Despite efforts by clinicians and scientists world-wide, coronary artery disease remains to be the leading cause of morbidity and mortality in industrialized nations. Development of a tissue engineered coronary by-pass graft with low thrombogenicity and immune responses, suitable mechanical properties, and a capacity to remodel to their environment could have a significant impact on the treatment of coronary artery disease. While many methods for the tissue engineering of blood vessels have been developed, one promising approach is the self-assembly method. Using autologous cells that produce an endogenous extracellular matrix (ECM), the potential for therapeutic success is high due to biocompatibility. However, despite these advantages, improvements can be made which will give the grafts an even higher rate of patency. This dissertation presents a study of the characterization of the biaxial mechanical properties of self-assembly tissue engineered blood vessels (SA-TEBV), as well as developing a framework for fabrication strategies of SA-TEBV. Native arteries are exposed to multiaxial mechanical loads, including (a pulsatile) blood pressure that causes the vessel to cyclically distend circumferentially, blood flow that induces a shearing load along the luminal surface, and an axial extending load; the latter is relieved upon excision, causing the vessel to retract. These mechanical loads introduce intramural wall stresses and flow induced wall shear stresses that play a key role in mechano-biological signaling and tissue homeostasis. Until now, the mechanical properties of SA-TEBV have only been characterized in the circumferential direction (i.e. burst pressure and circumferential elastic modulus). The objective of this work is to characterize the biaxial mechanical properties of SA-TEBV to quantify their mechanical behavior and local intramural stresses under physiological loading. The work will show that while the global mechanical response of the SA-TEBV is similar to that of native arteries (and potentially sufficient), the local intramural stresses (using the current fabrication techniques) differ greatly from native coronary arteries. Therefore, a novel approach to fabricate the self-assembly derived tissue sheets is developed and tested which utilizes biaxial mechanical stimulation to alter the microstructure, thereby controlling their mechanical response.

Planar supported, polymerized phospholipid bilayers (PPBs) composed of 1,2-bis[10-(2’,4’-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC or BSPC) were generated by a redox polymerization method. The PPBs were supported by a silicon substrate. The PPBs were characterized and tested for uniformity and stability under physiological conditions. The PPBs were analyzed in vitro with murine derived cells that are pertinent to the host response. Cellular attachment and phenotypic changes in RAW 264.7 macrophages and NIH 3T3 fibroblasts were investigated on PPBs and compared to bare silicon controls. Fluorescent and SEM images were used to observe cellular attachment and changes in cellular behavior. The PPBs showed much lower cellular adhesion for both cell lines than bare silicon controls. Of the cells that attached to the PPBs, a very low percentage showed the same morphological expressions as seen on the controls. The hypothesis generated from this work is that defects in the PPBs mediated the cellular attachment and morphological changes that were observed. Finally, a layer-by-layer (LbL) deposition of a poly(acrylic acid) (PAA) and poly(N-vinylpyrrolidone) (PNVP) alternating bilayer was attempted as a proof of concept for future modification of this system.

Fibrosis of vital organs remains one of the leading causes of death in the developed world, where it occurs predominantly in soft tissues (liver, lung, kidney, heart) through fibroblast proliferation and deposition of extracellular matrix (ECM). In the process of fibrosis, remodeling and deposition of ECM results in stiffening of cellular microenvironment; cells also respond to these changes in the stiffness through engagement of their cytoskeleton and signaling via cell-ECM contacts. Thus, understanding to what extent the stiffness of the cellular microenvironment changes as a consequence of fibrotic progression, and how cells respond to this change, is critical. In this thesis, we quantitatively measured stiffness of the lung parenchyma and its changes during fibrosis. We find that the average stiffness increases by approximately 10-fold. We then investigated how changes in ECM rigidity affect the cytoskeletal phenotype of lung fibroblasts. We find a complex relation between expression of the glycoprotein Thy-1 (CD90) and ECM rigidity-dependent cytoskeletal phenotype (i.e. “mechanotransduction”). Finally, we investigate a mechanism for the regulation of rigidity sensing by Thy-1 and its involvement in intracellular signaling through cell-ECM contacts. Taken together, this work helps define in vivo parameters critical to the fibrogenesis program and to define unique cellular phenotypes that may respond or contribute to mechanical homeostasis in fibrotic diseases.

Tissue regeneration in salamanders is a robust process that is not easily interrupted or altered. Therefore, inhibiting regeneration provides a means to interrogate the underlying cellular and molecular mechanisms regulating this complex event. Here we show that application of a relatively low concentration of beryllium nitrate solution (100mM) causes a delay in skin regeneration and severely alters normal limb regeneration. We provide evidence showing a beryllium-induced reduction in dermal fibroblast migration in vivo and in vitro. We link this phenomenon to delayed regeneration of the skin and abnormal blastema formation resulting in limb patterning defects during regeneration. Though our results show a slight reduction in fibroblast proliferation during the early stages of limb regeneration, we attribute this to an overall reduction in fibroblast presence at the site of injury. Keratinocytes appeared unresponsive to beryllium treatment with the rates of re-epithelialization and proliferation not significantly different between treatment and control groups. Taken together, these data reinforce a necessary role for fibroblasts during tissue regeneration and show that beryllium nitrate inhibits normal fibroblast behavior.

