Chemical & Biomolecular Engineering Theses, Dissertations, & Student Research Copyright (c) 2013 University of Nebraska - Lincoln All rights reserved. http://digitalcommons.unl.edu/chemengtheses Recent documents in Chemical & Biomolecular Engineering Theses, Dissertations, & Student Research en-us Thu, 24 Jan 2013 13:10:28 PST 3600 Application of Response Surface Methodology and Central Composite Design for 5P12-RANTES Expression in the <i>Pichia pastoris</i> System http://digitalcommons.unl.edu/chemengtheses/16 http://digitalcommons.unl.edu/chemengtheses/16 Thu, 06 Dec 2012 12:46:30 PST Pichia pastoris has demonstrated the ability to express high levels of recombinant heterologous proteins. Protein expression is enhanced during fermentation at high cell density. However, the level of expression is mainly regulated by fermentation operation factors. This research is directed to investigate the effect of methanol growth rate, temperature and pH in the expression of the total 5P12-Rantes concentration, expression of active 5P12-Rantes and Specific yield using the response surface methodology and central composite design.

The response surface methodology, RSM, has been used successfully used by Zhang, W. and Ian, M. to optimize the cell density and fermentation process. The RSM is equipped with statistical tools to determine the significance of a factor over a response. The evaluation of factors using the RSM uses experimental design in order to distribute the selected variables within the boundaries of the design.

The central composite design, CCD, allows allocation of Methanol growth rate, temperature, and pH to evaluate their effect in the expression of 5P12-Rantes. The effect of methanol growth rate is evaluated from a minimum value of 0.01 h-1 to a maximum of 0.4 h-1, 24.06 °C to 29.94 °C for temperature and 1.99 to 4.51 for pH.

The methanol growth rate has demonstrated to have no effect in any of the responses. In contrast, Temperature and pH has a significant effect in all the responses. However, the lack of fit of the proposed model doesn’t allow good estimations of predicted responses. In order to minimize the lack of fit of the propose models, the methanol growth rate has been excluded from all the three models. A new 2 factor second order model is proposed to analyze the significance of temperature and pH. The lack of fit decreased and the optimal operating conditions for temperature and pH was determined at 27.14 °C and 3.16 which results in a maximum of 26.52 g/L of active 5P12-Rantes and 0.16 mg Rantes/g dry cell.

Adviser: William H. Velander

]]> Frank M. Fabian Optimization of Biodiesel Production Plants http://digitalcommons.unl.edu/chemengtheses/15 http://digitalcommons.unl.edu/chemengtheses/15 Fri, 26 Oct 2012 10:45:28 PDT A conventional biodiesel plant utilizing two distillation columns to purify unreacted reactants and products is considered in this study. Thermodynamic analyses are used to assess the performance of the existing distillation columns, and reduce the costs of operation by appropriate retrofits in a biodiesel production plant. After the retrofits, the overall exergy loss for the two columns has decreased from 2430.87 kW to 1674.12 kW.

A reactive distillation is developed for esterification of lauric acid with methanol using equilibrium and nonequilibrium models. Equilibrium modeling dominated during last few decades due to their straightforward mathematical modeling. In reality, separation depends on the heat and mass transfer rates between liquid and vapor phases and a more sophisticated nonequilibrium modeling is more suitable to describe the separation process. Further, thermally coupled side-stripper reactive distillation sequence is used to reduce the overall energy consumption of the reactive distillation column and the methanol recovery column of the equilibrium design. The total exergy losses for the columns are reduced by 281.35 kW corresponding to 21.7% available energy saving.

In order to design a new generation biodiesel plant, direct carboxylation and glycerolysis routes are developed to convert a by-product, glycerol, of the biodiesel production plant into a value-added product, glycerol carbonate, to reduce the unit cost of the biodiesel production plant. A direct comparison of the economic analysis based on deterministic and stochastic models of the conventional biodiesel plant, biodiesel-glycerol carbonate production by direct carboxylation plant and biodiesel-glycerol carbonate production by glycerolysis plant is presented. The results show that either route can be used to reduce the unit cost of the biodiesel production plant.

