Browsing by Author "Zygourakis, Kyriacos"
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Item A Novel Human Adipocyte-Derived Basement Membrane for Tissue Engineering Applications(2012-09-05) Damm, Aaron; Nagrath, Deepak; Zygourakis, Kyriacos; Gonzalez, Ramon; Sabek, OmaimaTissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.Item Automated tracking of tumor invasion in three dimensional extracellular matrix analogs and a novel stochastic analysis of the cell trajectories(2001) Demou, Zoe NM; McIntire, Larry V.; Zygourakis, KyriacosTumor cell migration and invasion of body tissues are prerequisite mediators for lymphatic or hematogenous cancer dissemination. To date, there is insufficient understanding of what triggers the metastatic cascade, and of how the interplay among cell receptors, the cellular and acellular components of the extracellular matrix and proteolytic enzymes mediate cancer migration, invasion, proliferation and survival. In addition to the inherent complexity of each one of the aforementioned phenomena is the lack of an experimental technique capable of dissecting the mechanisms that mediate the dynamic invasive and migratory behavior at the cellular level and with respect to the properties of the cell environment. The goal of my thesis was to develop an automated system for cell tracking in three dimensions and use it to model the dynamics of cancer invasion and migration. Therefore the hardware and software were designed for a fully automated optical 3D cell tracking system that quantified long-term invasion and migration of cancer cells infiltrating 3D extracellular matrix analogs. The quantitative analysis of the cell trajectories employed a novel formulation of the continuous Markov model that evaluated the potential for invasive or lateral motion and cell stops. The infiltration of human HT1080 fibrosarcoma and human MDA-MB-231 adenocarcinoma cells, was monitored in plain or Matrigel-containing collagen type I gels. Parameters such as the speed subpopulations, the persistence of motion in certain directions, the turning frequency of the cells, the preferred directions of motion, and the invasion depth profiles over time quantified infiltration at the cellular level. Distinct migratory and invasive phenotypes significantly dependent on the gel composition were identified for the two cell types. The HT1080 cell line expressed a high motility phenotype and well-preserved lateral motion on the plain collagen gel surface. The basement membrane components transformed the HT1080 cells to robust invaders by significantly enhancing the matrix infiltration and the turning frequency. The low motility, slow invasion and low turning behavior of MDA-MB-231 cells indicated that their invasiveness may depend on matrix-degrading activity. To the best of my knowledge this is the first study employing a detailed set of quantitative descriptors to demonstrate that tumor invasion and migration are dynamic processes of individual cells that depend significantly on the cell type and the tumor microenvironment.Item Biochar and Microbial Signaling: Production Conditions Determine Effects on Microbial Communication(American Chemical Society, 2013) Masiello, Caroline A.; Chen, Ye; Gao, Xiaodong; Liu, Shirley; Cheng, Hsiao-Ying; Bennett, Matthew R.; Rudgers, Jennifer A.; Wagner, Daniel S.; Zygourakis, Kyriacos; Silberg, Jonathan J.Charcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell–cell communication varied, with the biochar prepared at 700 °C (surface area of 301 m2/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300 °C (surface area of 3 m2/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops.Item Carbon Sequestration through Biochar Soil Amendment: Experimental studies and mathematical modeling(2012-09-05) Sun, Hao; Zygourakis, Kyriacos; Masiello, Caroline A.; Chapman, Walter G.; Hockaday, William C.Intentional amendment of soil with charcoal (called biochar) is a promising new approach to sequester atmospheric carbon dioxide and increase soil fertility. However, the environmental properties of biochars can vary with production conditions, making it challenging to engineer biochars that are simultaneously optimized for carbon sequestration, nutrient storage, and water-holding capacity. For this reason, I have undertaken a systematic study to (a) determine the pyrolysis conditions that lead to biochars with desired chemical and physical properties, and (b) find how these properties affect the water-holding capacity and nutrient adsorption in biochar-soil mixtures. First, a library of biochars was produced in a custom-built pyrolysis reactor under precisely controlled conditions. The chemical and physical structures of the produced biochars were characterized with various analytical techniques including 13C NMR, XPS, EA and BET pore surface analysis. My results suggest that the chemical composition and pore structure of biochars are determined not just by the maximum heat treatment temperature, but also by several other factors that include the pyrolysis heating rate, treatment time at the maximum temperature and particle size. I also tested a new approach that combines thermogravimetric reactivity measurements, diffusion-reaction theory and structural models to achieve a better characterization of the complicated multi-scale pore structure of biochars. The structural models treat biochars as porous solids having micro- and macropores of different shapes and exhibiting widely ranging pore-size distributions. Simulations results are then compared to experimental data to identify the presence of ordered or random pore networks and test their size distributions and connectivity. I