm size range. These observations raise important questions regarding the mechanisms governing similar processes and cell behaviours documented during viral infections. Whether such biological phenomenon are evolutionarily conserved as natural defense mechanisms to counter foreign invasion, or whether many of the known viruses are naturally selected to breach the primary defense of cells - the plasma membrane, remains to be elucidated. In summary, nanotechnology offers great promises for biological research and medicine. This thesis demonstrates the use of semiconductor and metallic nanostructures for imaging, detecting and administrating therapeutics in cancerous cells, tissues and animal models. Although the results presented in this thesis are preliminary, and the technologies demonstrated are still years away from practical use, these studies nevertheless, pave the way for future experimental researches within the field of nanomedicine, and provide insights into the understanding of the most fundamental yet highly complex processes in cell biology.