Advisor: Yasar Demirel

]]> Nghi T. Nguyen Fabrication and Characterization of Thermomechanically Processed Sulfur and Boron Doped Amorphous Carbon Films http://digitalcommons.unl.edu/chemengtheses/14 http://digitalcommons.unl.edu/chemengtheses/14 Thu, 23 Aug 2012 13:35:28 PDT Small scale, high power density, reliable, and long-life power supplies would be useful or even critical for space missions or the growing number of microdetectors, microsensors, and miniature vehicles. Alpha or beta particle voltaic devices could satisfy these requirements but have been shown to degrade quickly due to radiation damage. Amorphous carbon (a-C) PN junctions or PIN devices could provide radiation hardness and sufficiently high efficiency. As the range of alpha and beta particles in a-C is ~20-120μm, much thicker films than are typical are needed to maximize collection of the particle energy.

In this work, the fabrication of thermomechanically processed p- and n-type doped a-C films were investigated as a first step in the future development of radiation hard voltaic devices. Boron carbide (B4C) powder was mixed with a-C nanopowders as a possible p-type dopant with sulfur powder utilized as a possible n-type dopant. Doping levels of 2.5at%, 5.0at%, and 10.0at% were investigated for both dopants with films pressed at 109°C over a pressure range of 0.3-5.0GPa. Initial attempts to fabricate rectifying PN junctions and PIN devices was unsuccessful.

Bonding properties were characterized using Raman spectroscopy with electronic properties primarily assessed using the van der Pauw method. Undoped a-C and boron-doped films were found to be slightly p-type with sulfur-doped films converting to n-type. All films were found to consist almost entirely of nano-graphitic sp2 rings with only slight changes in disorder at different pressures. Sulfur doped films were less brittle which is indicative of crosslinking.

Boron doping did not significantly change the film electronic properties and is not an effective dopant at these temperatures and pressures. Sulfur doping had a greater effect and could likely be utilized as basis for an n-type material in a device.

Initial irradiation studies using alpha particles showed that boron and undoped films became more p-type with sulfur films converting to p-type. The sulfur doped films returned to n-type after isothermal annealing.

]]> Lonnie Carlson PURIFICATION OF FIBRINOGEN FROM HUMAN PLASMA http://digitalcommons.unl.edu/chemengtheses/13 http://digitalcommons.unl.edu/chemengtheses/13 Mon, 30 Jul 2012 10:20:36 PDT A solvent detergent treated fibrinogen was purified from human plasma by cryoprecipitation (cryo) followed by chemical precipitation using ethanol (EtOH) or ammonium sulfate (AS) as precipitating agents. Amounts of fibronectin (FN), factor XIII A-subunit (FXIIIA), factor XIII b-subunit (FXIIIB), and alpha2-antiplasmin (α2-AP) in the isolated fibrinogen were quantified. Thromboelastography (TEG) analysis was used to evaluate the clot strength of the isolated fibrinogen and to determine the ability of the ethanol and ammonium sulfate precipitations to eliminate the solvent detergent. Sodium dodecylsulfate-polyacrylamide gel analysis indicated that fibrinogen produced by each of these precipitation methods had similar purity. Quantitative western blot analysis revealed that fibrinogen produced by ammonium sulfate precipitation contained increased amounts of FN, FXIIIB, and α2-AP. TEG analysis showed that ammonium sulfate precipitated fibrinogen yielded a fibrin clot with the highest maximal strength. In addition, a single ethanol precipitation was sufficient to remove the solvent detergent while a single ammonium sulfate precipitation was not effective in removing the solvent detergent mixture Tri (n-butyl) phosphate (TNBP) and Triton X-100 as judged by the ability of the material to form a clot.