then developed a multi-solid one-dimensional model that can use experimentally determined biochar properties to predict their field performance in beds packed with soil/biochar mixtures. The model used a system of coupled partial differential equations to describe the dynamic adsorption/elution of ammonium nitrate, a model fertilizer, in columns packed with biochar/soil mixtures and perfused with aqueous solutions of the fertilizer. The PDE system was solved using orthogonal collocation on finite elements. My chromatographic model accounted for all the important processes occurring in this system, including external mass transfer between the fluid phase and the solid particles, as well as intraparticle diffusion and adsorption of the solute on the pore surface area of the sorbents. To our knowledge, this is the first chromatographic model that accounted explicitly for the presence of two solid phases with widely different pore structures and adsorption capacities. A systematic parametric study was carried out to determine the importance of each system parameter. The adsorption equilibrium parameters and the intraparticle effective diffusivity of ammonium had the most significant effect on environmental performance. To complete the theoretical analysis, I also developed a model to describe the saturation and drainage of water from the packed column. The model accounted for all the important processes occurring in this system: (a) water exchange between the interstitial pore region and two different smaller pore regions and (b) water flow inside the larger pore region and the two different smaller pore regions. The transient mass balances led to a system of partial differential equations that was solved using block centered finite difference.Item Change in cell motility and metabolism following culture under low shear conditions(2008) Lennon, Sean Patrick; Zygourakis, KyriacosRecent studies suggest that the human immune system, and in particular lymphocyte function, may be suppressed during space flight. However, the mechanisms by which mechanical forces affect the function of immune cells are still poorly understood. Research performed in our laboratory indicates that culturing lymphocytes under low shear conditions (in rotating wall vessels) leads to an increase in cell motility and altered cell morphology. We have hypothesized that fundamental changes in the cytoskeleton, caused by changes in external forces, could lead to secondary changes in cellular metabolism, which could in turn be reflected by altered membrane structure. Utilizing NMR to investigate changes in lymphocyte function in an altered mechanical environment, we observed significant changes in cell metabolism following cell culture in the RWV, as compared to cells cultured statically. Our research seeks to advance the understanding of how microgravity affects the function of individual cells and how these cells interact with their environment.Item Co-Cultures of Articular Chondrocytes and Mesenchymal Stem Cells for Cartilage Tissue Engineering(2014-04-24) Dahlin, Rebecca L.; Mikos, Antonios G.; Kasper, Kurt; Ludwig, Joseph A.; Zygourakis, KyriacosArticular cartilage lines the surfaces of synovial joints to protect underlying bone and provide a smooth surface for articulation. Damage to articular cartilage typically leads to long-term pain and disability, as current treatments are unable to fully restore the functional tissue. Thus, tissue engineers seek to develop technologies to enhance cartilage repair. This thesis investigated two strategies for cartilage engineering: flow perfusion bioreactor culture and co-cultures of chondrocytes with mesenchymal stem cells (MSCs). First, we designed a novel bioreactor and then investigated the effect of flow perfusion on chondrocytes when combined with chondrogenic stimuli, including hypoxia and transforming growth factor-β3 (TGF-β3). We demonstrated that the combination of flow perfusion and hypoxic conditions enhanced proliferation, cartilage-like extracellular matrix production, and chondrogenic gene expression compared to perfusion alone. However, these results also demonstrated the need for a more potent chondrogenic stimulus, and thus the effect of perfusion with TGF-β3 was investigated on both chondrocytes and co-cultures of chondrocytes and MSCs. Here, we described the advantages of using exogenous growth factors in flow perfusion cultures, and the utility of flow perfusion for creating large tissue-engineered constructs. The second part of this thesis investigated co-cultures of chondrocytes and MSCs having the potential to reduce the demand for chondrocytes, which overcomes a significant challenge to current approaches toward cartilage repair. We first investigated the sensitivity of this cell population to TGF-β3 and then investigated the stability of the cell phenotype resulting from growth factor supplementation. The results demonstrated that co-cultures of chondrocytes and MSCs enable a reduced concentration and duration of TGF-β3 exposure to achieve an equivalent level of chondrogenesis compared to chondrocyte or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-β3. The final section of this thesis investigated the ability of such co-cultures to repair cartilage in a rat osteochondral defect model. Here, it was demonstrated that co-cultures achieved equivalent cartilage repair compared to the chondrocytes, thus demonstrating the potential use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.Item Computer-assisted analysis of endothelial cell migration and proliferation(1995) Lee, Yih; Zygourakis, KyriacosThis work presents some important results from experimental studies aimed