published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Tissue engineering is a recently developed field that combines material science, cell biology, and engineering to create or improve functional tissues/organs. The field of tissue engineering has progressed from a fledgling science to an emerging technology, in large part due to parallel advances in the application of biomaterials and understanding stem cell behavior. Current studies have evaluated certain types of natural and synthetic biomaterials for feasibility of replicating the physio-chemical microenvironments of stem cells. Furthermore, technologies derived from micro-machining and solid free-form fabrication industries have utilized these biomaterials to create scaffolds that resemble tissue-like structures. Recent scaffold fabrication methods have attempted to overcome certain challenges in engineering tissues and organs. One of the fundamental limitations in current tissue engineering efforts has been the inability to develop multiple tissue types (i.e. bone, cartilage, muscles, ligaments) within a single scaffold structure in a predesigned manner. The differentiation of multiple cells within a three-dimensional (3D) scaffold using a single stem cell population has yet to be developed due to challenges in integrating various biochemical factors in a spatially-patterned method. This dissertation discusses scaffold micro-fabrication techniques that use layerby-layer, ultraviolet-based (UV) stereolithography systems. These approaches in microfabricating scaffolds provide an optimal, biomimetic environment for the pre-patterned differentiation of mesenchymal stem cells into skeletal-type tissues. We demonstrated both laser-based and digital micromirror device-based stereolithography systems for creating intricate scaffold architectures with multiple bio-factors encapsulated in predetermined regions. We showed that micro-stereolithography has the powerful capability of building 3D complex scaffolds with specific pore sizes and shapes in a layer-by-layer fashion using photo-crosslinkable monomers. These polymer-based scaffolds were functionalized with specific signaling proteins to create a biomimetic niche in which stem cells can respond, attach, and differentiate. The ultimate goal of this project is to integrate novel concepts of micro-manufacturing along with polymer-controlled release kinetics and stem cell biology to attain pre-designed architectures of tissue structures. / text

Plant tissue culture regeneration is chiefly regulated by exogenous phytohormones. To stop regeneration and induce undifferentiated callus growth auxins are used. Unfortunately auxins influence many plant responses, most unrelated to development. Using the thymidine analogue 5-bromodeoxyuridine (BrdU) a phytohormone independent means for differentiation inhibition has been developed. Studies were focused on the target site and mechanism of BrdU action. The BrdU inhibited step in development is indicative of a plant response necessary for normal differentiation. BrdU (5-30 uM) interrupts callus growth in all tested plants. Exogenous cytokinin does not restore growth while thymidine and deoxycytidine rescue plant growth and differentiation in the presence of BrdU. Endogenous cytokinin levels are not greatly affected by subtoxic BrdU levels and indicate that cytokinin and BrdU act upon independent sites. Domestic carrot cells were used in further studies. Normally carrot cells are undifferentiated in medium with the auxin 2,4D. When 2,4D is removed, somatic embryogenesis takes place. By including 5 uM BrdU in the hormoneless medium, the cells fail to differentiate. The growth of carrots in 2,4D is not affected by 5 uM BrdU. Thus, BrdU influences growth during differentiation to a greater extent than the growth of callus cells. BrdU is effective in halting development when applied 0-24 hours after differentiation induction. An event required for differentiation (the first and second replications) must take place at this time. BrdU action begins with DNA incorporation. The consequent cellular replication becomes slowed and DNA repair results. At the same time RNA and protein levels are similar in BrdU treated and untreated cultures. BrdU thymidine substitution into DNA increases from 28% (2 days) to 68% (3 days) after embryogenic induction. A second BrdU effect follows DNA incorporation. Factors (MIFs) in the medium of BrdU treated cells arrest differentiation. After BrdU is repaired from the DNA, the cells are only able to differentiate after a medium change. Understanding MIF production could explain why some plants differentiate more readily than others. BrdU provides the means for further study of MIF's in the auxin-free inhibition of development.