Adviser: William H. Velander

]]> Ayman E. Ismail Technological Aspects of Molecular Diagnosis of Bacterial Infectious Diseases http://digitalcommons.unl.edu/chemengtheses/12 http://digitalcommons.unl.edu/chemengtheses/12 Mon, 23 Apr 2012 14:10:12 PDT The polymerase chain reaction (PCR) is continually growing in its application, particularly in the field of molecular diagnosis of disease from clinical specimens. The main focus has been in the detection and identification of pathogens. However, quantitative PCR is increasingly utilized to determine initial pathogen load. A well-designed PCR protocol is required in all of these instances. Just as importantly, in the context of disease diagnosis; is the design of the sample processing methodology. The ideal method should concentrate the DNA and effectively isolate a high-quality DNA product, free of PCR inhibitors, while also being simple, reproducible and safe.

The aim of this work is to address the research challenges posed in the preceding paragraph. A previously developed prototype diagnostic system is used to analyze and suggest improvements and an application of the technology is also described. Briefly, the system includes a polystyrene strip that is inserted into a lysis microreactor (LMR) that is fitted with an impeller and temperature control to lyse DNA. The DNA binds noncovalently to the strip and is transferred through a wash step to the thermocycler cuvette for amplification.

The research challenges were addressed by the following:

  1. An analytical model was developed to determine the efficiency of each process comprising a PCR cycle. Using this model, reaction conditions can be directly linked to the overall yield and initial template concentration can be determined from real-time PCR data.
  2. The flow characteristic of the LMR was solved by computational fluid dynamics to determine the DNA capture efficiency as a function of initial position.
  3. Improvements to the use of a non-specific strip for DNA binding were explored by attaching target-complimentary oligonucleotides to a surface.
  4. The prototype system was evaluated on a bank of frozen clinical stool samples. Samples were tested for Clostridium difficile genomic DNA and the results compared with standard C. difficile testing methods used routinely by a hospital clinical laboratory. The prototype system showed 97.5% concordance with standard testing methods.

Adviser: Hendrik J. Viljoen

]]> Christine S. Booth Real-time characterization of ultra-thin organic layers via simultaneous spectroscopic ellipsometry and piezoelectric nanogravimetry http://digitalcommons.unl.edu/chemengtheses/11 http://digitalcommons.unl.edu/chemengtheses/11 Mon, 23 Apr 2012 12:38:36 PDT Analysis techniques are needed to determine the quantity and structure of materials composing an organic layer that is below an optical ultra-thin film limit and in a liquid environment. Neither optical nor acoustical techniques can independently distinguish between thickness and porosity of ultra-thin films due to parameter correlation. A combined optical and acoustical approach yields sufficient information to determine both thickness and porosity. The author describes application of the combinatorial approach to measure single or multiple organic layers when the total layer thickness is small compared to the wavelength of the probing light. The instrumental setup allows for simultaneous in-situ spectroscopic ellipsometry and quartz crystal microbalance dynamic measurements, and it is combined with a multiple-inlet fluid control system for different liquid solutions to be introduced during experiments. A virtual separation approach is implemented into an analysis scheme, differentiated by whether or not the organic adsorbate and liquid ambient densities are equal. The analysis scheme requires that the film be assumed transparent and rigid (non-viscoelastic). The author presents and discusses applications of the approach to studies of organic surfactant adsorption, self-assembled monolayer chemisorption, and multiple-layer target DNA sensor preparation and performance testing.