at elucidating the fundamental mechanisms of endothelial cell migration and proliferation. To continuously monitor populations of migrating and proliferating cells, we used video microscopy coupled with a novel computer-automated digital time-lapse recording technique. Migrating cells were identified and their positions at each time instant were obtained using digital image processing. We also developed a modified nearest neighbor tracking algorithm to reconstruct approximations to the cell trajectories. Our experimental studies on the locomotion of bovine pulmonary artery endothelial (BPAE) cells have shown that these cells execute persistent random walks in culture. Cells change their direction of migration either in response to some intracellular signal or because they collide with other cells. Cells slow down as they approach other cells and then turn and move away from each other with increasing speeds. The temporal evolution of population-average speed of locomotion reveals that increases in cell density due to proliferation are immediately accompanied by a decrease in the average cell speed. Markov chain analysis on cell trajectories has shown that the enhanced motility of the BPAE cells cultured with basic fibroblast growth factor (bFGF) seems to be derived not only from fewer visits to the stationary state, but also from a decrease in the waiting time for each visit to the stationary state. BPAE cells execute persistent random walks when they are cultured without or with added bFGF, although the addition of bFGF does make them more motile. An independent set of cell proliferation experiments indicates that bFGF concentrations that increase cell motility also increase cell proliferation rates. A model based on a cellular automaton was developed to describe the proliferation of migrating cells. Our cellular automaton models asynchronous proliferation of cells executing persistent random walks and accounts for changes in the direction of movement when two cells collide. The simulation results reveal that cell motility reduces the adverse effects of contact inhibition on cell proliferation rates. Excellent agreement between model predictions and experimental data was observed indicating that this discrete model can accurately describe the dynamics of populations of migrating and proliferating cells.Item Contaminant Removal from Liquid Fuels and Oil-Contaminated Soils: Elucidating the Fundamental Mechanisms of Adsorptive Desulfurization and Pyrolytic Remediation(2021-12-03) Dias da Silva, Priscilla da; Zygourakis, Kyriacos; Wong, Michael S.Adsorptive desulfurization of liquid fuels is an emerging technology that may allow for the portable generation of electricity using jet fuel-powered fuel cell systems. Pyrolytic remediation of oil-contaminated soils is a thermal treatment that may sustainably detoxify oil-contaminated soils using less energy and preserving soil integrity. This work investigates the underlying mechanisms of these processes through advanced thermo-analytical techniques and mathematical analysis. First, adsorptive desulfurization with CuNa-Y zeolite was studied at temperatures between 30 °C and 180 °C as an approach to remotely remove sulfur present in liquid fuels for fuel cell applications. The amount of sulfur selectively removed from jet fuel and adsorbed onto the Cu sites of the zeolite increased by almost 14x as the temperature was raised from 30 °C or 80 °C to 180 °C. Elevated temperatures promoted the formation of covalent sulfur-metal bonds that displaced other aromatics that do not contain sulfur, which are present at much higher concentrations and compete for adsorption. Operating a flow-through adsorber at 180 °C could effectively reduce the sulfur content in jet fuel to ultra-low levels (1-10 ppmw) over a broad range of liquid hourly space velocities (0.13 to 3.24 h^(-1)). Detailed characterization revealed that desulfurization occurs in two stages. Sulfur is initially removed via adsorption (chemisorption) on the CuNa-Y zeolite, an assertion supported by simulations with a transient heterogeneous model. As the adsorbent becomes saturated, however, surface chemical reactions start taking place leading to the decomposition of benzothiophenes. As a result, the process continues to remove sulfur from the jet fuel feed even after it has exceeded its theoretical adsorption limit. Second, pyrolytic remediation of soils contaminated with heavy hydrocarbons, including polycyclic aromatic hydrocarbons (PAHs), was studied at temperatures between 300 °C and 420 °C. Our team developed a novel methodology that combines thermo-analytical measurements and mathematical methods to inform the reliable pyrolytic temperatures for specific soil/contaminant systems. To achieve that, we characterized the complex network of soil and contaminant transformations using thermogravimetry coupled with evolved gas analysis. Additionally, we investigated the contribution of clays during pyrolytic remediation of soils. Clays were found to be the primary component in soil that retains PAHs such as pyrene and therefore sets the remediation intensity requirements. Bentonite modified with Fe(III), Fe-bentonite, performed as a catalyst under pyrolytic conditions, decreasing the temperature at which hydrocarbon decomposition reactions were triggered. The addition of 10%wt Fe-bentonite decreased the residual total petroleum hydrocarbon (TPH) content by 65.9% for treatments at 370 °C and by 79.3% for treatments at 300 °C. Moreover, treatment at 300 °C with the addition of Fe-bentonite resulted in a similar residual TPH when compared to the treatment at 370 °C with no additives. Using such earth-abundant amendments