Osteoclasts are multinucleated, terminally differentiated cells found in bone tissue, which have the unique ability to resorb calcified substrates. The study of human osteoclasts has been restricted in the past due to difficulties in obtaining these cells. Recently, however, cell culture techniques have been devised that allow human osteoclasts to be grown in culture from their mononuclear precursors found in peripheral blood, therefore providing a constant supply of these cells. These cultures are based on the discovery of RANKL (receptor activator for NFJ(B ligand), which has recently been shown to playa central role in osteoclast formation and activation. This thesis has initially characterised cells grown in such cultures to confirm that they are authentic human osteoclasts, possessing osteoclast markers and with the ability to resorb calcified substrates. Once these cultures were established, and the authenticity of human osteoclasts grown in these cultures was confirmed, these cells were used to study the effects of extracellular nucleotides on human osteoclast acti vity. Adenosine-5' -triphosphate (ATP) is well known as an intracellular energy source. This and other nucleotides, however, also exist extracellularly, where they are agonists at a group of receptors termed P2 receptors. This receptor family is subdivided into P2X ligand gated ion channels, and P2Y G-protein coupled receptors. Bone-forming osteoblast cells, and boneresorbing osteoclast cells both express multiple SUbtypes of these receptors. Studies examining the effects of extracellular nucleotides on osteoclasts have been largely limited to non-human cells in the past due to the difficulty in obtaining human osteoclasts. This thesis, therefore, has examined the effects of extracellular nucleotides on human osteoclast activity. Human osteoclasts and their precursors were shown to express mRNA for nearly all of the P2Y and P2X receptor subtypes. ATP was found to both activate human osteoclast formation, by acting directly on P2X receptors expressed by osteoclast precursors, and to stimulate osteoclast resorption indirectly by acting at osteoblast-expressed P2Y) receptors to induce elevated RANKL expression by these cells. Activation of P2Y receptors expressed by osteoclasts was shown to result in downstream activation of MAPKinase pathways. Parathyroid hormone (PTH) is considered to be one of, if not the most important systemic factor in the regulation of bone. Co-stimulation of UMR-I 06 osteoblast-like cells with this hormone and P2Y) agonists resulted in potentiation of P2Y) agonist-induced [Ca2+Ji release by PTH, while PTH alone produced no [Ca2+]j elevation at all. The mechanism of this potentiation was attributed to Gs activation following PTH receptor stimulation, Gq having no involvement. Co-stimulation of these cells by PTH and P2Y) agonists also resulted in synergistic immediate early gene expression. These findings suggested that extracellular nucleotides are able to sensitize osteoblasts to the actions of PTH, providing a mechanism for localizing the response to this systemic hormone in bone, consistent with the discrete pattern of remodelling observed in the skeleton. Finally, the involvement of PTH on osteoclast formation was investigated. PTH was found to inhibit this process via activation of contaminating T lymphocytes present in osteoclast cultures. In conclusion this thesis presents evidence to suggest that extracellular nucleotides are important local signaling molecules in bone, affecting both osteoclast and osteoblast activity, alone and in combination with systemic factors such as PTH. Additionally, a novel action of PTH acting via lymphocytes to affect osteoclast formation is described.

The G-protein coupled receptor (GPCR) Cysteine (C)-X-C Receptor 4 (CXCR4) plays an important role in prostate cancer metastasis. CXCR4 is regarded as a plasma membrane receptor, that it transmits signals that support transformation, progression and metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutic approaches such as antagonists and monoclonal antibodies have focused on receptors the located at the plasma membrane. An emerging concept for GPCRs is that they can localize to the nucleus where they may retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 is highly expressed in high grade metastatic prostate cancer tissues. Increased expression of CXCR4 is also detected in several prostate cancer cell lines as compared to normal prostate epithelial cells. Our studies identify a nuclear pool of CXCR4 and also define a mechanism for nuclear targeting of CXCR4. A classical nuclear localization sequence (cNLS), "RPRK", in CXCR4 can contribute to nuclear localization. In addition, CXCR4 interacts with the nuclear transport receptor, Transportin βi, to promote nuclear accumulation of CXCR4. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicate that nuclear CXCR4 is functional and can participate in G-protein signaling revealing that the nuclear pool of CXCR4 can retain function. Localization of functional CXCR4 to the nucleus may be a mechanism by which prostate cancer cells evade treatment, thus contributing to increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4. This study addresses the mechanism of nuclear targeting for CXCR4 and demonstrates that CXCR4 can retain function within the nucleus and provides important new information to illuminate what have previously been primarily clinical observations of nuclear CXCR4.

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Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and hence plays a central role in tissue morphogenesis and a number of pathologic processes. My main goal has been to investigate the mechanisms of versican gene regulation, focusing on the signal transduction pathways, promoter regions, cis-acting elements, and trans- factors. This thesis puts forth new knowledge regarding transcriptional regulation of the human versican gene. In chapter III, I present the cloning of a 752-bp fragment of the human versican promoter (- 634/+118 bp) and nine stepwise 5' deletion fragments in the PGL3-luciferase reporter plasmid. Furthermore, I identify three potential enhancer and two repressor regions in this promoter. I also demonstrate that both cAMP and C/EBPβ enhanced and repressed versican transcription in HeLa cells and rat aortic smooth muscle cells (SMC), respectively, suggesting that versican transcription is differentially regulated by the respective mediator and transcription factor in epithelial cells and SMC. In chapter IV, I reveal the role of PI3K/PKB/GSK-3β signaling pathway in regulating versican promoter activity and transcription. Furthermore, I identify that the β-catenin/TCF-4 transcription factor complex, one of the downstream targets of GSK-3β, mediates versican promoter activity and transcription. In chapter V, I identify that variations in C-terminal regions of TCF family members determine their repressor or enhancer properties on Wnt target genes. Furthermore, I show that curcumin is a strong inhibitor of the β-catenin/TCF-p300 mediated gene expression. In chapter VI, I demonstrate that the androgen receptor trans-activates versican transcription in prostate cancer cells. Furthermore, I show cross-talk between the androgen receptor and β-catenin in regulating versican transcription in prostate stromal fibroblasts. Overall, this study charts previously uncharacterized promoter elements, transcription factors, and signal transduction pathways involved in regulation of the versican gene.