Advisor: Mathias Schubert

]]> Keith B. Rodenhausen Jr. Nanoparticle Necklace Network Arrays Exhibiting Room Temperature Single-Electron Switching http://digitalcommons.unl.edu/chemengtheses/10 http://digitalcommons.unl.edu/chemengtheses/10 Thu, 01 Dec 2011 11:36:46 PST A single nanoparticle is one of the most sensitive electronic devices for sensing chemicals in a gas or liquid. The conductivity of a single Au nanoparticle is significantly modulated by the binding of a molecule that alters charge by just one electron. However, the single-electron sensitivity requires cryogenic temperatures and interconnection is not easy. A patterned two-dimensional network of one-dimensional nanoparticle necklaces made up of 10 nm Au particles are fabricated and shown to exhibit similar single-electron effect at room temperature. Furthermore, the long range conductivity of over 10’s of microns makes the structure easy to self-assemble onto conventional microelectronics circuitry. A device exhibiting single-electron effect is characterized by highly non-linear current-bias behavior where at bias, V > VT current rises rapidly and scales as (V/VT – 1)ζ, where ζ ≥ 1 is the critical exponent and VT is the threshold voltage. Below VT, current does not flow. Thus, VT is the switching voltage and larger ζ signifies sharper switching characteristics. While arrays of one and two dimension are well known to exhibit appreciable VT at cryogenic temperatures, at ambient temperatures the blockade effect vanishes. The unique architecture of the necklace network results in a weak dependence of VT on temperature which leads to room temperature single-electron effect. The high sensitivity of the nanoparticle necklace network array at room temperature allows coupled live cells to electronically switch, or gate, the device through cellular metabolic activity. Additionally, the critical exponent, ζ, which is a measure of how current will rise during switching, can be significantly enhanced by cementing the necklaces with the dielectric material CdS, thereby greatly increasing the switching gain and sensitivity of the device. Given robust room temperature single-electron switching, enhanced ζ values, cellular coupling capability, and natural integrability with microelectronics circuitry, nanoparticle necklace network arrays have the potential to be implemented in a wide range of applications, such as, chemical sensors, biofuel cells, biomedical devices, and data storage devices.

Adviser: Ravi F. Saraf

]]> Jennifer L. Kane Expression and Characterization of Protein Chimera with Embedded Activated Protein C Generation http://digitalcommons.unl.edu/chemengtheses/9 http://digitalcommons.unl.edu/chemengtheses/9 Fri, 08 Jul 2011 11:14:54 PDT Increasing demand of medical implants has lead researchers to develop biomaterial surfaces that offer improved performance and lower thrombogenecity. There have been attempts to model biomaterial surfaces after the native endothelium as it represents an optimal non-thrombotic surface. Typically, when biomaterials are contacted with blood, they often invoke the activation of clotting cascades. Thus, to overcome surface mediated thrombotic events, proteins and molecules with anticoagulant attributes of the endothelium have been immobilized onto biomaterial surfaces. Protein C (PC) plays a main role in blood coagulation and acts as an anticoagulant when it is converted into activated protein C (APC). Activation of PC can be initiated only by thrombin (TR)- thrombomodulin (TM) complex with a relatively slower reaction, and is enhanced 20- fold when EPCR, the co-factor to PC, is also present. The objective of this thesis is to generate a chimeric protein that includes the functional domains of both EPCR and TM in an attempt to generate APC in-situ. The synthetic genes encoding the functional domains of EPCR (1-193) and TM (224-462) with either a myc or flag linker along with a histidine tag were first assembled by PCA/PCR method, sequenced and the synthetic genes encoding the correct sequences for chimera I, chimera II, and TM2-His were successfully expressed in yeast Pichia host X-33 strain. Expressed protein was analyzed by Western blotting analysis, purified using Ni-chelate chromatography, and the ability of EPCR-TM chimera to catalyze PC activation was tested in solution phase. Overall, the expression level of chimera I, chimera II, and TM2-His was low, and mature proteins were partially secreted into the medium, and His-tag purification yielded partially pure products. Chimera containing EPCR and TM was noted to generate 1.8-fold higher APC compared to standard TM at comparable concentrations.