during pyrolytic remediation of oil-contaminated soils could improve energy usage, reduce associated carbon dioxide emissions and lessen unwanted soil transformations. Overall, this work elucidates the extent and the mechanism of separation and transformation of contaminants like benzothiophenes, heavy hydrocarbons and PAHs at selected temperatures. The findings reported here contribute to the development of efficacious approaches to remove such contaminants from sensitive environments.Item Design of artificial genetic networks to regulate the biosynthesis of polyhydroxyalkanoate copolymers with desirable structures(2008) Iadevaia, Sergio; Zygourakis, KyriacosThe design of artificial genetic networks constitutes a powerful tool to regulate cellular physiology. Simple regulatory structures comprised of a few interacting genes can be assembled to engineer desirable phenotypes and control the biosynthesis of end products of biomedical and/or biotechnological interest. This doctoral thesis has focused on the in silico design of artificial genetic networks to drive the biosynthesis of a specific product of biotechnological interest, namely, PHA copolymer chains with desirable structures. In order to understand this complex process, a mathematical model was developed to describe the coupling between the dynamics of polymer and monomer formation and those of the genetic networks. The modeling studies have focused on the utilization of two synthetic networks, known as the genetic toggle and repressilator. The results indicate that the bistable toggle allows regulating the monomer composition of PHA copolymers. The use of the repressilator offers a higher level of control, as it enables the synthesis of PHA block copolymers with different length and composition of each of the blocks that comprise the chains. Additional computational studies have revealed the possibility to achieve superior performance than that of the repressilator, through the design of a novel genetic network that exhibits oscillatory dynamics with minimal overlap amongst gene expression levels. The oscillations were also found to be robust to stochastic fluctuations. Finally, an existing mathematical model was modified to explain the discrepancy of the original repressilator model with experimental data. The modeling studies support the hypothesis that non-specific interactions may also be present in addition to the original three promoter-repressor interactions, which the repressilator was designed to include.Item Development of a 3D Tissue Engineered Bone Tumor Model(2013-09-16) Burdett, Emily; Mikos, Antonios G.; Ludwig, Joseph A.; Kasper, Kurt; Jacot, Jeffrey G.; Zygourakis, Kyriacos3D ex vivo tumor models are required which better replicate the microenvironment encountered by tumor cells in vivo. In this study, we applied bone tissue engineering culture techniques to develop an ex vivo 3D bone tumor model. Ewing sarcoma cells were cultured on poly(ε-caprolactone) (PCL) microfiber scaffolds, and cellular growth kinetics, morphology, and infiltration were assessed. Cell/scaffold constructs were then exposed to anticancer drugs for up to 16 days and drug response was compared to 2D controls. Ewing sarcoma cells were capable of attachment and proliferation on PCL scaffolds and dense scaffold infiltration up to 200 micrometers. Constructs could be maintained in culture for up to 32 days, and high density 3D cell growth conferred an increased resistance to anticancer drugs over 2D controls. This 3D tumor model shows potential for use in future studies of bone tumor biology, especially as it pertains to the development of new anticancer drugs.Item Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations(2002) Payne, Richard Grady; Zygourakis, KyriacosAn injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations was developed. Specifically, a system for encapsulating marrow stromal osteoblasts in gelatin microspheres has been implemented with the goal of incorporation into a crosslinking composite based on poly(propylene fumarate) (PPF). Initially, the microparticle formation procedure was evaluated for effects on the marrow stromal cells. It was determined that the encapsulation procedure had only minor effects on the viability, proliferation, and phenotypic expression through 28 days. The surfaces of the microparticles were treated to provide mechanical integrity at body temperature. The gelatin microparticles were exposed to two levels of a crosslinker in order to assess the effect of crosslinker concentration on cell viability, proliferation, and phenotypic expression. The results indicated that exposure to a relatively high concentration of the crosslinker (5 mM) for a relatively short amount of time (5 min) produced microparticles which maintained their mechanical integrity in 37°C media for about one hour before dispersing. It yielded only minor reductions in the measured properties over 28 days. Physical properties of the crosslinked microspheres were measured. Based on these observations, it was concluded that the encapsulation procedure we had developed was a candidate for use with the crosslinking PPF composite in the next study. Cells encapsulated in crosslinked microparticles were placed on fully crosslinked PPF composites and on composites in various stages of crosslinking. The results showed that encapsulated cells retained their viability and proliferation to a much greater extent than nonencapsulated cells when placed on crosslinking substrates. A final study was performed using one of the crosslinking composite addition times, and varying the formulation of the composites by adjusting the polymer to monomer ratio. The