published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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No description available.

No description available.

This thesis explores the unequal partitioning of damaged proteins during mitosis and its implications for cell fate. Initially described in unicellular organisms, it was unclear if this method was used in vivo in multicellular organisms and had functional consequences in mammalian cells. To determine if this asymmetry was conserved in multicellular organisms, I studied three stem/progenitor populations in Drosophila: the larval neuroblast, adult female germline stem cell, and adult intestinal stem cell. Each cell type was found to asymmetrically segregate damaged proteins identified by the 2,4-hydroxynonenal (HNE) modification, which are associated with oxidative stress and age. Both the larval neuroblast and female germline stem cell were found to retain damaged proteins during division, whereas the intestinal stem cell segregated damaged proteins to differentiating progeny. I suggest that functional lifespan, and not cell type, determines the cell that receives the majority of damaged proteins during division. In each cell type, damaged proteins were associated with DE-Cadherin, a common component of the stem cell niche and removal from the niche was associated with reduced damaged protein polarization. Interestingly, when larval neuroblasts were mechanically dissociated from their niche and placed in culture, the internal polarization of damaged proteins was found to increase with progression through the cell-cycle. Therefore, I suggest that both the niche and intrinsic factors play a role in the asymmetric division of damaged proteins. To determine if an asymmetric division of damaged proteins influenced cell fate, I used a mammalian cell line with inducible expression of misfolded Huntingtin, which shares similar properties to damaged proteins. This study also revealed that the conformation of damaged proteins impacts cell fate: cells with diffuse Huntingtin displayed greater proliferation and reduced resistance to stress. Tracking cells containing an aggregate with live-imaging revealed that the cell that inherits the aggregate has a longer cell-cycle and an enhanced capacity to differentiate. Therefore, the asymmetric inheritance of damaged proteins impacts cell fate. In the final chapter of this thesis, I discuss the implications of an asymmetric division of damaged proteins on cell fate and how this information can be applied to cancer treatments.

This thesis explores the unequal partitioning of damaged proteins during mitosis and its implications for cell fate. Initially described in unicellular organisms, it was unclear if this method was used in vivo in multicellular organisms and had functional consequences in mammalian cells. To determine if this asymmetry was conserved in multicellular organisms, I studied three stem/progenitor populations in Drosophila: the larval neuroblast, adult female germline stem cell, and adult intestinal stem cell. Each cell type was found to asymmetrically segregate damaged proteins identified by the 2,4-hydroxynonenal (HNE) modification, which are associated with oxidative stress and age. Both the larval neuroblast and female germline stem cell were found to retain damaged proteins during division, whereas the intestinal stem cell segregated damaged proteins to differentiating progeny. I suggest that functional lifespan, and not cell type, determines the cell that receives the majority of damaged proteins during division. In each cell type, damaged proteins were associated with DE-Cadherin, a common component of the stem cell niche and removal from the niche was associated with reduced damaged protein polarization. Interestingly, when larval neuroblasts were mechanically dissociated from their niche and placed in culture, the internal polarization of damaged proteins was found to increase with progression through the cell-cycle. Therefore, I suggest that both the niche and intrinsic factors play a role in the asymmetric division of damaged proteins. To determine if an asymmetric division of damaged proteins influenced cell fate, I used a mammalian cell line with inducible expression of misfolded Huntingtin, which shares similar properties to damaged proteins. This study also revealed that the conformation of damaged proteins impacts cell fate: cells with diffuse Huntingtin displayed greater proliferation and reduced resistance to stress. Tracking cells containing an aggregate with live-imaging revealed that the cell that inherits the aggregate has a longer cell-cycle and an enhanced capacity to differentiate. Therefore, the asymmetric inheritance of damaged proteins impacts cell fate. In the final chapter of this thesis, I discuss the implications of an asymmetric division of damaged proteins on cell fate and how this information can be applied to cancer treatments.

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.

Developing an optimized growth environment for adipose-derived stems cells (ASCs) to obtain clinically useable cell quantities from relatively small tissue biopsies would significantly impact the field of tissue engineering. To date, ASCs have been differentiated into adipose, bone, cartilage, smooth muscle, endothelial, skeletal muscle, nervous, and cardiac tissue. Therefore, ASCs have potential for use in the treatment of a wide variety of clinical conditions ranging from myocardial infarction, to musculoskeletal disorders, and the repair of soft tissue defects. In this work, a custom-designed, 3-dimensional (3-D) scaffold-based perfusion bioreactor system was investigated in the culture of ASCs. Decellularized adipose tissue (DAT) was used to provide a 3-dimensional scaffold, as it possesses the native extracellular matrix (ECM) architecture and composition of human adipose tissue. The DAT had a permeability of 149 m2, based on a perfusion rate of 1.5 mL/min over a 200 mg DAT sample, and the culturing medium was evenly perfused throughout the DAT, thereby permitting possible cell growth within the central regions. Initial culturing studies of human ASCs on tissue culture polystyrene (TCPS) demonstrated that hypoxic (5% O2) conditions decreased the doubling time, and resulted in enhanced cell proliferation, as compared to normoxic (21% O2) conditions. The cell imaging and DNA quantification results showed that suspension seeding of the ASCs permitted cell attachment to the DAT scaffold, but did not support long-term ASC growth. In contrast, when the ASCs were seeded as multicellular aggregates, the cells attached and underwent measurable proliferation. The optimal seeding density observed was 1 x 106 ASCs/scaffold; or 50 aggregates (20,000 ASCs/aggregate) per scaffold. Based on the confocal imaging, the ASCs remained spherical in morphology during the entire culturing period. Moreover, results illustrated that the perfusion bioreactor provided an improved culturing environment for ASCs over traditional static culturing. Hypoxic (5% O2) conditions showed improved proliferation over normoxic (21% O2) conditions, within the bioreactor system. After a 14-day hypoxic culturing period in the perfusion bioreactor, the seeded ASCs retained the ability to undergo adipogenesis, as indicated by Glycerol-3-Phsophate Dehydrogenase (GPDH) enzymatic activity measurements, demonstrating the promise of this approach for soft tissue engineering applications / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-11-09 20:28:34.252

There is emerging evidence to suggest that cardiac myofibroblasts (CMyfbs) participating in cardiac fibrosis represent a heterogeneous population in origin. We hypothesized that bone marrow derived mesenchymal stem cells (MSCs) readily adopt a myofibroblastic phenotype in culture. We assessed and compared human primary MSCs and human CMyfbs with respect to their phenotypic and functional characteristics by examining their gene expression profile, ability to contract collagen gels, and ability to synthesize collagen. We also examined the role of non-muscle myosin II (NMMII) in modulating the myofibroblast function using siRNA and blebbistatin to inhibit NMMII activity. The data revealed that MSCs adopt a myofibroblastic phenotype in culture and demonstrate the capability to contract collagen gels and synthesize collagen similar to human CMyfbs. Inhibition of NMMII activity with blebbistatin completely inhibits gel contractility without affecting cell viability. Thus, MSCs exhibit similar physiological and functional characteristics as CMyfbs, and may contribute to cardiac fibrosis.

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.