Advisor: Anuradha Subramanian

]]> MinJeong Schneider DE NOVO GENE SYNTHESIS BY RAPID POLYMERASE CHAIN ASSEMBLY COUPLED WITH IMMUNOAFFINITY PURIFICATION: A NOVEL PROCESS AND WORKSTATION http://digitalcommons.unl.edu/chemengtheses/8 http://digitalcommons.unl.edu/chemengtheses/8 Tue, 12 Apr 2011 07:08:17 PDT The de novo synthesis of genes is emerging as a powerful tool in biotechnology. The ability to synthesize genes of any desired sequence opens the door to seemingly unlimited research possibilities. Major advances have been made recently in de novo gene synthesis, with Polymerase Chain Assembly (PCA) routinely used to construct functional sequences from short single-stranded oligonucleotides. However, current PCA techniques are lacking in speed and fidelity. Additionally, substantial undesired reactants/products are present in the final reaction. A novel process and accompanying workstation has been developed that incorporates rapid PCA synthesis coupled with subsequent affinity purification of the synthesis mixture. The system enables fast and accurate PCA synthesis and isolation of the full length DNA of interest.

In the synthesis step, the desired sequence is assembled and PCR amplified in a fast thermocycler to generate a high yield of product with minimal runtime and errors. A traditional 2-step PCA-PCR approach is utilized to assemble and amplify the full-length gene. Alternatively, integration of PCA and PCR into a single rapid reaction is also employed, working reliably up to about 1 kb. For the synthesis of genes longer than 1.5 kb, a convergent rapid synthesis strategy is proposed in which the full-length sequence is assembled by a series of synthesis steps from smaller fragments. In this work, a variety of genes ranging from 600 bp up to 3.8 kb in length are synthesized by rapid PCA techniques.

The second section of the workstation employs two affinity columns to isolate the desired full-length product from shorter unwanted reactants/products inherent in the PCA reaction. During PCR amplification, labels are incorporated into the desired product on both ends via PCR primers. Undesired products contain only one of these labels, or no label at all. The first column interacts with one of the labels to partially purify the mixture. The intermediate product is then subsequently purified via the second column to isolate the full-length sequence. An initial prototype workstation was developed, while the 2nd generation instrument consisted of process refinements using two antibody columns for immunoaffinity purification.

]]> Joel R. TerMaat An integrated approach for phytate degradation and recovery of myo-inositol and phosphate as valued-added products from the by-products of corn ethanol industry http://digitalcommons.unl.edu/chemengtheses/7 http://digitalcommons.unl.edu/chemengtheses/7 Tue, 28 Dec 2010 08:39:52 PST An integrated process was developed to hydrolyze the phytates in light steep water (LSW) and to simultaneously isolate inorganic phosphate (Pi) and myo-inositol products. The proposed integrated process is helpful in resolving the environmental and nutritional concerns in the use of corn gluten feed (CGF) in the animal diets. This process comprised of partial and total hydrolysis of LSW and intermediate anion exchange separation technique. The phytates in LSW were initially degraded to negatively charged myo-inositol phosphates (InsP2 - InsP5). The optimized experimental parameters for the partial hydrolysis of LSW were determined to be 2 h hydrolysis with 1 FTU A. niger/g substrate at 35°C. The negatively charged species of the partially hydrolyzed substrate were separated on a strong base anion exchange resin. The negatively charged species, retained by the resin, were eluted with 1M NaCl solution and were subjected to complete hydrolysis with E. coli, A. niger-derived phytases and their respective combinations. The maximum amount of myo-inositol released from the anion exchange column was detected after 48 h reactions catalyzed by 100 FTU E. coli, 150 FTU E. coli, and 150 FTU the combination of A. niger and E. coli. The time course of Pi released showed a similar trend to that of myo-inositol and the released Pi reached a maximum amount after 48 h incubation at the enzyme loadings for which the maximum concentration of myo-inositol were reached.

An isocratic HPLC method was developed for routine analysis of myo-inositol and Pi. For myo-inositol, the limit of detection (LOD) was 0.01 mg/ml, and the limit of quantification (LOQ) was 0.04 mg/ml. The linear range of this method for myo-inositol was 0-20 mg/ml. The HPLC method is also a fast method for Pi quantification in the hydrolysate. The linear range of this method for Pi was 0-10 mg/ml. The LOD and LOQ were 0.05 and 0.17 mg/ml, respectively.