results of this 28 day experiment indicated that encapsulation of cells allowed them to remain viable and express the osteoblastic phenotype when placed on crosslinking PPF based composites. Nonencapsulated cells, however, did not retain their viability on those same crosslinking substrates. The outcome of this work is that the resulting polymeric cell delivery system, which is injectable and in situ crosslinkable, holds promise for bone regeneration and orthopaedic tissue engineering.Item Development of Cell-laden Hydrogel Composites for Osteochondral Tissue Engineering(2015-12-14) Lam, Johnny; Mikos, Antonios G.; Grande-Allen, Kathryn J; Zygourakis, Kyriacos; Kasper, Fred KArticular cartilage is a flexible connective tissue that enables the frictionless and painless articulation of bones in synovial joints throughout the body. Given its avascular nature, articular cartilage tissue inherently exhibits a compromised endogenous capacity for regeneration upon damage. Defects caused by disease or trauma often lead to chronic pain and osteoarthritis as current clinical treatments are still unable to achieve long-term repair. Hence, tissue engineers are developing innovative technologies to provide strategies for successful cartilage repair and regeneration. This thesis focuses on the development and evaluation of cell-based osteochondral tissue engineering solutions based on injectable and biodegradable polymer biomaterials for bone and cartilage repair. First, we developed and characterized oligo(poly(ethylene glycol) fumarate) (OPF)-based hydrogels for osteochondral tissue engineering applications. We investigated the main effects of five main hydrogel fabrication parameters (the poly(ethylene glycol) molecular weight (PEG MW), the crosslinker-to-OPF carbon-carbon double bond ratio (DBR), the crosslinker type, the crosslinking density of encapsulated gelatin microparticles, and the incubation medium composition) as well as their interaction effects on the swelling behavior and degradation of OPF hydrogel composites. We found that increasing the PEG MW increased the mean swelling ratio and decreased the mean mass remaining %, while changing the crosslinker type from methylene bisacrylamide (MB) to PEG diacrylate yielded the opposite effect. Additionally, we found that the swelling of hydrogels fabricated with higher PEG MW or with MB were more sensitive to increases in DBR. From these results, we showed that the swelling and degradation properties of OPF-based hydrogels can be precisely tuned through the modulation of these five fabrication parameters. The second part of this thesis investigated the potential of bilayered OPF hydrogel composites encapsulating chondrogenically and osteogenically pre- differentiated MSCs in a spatially controlled fashion for osteochondral tissue repair. We demonstrated that MSCs that underwent 7 days (CG7), but not 14 days (CG14), of chondrogenic pre-differentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenically pre-differentiated (OS) cells in a bilayered OPF hydrogel. We found that the respective chondrogenic and osteogenic phenotypes of encapsulated MSCs were maintained for up to 28 days in vitro without the need for external growth factors. When taken in vivo, the delivery of CG7 cells, as opposed to CG14 cells, in combination with OS cells via a bilayered OPF hydrogel composite stimulated morphologically superior cartilage repair. Indeed, the present work showed that cartilage regeneration in osteochondral defects can be enhanced by MSCs that are chondrogenically and osteogenically pre-differentiated prior to implantation. Longer chondrogenic pre-differentiation periods, however, resulted in diminished cartilage repair. The final section of this thesis investigated the use of poly(L-lysine) (PLL), previously shown to up-regulate condensation during cartilage development in vitro, as an early chondrogenic stimulant of MSCs encapsulated in OPF hydrogels. We showed that PLL incorporation resulted in early enhancements of type II collagen and aggrecan gene expression as well as increased type II/type I collagen expression ratios when compared to blank controls. We also demonstrated that PLL enhanced N-cadherin gene expression of encapsulated MSCs under certain conditions, suggesting that PLL also likely induced pre-cartilaginous condensation.Item Development of Osteoinductive Tissue Engineering Scaffolds with a Bioreactor(2013-07-24) Thibault, Richard; Mikos, Antonios G.; Grande-Allen, K. Jane; Zygourakis, Kyriacos; Drezek, Rebekah A.; Kasper, Kurt; Jansen, JohnThe conventional treatments for craniofacial bone defects currently are unsatisfactory due to several drawbacks. Replacement of lost bone by autografts typically causes donor site morbidity while allografts, xenografts, and demineralized bone matrix all have a chance of disease transmission. Current synthetic implants placed within the defect site generally lack osseointegration and biodegradability. There are several methods of generating a hybrid extracellular matrix (ECM) and synthetic material construct. These include coating the synthetic material scaffold with collagen and calcium phosphate, incorporating acellular biological tissue within the scaffold material, and using cells to generate an ECM coating on the synthetic material scaffold. The research performed for this thesis developed and characterized mesenchymal stem cell (MSC)-generated extracellular matrix poly(ε-caprolactone) constructs (PCL/ECM) for the replacement of bone tissue. The osteogenic potential of the PCL/ECM constructs was explored by culturing i) MSCs and ii) whole marrow cells combined with MSCs onto the construct with or without the