Chemical and Biomolecular Engineering Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/2751

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Start date: 2000End date: 2009

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    ENGINEERING THE B1 DOMAIN OF STREPTOCOCCAL PROTEIN G: STRUCTURAL INVESTIGATIONS BY MULTlDIMENSIONAL HETERONUCLEAR NMR
    (2000) Frank, Mary Kirsten; Thirumalai, Devarajan; Institute for Physical Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, MD)
    The B1 domain of streptococcal protein G provides a well-characterized system for structural investigations of proteins. In this thesis, the urea-unfolded state has been characterized, the tolerance towards hydrophobic substitutions in the core has been surveyed, the hydrogen exchange behavior of the backbone amides has been elucidated, and structural information on a tetrameric mutant of this domain has been gathered. The chemical shifts of the urea-unfolded state were assigned. The secondary chemical shifts, the 3JHNa coupling constants and the short-range NOEs gave no indication of residual structure. Measurement of the backbone 15N relaxation parameters revealed a region of restricted motion in the β3- β4 turn of the native protein. Motion in the rest of the protein was uniform, with the exception of 3-4 residues at either end of the chain. A series of hydrophobic substitutions were made in the hydrophobic core. The resulting mutants were assayed for stability and overall fold . The core of the protein is particularly sensitive to substitutions at position 26. One of the mutants was unable to adopt the GB1 fold and optimized its stability by adopting a homotetrameric form. Hydrogen exchange in the backbone amides was measured at 25 °C. Rates of hydrogen exchange were inversely correlated with burial of the amide nitrogen. The slow-exchanging backbone amides did not correlate with the hydrogen bonds formed early in protein folding. Hydrogen exchange rates from NH to ND and from ND to NH were similar. The ratio between these two rates does not correlate with any obvious physical parameters of the hydrogen bonds. Chemical shifts for the tetrameric mutant (HS#124) were determined using three-dimensional heteronuclear NMR techniques. Measurement of the backbone dynamics revealed a highly flexible region between positions 8 and 22. The secondary structure and β-sheet interactions of this mutant were characterized. The β-sheet interactions were intermolecular and only one of the three β-strand pairings was similar to the β-strand pairings found in wild type GB1 . The novel pairing is between β1 of one monomer and β1 of another monomer and a shift in register is observed for the β3-β4 pairing.
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    SLIDING MESH COMPUTATIONAL FLUID DYNAMICS SIMULATION OF A WIDE AND NARROW GAP INLINE ROTOR-STATOR MIXER
    (2001) Kevala, Karl Rustom; Calabrese, Richard V.; Chemical and Biomolecular Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md)
    The FLUENT™ Computational Fluid Dynamics code was used to simulate the flow of water within an inline rotor-stator mixer. Two devices were simulated. Both had identical dimensions, except for the width of the shear gap: 4 mm for the 'wide gap' model and 0.5 mm for the 'standard gap' model. A two-dimensional approximation was used in conjunction with a RANS turbulence model and sliding mesh technique. Simulated turbulence intensities and mass flow rates are more evenly distributed in the standard gap model. Further, turbulence and mean shear levels within the gap are minimal and probably not important for the production of dispersions. The most intense turbulence is near the downstream stator slot wall, and it is due to fluid impingement. For the standard gap model, this region is more focused and of higher intensity. It is concluded that a narrow gap clearance is needed to produce high intensity stagnation flows on the stator teeth. Simulation results are also compared with previously reported measurements acquired via Laser Doppler Anemometry. With respect to mean velocity, qualitative agreement is good. Quantitatively, neither the mean velocities nor turbulence values are well predicted. This discrepancy is believed to be due in large part to leakage flow over the top of the rotor and stator teeth and to the three-dimensional nature of the flow. Future simulations should be carried out in three dimensions using more sophisticated turbulence models. Additionally, algorithms should be developed to decrease computation time by exploiting the periodic nature of rotor-stator flows.