A size-exclusion chromatography packed was developed for isolating and purifying myo-inositol and Pi, from the mixture with E.coli phytase and Cl-.

Adviser: Hossein Nouredddini

]]> Jun Dang FIBRONECTIN FOR USE IN HEMOSTATSIS AND WOUND STABILIZATION IN TRAUMA http://digitalcommons.unl.edu/chemengtheses/6 http://digitalcommons.unl.edu/chemengtheses/6 Tue, 30 Nov 2010 13:09:00 PST Exploring the phase during wound healing when fibrinogen (FBG) and fibronectin (FN) interact forming a small thin layer for cells to migrate and proliferate at the injury site is necessary for the primary building blocks and initial stages of skin recovery. The aim of this thesis is to illustrate that administrating a reasonable amount of (FN) and FBG, in a semi-organizational technique, to the wound site will encourage cell population and ECM formation, as well as, improve the wound healing process. WE hypothesized that FBG and FN interactions will configure the construction of dermal cells and their final fate to remodel and flourish the wound.

One section of this thesis will compare the functional integrity of the different forms of FNs. Plasma derived fibronectin (Pd-FN), although different in structure and composition could function similarly to the cellular-derived fibronectin (c-FN).

The short peptide sequence Arg-Gly-Asp-Ser (RGDS), expressed on the structure of FN, helps the molecule to interact with a wide variety of crucial dermal cell receptors known as integrins, The α,β combinations of integrins expressed on the cell’s surface are triggered by external stimulus. The interactions are the initial steps in skin regeneration and recovery. They are responsible for cell migration, differentiation, and proliferation. The interactions are also responsible for ECM formation and anchoring cells to the ECM.

]]> Mohammed Halhouli Recombinant Factors for Hemostasis http://digitalcommons.unl.edu/chemengtheses/5 http://digitalcommons.unl.edu/chemengtheses/5 Wed, 28 Jul 2010 09:08:23 PDT Trauma deaths are a result of hemorrhage in 37% of civilians and 47% military personnel and are the primary cause of death for individuals under 44 years of age. Current techniques used to treat hemorrhage are inadequate for severe bleeding. Preliminary research indicates that fibrin sealants (FS) alone or in combination with a dressing may be more effective; however, it has not been economically feasible for widespread use because of prohibitive costs related to procuring the proteins. To meet future demands for hemostatic therapies, FS will likely include recombinant human fibrinogen (rFI) and recombinant human Factor XIII (rFXIII). The underlying hypothesis of the research presented in this dissertation is that a liquid fibrin sealant (LFS) composed of recombinant FI, FXIII and FIIa in optimized proportions can assist hemostasis in the presence and absence of a bioresorbable bandage while using considerably fewer biologics than commercial products currently available. This dissertation characterized rFI produced in the milk of transgenic cows, plasma-derived thrombin (pdFIIa) activated by sodium citrate and rFXIIIa expressed in genetically engineered Pichia pastoris with respect to their capacity to serve as components in a LFS. The ratios of these factors were optimized to yield a LFS with a rapid clot formation rate and high viscoelastic strength. This optimized LFS was preliminarily tested ex vivo and in vivo. The clotting kinetics and viscoelastic strength of our optimized LFS was equivalent to those of a commercially available LFS; however, it uses approximately 75% less fibrinogen and thrombin. Our optimal LFS successfully achieved hemostasis in a significant number of the wounds that included extensive tissue and vascular damage. LFS applied without the assistance of a dressing was able to stop bleeding of oozing wounds or those with small vessels; however, a scaffold was needed when wounds contained large vasculature.