osteogenic differentiation supplement, dexamethasone. It was established that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs was maintained even in the absence of dexamethasone and that the co-culture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow. The osteogenicity of the constructs encouraged the characterization of the protein and mineral composition of the ECM coating on the PCL/ECM constructs. Characterization revealed that at short culture durations the MSCs used to generate the ECM deposited cellular adhesion proteins that are a prerequisite protein network for further bone formation. At the later culture durations, it was determined that the ECM was composed of collagen 1, hydroxyapatite, matrix remodeling proteins, and regulatory proteins. The prior studies on the PCL/ECM constructs persuaded exploration of the effect of various devitalization and demineralization processes on the retention of the ECM components within and the osteogenicity of the PCL/ECM constructs. Analysis demonstrated that the freeze-thaw technique is a milder method of devitalization of cell-generated ECM constructs as compared to other methods, but it reduced the osteogenicity of the constructs. In addition, it was elucidated that void spaces in the surface of the constructs are important for allowing access of MSCs into the interior of the constructs.Item Elucidating the connection between cell population heterogeneity and genetic regulatory architecture in specific artificial networks(2009) Portle, Stephanie; Zygourakis, KyriacosUnderstanding the expression patterns of simple, synthetic gene regulatory networks will not only shed light into the complexity of naturally occurring networks, but it will also provide a platform for expression control that can be valuable in biotechnological applications. The expression of regulatory networks is influenced by the fact that the intracellular environment varies among the cells of a population. In turn, this variability is tightly related to the architecture of such networks. The relationship between the architecture of synthetic regulatory networks and cell population heterogeneity was studied using two model regulatory networks: a gene-switching system and an oscillatory system. A green fluorescent protein (GFP) served as the reporter for both systems, which were expressed from plasmids in the Gram-negative bacterium Escherichia coli. Inducer concentrations were varied in shake flask cultures, and GFP distributions were monitored over time with flow cytometry. In studying the effect of GFP half-life on the gene-switching network behavior, we observed how it influences the view of the network behavior: using a lower half-life GFP reduced the inducer concentration range at which we could distinguish between network states due to lower GFP expression, but its use also showed better evidence of the fast-switching transient behavior predicted by the network architecture through wider separation of states. The oscillatory network was shown to exhibit three steady states, bi-threshold behavior, and multiplicity, contrary to behavior predicted by an existing model. We experimentally discovered four significant nonspecific interactions between promoters and repressors within the network that, through modeling, can be shown to qualitatively create the behavior experimentally observed. Beyond the understanding of network behavior gained through the combination of average and population-level data, the distributions demonstrated a connection between the network architecture and heterogeneity. We found heterogeneity expanded at intermediate inducer levels in both networks, when the distribution was bimodal (gene-switching network) or individual cells were displaying oscillatory behavior (oscillatory network). Both the oscillatory behavior and bimodal distributions are a result of the network architectures. We had the ability to restrict heterogeneity with multiple inducers in the oscillatory network. However, there were observable limits in doing so.Item Fundamental mechanisms of coal pyrolysis and char combustion(1992) Matzakos, Andreas N.; Zygourakis, KyriacosCoal pyrolysis and combustion have been systematically investigated at high temperatures where external and intraparticle transfer limitations become important. A thermogravimetric reactor equipped with in-situ video imaging capabilities provided the reaction rate measurements while its video microscopy system simultaneously allowed observation of the pyrolyzing or combusting coal particles. Video microscopy permitted direct observation of several transient phenomena occurring during combustion (particle ignition, macropore opening, particle fragmentation) or pyrolysis (particle swelling and bubbling) and these phenomena have been correlated with the combustion or devolatilization rate measurements. Particle ignition causes a sharp increase in the char combustion rates. The probability of particle ignition increases with increasing particle size, increasing porosity, increasing oxygen concentration and decreasing gas flow rate. Macropore opening also enhances char reactivity. During pyrolysis, the most vigorous bubbling of the particles occurred when the devolatilization rate was at its maximum. Pyrolysis conditions also affect char ignition behavior. Increasing pyrolysis heating rates result in chars with more open macropore structure and higher reactivity in the diffusion-limited regime. However, heating rates do not affect reactivity in the kinetic control regime. Chars pyrolyzed in 5% oxygen are more swollen and more porous than chars produced in pure nitrogen and are also more reactive in the diffusion-limited