Cancer is a set of complex genetic diseases driven by diverse genomic alterations. The genomic study of cancer has enabled the discovery of novel, targetable events in almost all cancer types and in turn, has led to the development of new, targeted cancer therapies benefiting patients; however, the recent explosion of genomic datasets has also resulted in huge lists of new oncogenic factors of unknown biological relevance, and uncertainty over how best to use the data appropriately to influence patient care. Some of the most pressing questions surround the use of statistical methods to identify actionable genomic alterations in cancer and the identification of driving oncogenes in the context of the genomic evolution of cancer cells, undergone before, during, and after prolonged treatment regimens.

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.

Basic fibroblast growth factor (FGF-2) is a trophic molecule involved in a number of functions within the central nervous system (CNS), including a prominent role in the regulation of CNS responses to injury. Prior studies suggest that rats recover differently from injury inflicted to different regions and at different ages throughout development, and that FGF-2 might underlie this phenomenon. This thesis examined whether the functional and structural outcome following bilateral injury to the motor cortex inflicted at postnatal day (P10) could be ameliorated by exogenous administration of a growth factor (FGF-2). Four complimentary studies were conducted that each assessed the role of FGF-2 in mediating recovery from bilateral motor cortex injury inflicted at P10. We found that FGF-2 improves physiological, anatomical, and functional outcome from bilateral lesions to motor cortex at P10. / xiii, 171 p. : ill. ; 28 cm.

Basic fibroblast growth factor (bFGF) has been implicated in the brain's trophic response to injury. This thesis examined the effects of endogenous bFGF on brain plasticity and recovery of behavioral function following cortical injury in adult rats. The first experiment investigated the post-lesion time course of the astrocytic expression of bFGF. Subsequent experiments examined the effects of injury-induced bFGF on neuroonal morphology, cortical morphology, and post-lesion behavioral deficits. Following motor cortex injury, endogenous bFGF prevented neuritic degeneration in layer V pyramidal neurons in Zilles' area Fr2 and promoted recovery of function in the Whishaw Reaching Task. Housing rats in an enriched environment prior to cortical injury enhanced the expression of bFGF but did not increase cortical thickness nor reduce post-lesion behavioral deficits (relative to laboratroy-housed rats). Collectively, these experiments indicate that injury-induced bFGF plays a role in potentiating recovery from brain damage. This implies that bFGF may be beneficial as a treatment following brain injury. / x, 123 p. ; 28 cm.

This thesis examined the role of exogenous basic fibroblast growth factor (FGF-2) in stimulating recovery after early cortical injury. Rats with medial prefrontal cortex (MFC), posterior parietal cortex (PPC), or sham lesions at postnatal day 3 (P3) received one of three variations of FGF-2 treatment: postnatal FGF-2 that was either pre-mixed or prepared daily, or prenatal FGF-2, and tested in adulthood. Behavioral tests used were: 1) the Morris Water task and, 2) the Whishaw Tray Reaching task. The level of functional recovery attained was dependent on FGF-2 preparation and the developmental period. MFC lesion rats showed good recovery but there was a differntial effect of pre and postnatal FGF-2 that appeared to be related to task. PPC rats showed greater recovery after postnatal rather than prenatal treatment. Anatomical changes were restricted to groups with relatively good functional recovery. These findings suggest a multifunctional role of FGF-2 in the injured brain. / xvi, 223 leaves : ill. ; 29 cm.

Podosomes are adhesive and invasive structures which may play an important role in numerous physiological and pathological conditions including angiogenesis, atherosclerosis, and cancer metastasis. Recently, the cysteine protease m-calpain (m-Capn) has been shown to cleave cortactin, an integral component of the podosomal F-actin core, as well as various proteins found in the peripheral adhesive region leading to the disassembly of these dynamic structures. In this study, I investigated whether Capn plays a role in the formation of podosomes downstream of c-Src. I show that: 1) phorbol-12, 13-dibutyrate (PDBu) as well as c-Src-Y527F expression induces podosome formation in mouse embryonic fibroblasts; 2) PDBu- and constitutively active c-Src-induced podosome formation is inhibited by the knockout of the m- and µ-Capn small regulatory subunit Capn4 in mouse embryonic fibroblasts (Capn4-/-), but is partially restored by re-expression of Capn4; 3) Capn4 localizes to podosomes; and 4) Inhibition of m- and µ-Capn proteolytic activity by the cell permeable calpain inhibitors has little effect on the formation of podosomes downstream of active c-Src. I conclude that Capn4 may play a role in the assembly phase of podosomes independent of calpain proteolytic activity. Work done in collaboration to determine a possible mechanism of action for the role of Capn4 in podosome assembly indicates that a possible binding partner of Capn4, β-PIX, co-localizes with, and shows in vivo association with Capn4. Furthermore, β-PIX and Capn4 bind directly in vitro in the presence of Ca2+. We conclude that Capn4 plays a role in podosome assembly, and this role may be through direct interaction with β-PIX in a calcium-dependent manner. / Thesis (Master, Biochemistry) -- Queen's University, 2008-09-26 16:16:00.768