]]> Jennifer Calcaterra Rapid Diagnosis of Tuberculosis in a Peripheral Setting http://digitalcommons.unl.edu/chemengtheses/3 http://digitalcommons.unl.edu/chemengtheses/3 Thu, 22 Apr 2010 12:19:07 PDT Tuberculosis is an ancient and worldwide epidemic affecting millions of people in mainly the developing world, killing almost 2 million people in 2008. Current diagnostic techniques are outdated and have proven insufficient to control the disease. Smear microscopy has poor sensitivity and culture is slow to yield results. Modern diagnostic techniques are making great strides in shortening time to result but are restricted by two qualities: 1) prohibitively high costs prevent implementation in resource poor areas, and 2) equipment and technician requirements limit application to centralized laboratories. There exists a divide between new technologies and the people that need them most. Here, a novel epidemiological model of tuberculosis in an urban community confirms the importance of improved diagnostics in lowering prevalence. The model highlights the importance of sensitivity and accessibility.

This work presents the development of a nucleic acid amplification test for tuberculosis diagnosis from sputum. The prototype system consists of 1) a sputum processing unit capable of extracting DNA within 5 minutes, and 2) a rapid PCR thermocycler which amplifies Mycobacterium tuberculosis complex specific sequences (IS6110 and IS1081) in under 15 minutes and detects product in real-time. Lysis protocol development was guided by a combined theoretical/experimental analysis of the kinetics of heat lysis of Mycobacterium smegmatis. The analysis revealed the activation energy of lysis (22.1 kcal/mole) and the minimum cell wall damage that result in cell distruction (14-17%). The PCR is capable of amplifying template amounts below smear microscopy concentrations.

The test was applied to 58 clinical samples from the Steve Biko Academic Hospital in Pretoria, South Africa. Sensitivity was 95% on smear positive culture positive samples and 70% on smear negative culture positive samples. Specificity was 86%.

In summary, the test moves toward an important niche of rapid (less than 30 minutes) and affordable ($5-10) diagnosis in a peripheral setting. Sensitivity of the test is comparable to other available systems, while specificity still needs improvement. However, turnaround times and costs are far below other tests currently being developed.

]]> Elsje Pienaar Synthesis of an Endothelial Cell Mimicking Surface Containing Thrombomodulin and Endothelial Protein C Receptor http://digitalcommons.unl.edu/chemengtheses/2 http://digitalcommons.unl.edu/chemengtheses/2 Fri, 16 Apr 2010 13:54:51 PDT Synthetic materials for use in blood contacting applications have been studied for many years with limited success. One of the main areas of need for these materials is the design of synthetic vascular grafts for use in the hundreds of thousands of patients who have coronary artery bypass grafting, many without suitable veins for autologous grafts. The design of these grafts is constrained by two common modes of failure, the formation of intimal hyperplasia (IH) and thrombosis. IH formation has been previously linked to a mismatching of the mechanical properties of the graft and has been overcome by creating grafts using materials whose compliance mimics that of the native artery. Several techniques and surface modification have been designed to limit thrombosis on the surface of these synthetic materials. One which has shown the greatest promise is the immobilization of Thrombomodulin (TM), a protein found on the endothelial cell membrane lining native blood vessels involved in the activation of the anticoagulant Protein C (PC). While TM immobilization has been shown to arrest thrombin formation and limit fibrous formations in in-vitro and in-vivo experiments, it has shown to be transport limiting under arterial flow. On the endothelial cell surface, TM is co-localized with Endothelial Protein C Receptor (EPCR), which increases PC transport onto the cell surface and increases PC activation via TM between 20-100 fold. This dissertation will describe the chemical modification of medical grade polyurethane (PU), whose compliance has been shown to match that of native arteries. This modification will enable the immobilization of two proteins on an enzymatically relevant scale estimated at less than 10 nm. This dissertation will further describe the immobilization of the proteins TM and EPCR, and analyze the ability of a surface co-immobilized with these proteins to activate the anticoagulant PC. Finally, it will compare the ability of this co-immobilized surface to delay fibrous clot formation when compared to unmodified PU, albumin or heparin coated surfaces.

]]> Karl E. Kador