regime. Finally, increasing soak times and heat treatment temperatures deteriorate char reactivity in all regimes. A cellular automaton algorithm was developed to simulate combustion of chars with complex macropore structures in the diffusion-limited regime. This model accounts for diffusional limitations by assuming a finite penetration length of gas inside the porous solid and by treating the closed pores as inaccessible to the reactants. Computational grids were generated to model the structure of chars prepared at three different heating rates. Simulation results suggest that char reactivity depends strongly on macroporosity and macropore specific surface area. In agreement with our experimental reactivity measurements, the simulations show significant reactivity differences of the studied chars, even under isothermal conditions. The simulations do not detect significant particle fragmentation at conversions as high as 81%. Small fragments were produced though, at all conversions and their number reached a maximum at about 95% conversion.Item Hybrid computational modeling of cell population and mass transfer dynamics in tissue growth processes(2005) Cheng, Gang; Zygourakis, KyriacosThis work presents a comprehensive hybrid computer model simulating the cell population and mass transfer dynamics during tissue growth processes. The model has three major components: (a) a discrete algorithm simulating individual cell activities and cell-cell interactions; (b) transient, three-dimensional partial differential equations (PDE's) describing the convection, diffusion, consumption and, possibly, secretion of nutrients or other important substances in tissue systems; and (c) equations describing how cell behavior is modulated by the local concentration fields. The hybrid model is first used to study the growth of bioartificial tissues under conditions leading to nutrient depletion. Simulation results indicate that large tissue size, low nutrient diffusivity, high cell uptake rate and low nutrient concentration in the culture media lead to severe transport limitations and have serious adverse effects on the growth rates and the structure of bioartificial tissues. The incorporation of perfusion channels is one of the proposed methods for alleviating diffusional limitations. However, the selection of optimal channel placement and size leads to an interesting optimization problem. Our results indicate the existence of an optimal channel diameter for each set of cell parameters and culture conditions. As diffusional limitations become more severe, larger perfusion channels are needed and the value of the achievable cell density decreases. Finally, the hybrid model is used to study the acid-mediated growth of solid tumors. With its ability to describe the complex, three-dimensional vasculature of tissues invaded by tumors, our model represents a significant extension of previous two-dimensional studies. In addition to a three-dimensional capillary network generated from literature data, tree-like capillary networks with adjustable overall vascularity are generated using a bifurcating distributive algorithm in order to study the effect of host vascularity on tissue growth. Our simulations produce tumor growth curves similar to those observed clinically. The predicted range of tumor cell acid production rate shows better agreement with experimental values than existing two-dimensional models. Our model can also predict the universal existence of necrotic regions in large tumors.Item Ignition of coal and char particles: Effects of pore structure and process conditions(1998) Perkins, Dosite Samuel, II; Zygourakis, KyriacosThis study reports the results from experimental and theoretical studies aimed at elucidating the effects of particle pore structure and process conditions on the ignition of coal and char particles. A novel reactor combining thermogravimetric analysis and video microscopy imaging (TGA/VMI) was used for our combustion studies. By allowing simultaneous observation of light emissions from igniting particles and measurement of sample reactivity from weight-loss data, the TGA/VMI reactor was very effective in detecting and characterizing particle ignitions. Investigations of ignition mechanism showed that the ignition of char particles typically occurs heterogeneously, while coal particles may ignite heterogeneously, homogeneously, or by a combination of both mechanisms. Homogeneous ignitions were favored by high oxygen concentrations and close particle interactions. Other transient phenomena such as multiple ignitions of a single particle were also observed. A second hot stage reactor was also used for our studies to achieve heating rates as high as 1000$\sp\circ$C/sec. We found that chars prepared at higher pyrolysis heating rates ignited more frequently and exhibited higher reactivity when combusted at temperatures leading to diffusional limitations. Due to inherent differences in the two reactors, chars prepared in the hot stage at the same conditions as those prepared in the TGA/VMI exhibited lower ignition and reactivity behavior. Despite these differences, chars prepared at the highest heating rates in the hot stage reactor clearly exhibited higher reactivity and ignited more frequently. Mathematical modeling efforts focused on the coupled transient mass and energy balances governing the diffusion-reaction problem in char combustion. Changes in particle size and pore structure with conversion were described using experimental data obtained in our laboratory. Combustion kinetics were also measured experimentally. Theoretical predictions agreed very well with experimentally observed trends. Important char structural properties which favored ignitions were larger particle radii, more open macropore structures, and larger macropore surface areas. Process conditions which increased particle ignitions were high pyrolysis heating rates, high combustion temperatures and increased oxygen concentrations during combustion.Item Influence of pyrolysis conditions on macropore structure of char particles(1993) Boissiere, Francois Patrice; Zygourakis, KyriacosA systematic analysis of the structure of char particles produced from an Illinois #6 coal was carried out. Coal particles were pyrolyzed in a hot-stage reactor under inert (N$\sb2$) and reactive (5% O$\sb2$ / 95% N$\sb2$) atmospheres at various heating rates (0.1, 1 and 10$\sp\circ$C/s) and final heat treatment temperatures (500 and 700$\sp\circ$C). Image analysis procedures were used to measure the size and macroporosity of the particles, and the surface area and size distribution of the macropores. High heating rates and the addition of oxygen resulted in larger particle swelling and increased the macroporosity, the surface area and the fraction of large macropores with thin walls. Heat treatment temperatures had smaller effects on char particle structure. Our data confirm the strong effects of pyrolysis conditions on char reactivity during combustion in the diffusion-limited regime. They also provide the necessary parameters for char combustion models.Item JP-8 Desulfurization by CuNa-Y Zeolite at Elevated Temperatures Has Two Distinct Stages: Chemisorption Followed by Surface Reactions(American Chemical Society, 2021) da Silva, Priscilla Dias; Wong, Michael S.; Zygourakis, KyriacosThis study evaluates the performance of continuous-flow adsorbers for adsorptive desulfurization. JP-8 fuel with 2230 ppmw of sulfur was treated in a flow-through adsorber packed with CuNa-Y zeolite pellets and operating at 180 °C and 200 psig with liquid hourly space velocities (LHSV) from 0.13 to 3.24 h–1. Our results showed that a flow-through adsorber operating under these conditions can effectively reduce the sulfur content of JP-8 to ultralow values (1–10 ppmw) over the entire LHSV range tested, although the overall performance of the adsorber declined with increasing flow rates as expected. We also observed that the total sulfur removal exceeded the theoretical adsorption limit of our zeolite adsorbent. Detailed characterization of the treated fuel and spent adsorbent via chromatographic and surface analysis techniques revealed that desulfurization occurs in two stages. Sulfur is initially removed via adsorption (chemisorption) on the CuNa-Y zeolite, an assertion supported by simulations with a transient heterogeneous model. As the adsorbent becomes saturated, however, surface chemical reactions start taking place, leading to the formation of hydrogen sulfide and polymerization products and depositing carbon residues on the zeolite. The spent adsorbent was regenerated by treating it with air at 550 or 600 °C, which restored the adsorption capacity of the material to about 90% of its initial value.Item Linking a mitotic oscillator to the extracellular environment: the importance of protein network structure and multisite phosphorylation(2010) Vargo, Ryan Christopher; Zygourakis, KyriacosThis thesis work contributes the first vital steps in the development of a biologically based proliferation model to advance a bioartificial tissue regeneration model. Specifically, this work presents a mitotic oscillator model incorporating ATP, which was linked to extracellular glucose. This model is the first mitotic oscillator linked to the extracellular environment. Furthermore, this work is the first to connect extracellular glucose to mitosis with ATP. Taking a bottom-up approach, a base mitotic model was developed using the latest biology. The reaction network structure of mitosis is not fully understood, and the role of multisite phosphorylation is uncertain. Therefore, using bifurcation analysis and transient simulations, the effect of the mitotic reaction network structure and multisite phosphorylation on system behavior was analyzed by varying the MPF activation network structure, the number of positive feedback loops, and the number of phosphorylations on the positive feedback loop proteins. The results suggest that the MPF activation network has evolved to efficiently utilize cyclin B and to generate switch-like transitions into mitosis. The behavior of the mitotic oscillator model was affected by the order and number of multisite phosphorylations, which are essential to generate sharp switch-like transitions into mitosis. Addition of multiple positive feedback loops into the model enhanced the signal to initiate mitosis. Next, ATP was incorporated into the network. The model was then tuned to a relative ATP concentration, which is generic and therefore applicable to different cell lines. Multiple Wee1 networks were analyzed to elucidate the function of the two inhibition mechanisms, kinase inhibition and increased degradation. The results suggest that the inhibition mechanisms are redundant. Therefore, the model incorporates the Wee1 mechanism that allows the cell to maintain maximum control over the initiation of mitosis. To generalize the mitotic model, the parameter set was tuned for to a relative ATP concentration and fibroblast division times. Finally, the relative intracellular ATP model was linked to the extracellular glucose. The model developed in this thesis work is the first to use ATP as the link between mitosis and the extracellular glucose, and the first mitotic model connected to the extracellular environment.
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