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    Proteomic Analysis of Human Urinary Exosomes
    (2009) Gonzales, Patricia Amalia; Wang, Nam Sun; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Exosomes originate as the internal vesicles of multivesicular bodies (MVBs) in cells. These small vesicles (40-100 nm) have been shown to be secreted by most cell types throughout the body. In the kidney, urinary exosomes are released to the urine by fusion of the outer membrane of the MVBs with the apical plasma membrane of renal tubular epithelia. Exosomes contain apical membrane and cytosolic proteins and can be isolated using differential centrifugation. The analysis of urinary exosomes provides a non-invasive means of acquiring information about the physiological or pathophysiological state of renal cells. The overall objective of this research was to develop methods and knowledge infrastructure for urinary proteomics. We proposed to conduct a proteomic analysis of human urinary exosomes. The first objective was to profile the proteome of human urinary exosomes using liquid chromatography-tandem spectrometry (LC-MS/MS) and specialized software for identification of peptide sequences from fragmentation spectra. We unambiguously identified 1132 proteins. In addition, the phosphoproteome of human urinary exosomes was profiled using the neutral loss scanning acquisition mode of LC-MS/MS. The phosphoproteomic profiling identified 19 phosphorylation sites corresponding to 14 phosphoproteins. The second objective was to analyze urinary exosomes samples isolated from patients with genetic mutations. Polyclonal antibodies were generated to recognize epitopes on the gene products of these genetic mutations, NKCC2 and MRP4. The potential usefulness of urinary exosome analysis was demonstrated using the well-defined renal tubulopathy, Bartter syndrome type I and using the single nucleotide polymorphism in the ABCC4 gene. The third objective was to study the normal variability between proteomes of female and male urinary exosomes, and to implement a normalization method to analyze urinary exosome samples. Only 19 proteins had a 2-fold change representing 4.9% of the total number of proteins identified which shows that there is high concordance between proteomes of urinary exosomes isolated from males and females. The normalization method, timed urine collection did not correlate as expected with the intensity signal of MVB markers, TSG101 and Alix. This research shows that the proteomic analysis of human urinary exosomes can be the basis for future biomarker studies as well as physiological studies.
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    Analysis of Air Quality with Numerical Simulation (CMAQ), and Observations of Trace Gases
    (2009) Castellanos, Patricia; Ehrman, Sheryl H; Dickerson, Russell R; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Ozone, a secondary pollutant, is a strong oxidant that can pose a risk to human health. It is formed from a complex set of photochemical reactions involving nitrogen oxides (NOx) and volatile organic compounds (VOCs). Ambient measurements and air quality modeling of ozone and its precursors are important tools for support of regulatory decisions, and analyzing atmospheric chemical and physical processes. I worked on three methods to improve our understanding of photochemical ozone production in the Eastern U.S.: a new detector for NO2, a numerical experiment to test the sensitivity to the timing to emissions, and comparison of modeled and observed vertical profiles of CO and ozone. A small, commercially available cavity ring-down spectroscopy (CRDS) NO2 detector suitable for surface and aircraft monitoring was modified and characterized. The CRDS detector was run in parallel to an ozone chemiluminescence device with photolytic conversion of NO2 to NO. The two instruments measured ambient air in suburban Maryland. A linear least- squares fit to a direct comparison of the data resulted in a slope of 0.960±0.002 and R of 0.995, showing agreement between two measurement techniques within experimental uncertainty. The sensitivity of the Community Multiscale Air Quality (CMAQ) model to the temporal variation of four emissions sectors was investigated to understand the effect of emissions' daily variability on modeled ozone. Decreasing the variability of mobile source emissions changed the 8-hour maximum ozone concentration by ±7 parts per billion by volume (ppbv). Increasing the variability of point source emissions affected ozone concentrations by ±6 ppbv, but only in areas close to the source. CO is an ideal tracer for analyzing pollutant transport in AQMs because the atmospheric lifetime is longer than the timescale of bound- ary layer mixing. CO can be used as a tracer if model performance of CO is well understood. An evaluation of CO model performance in CMAQ was carried out using aircraft observations taken for the Regional Atmospheric Measurement, Mod- eling and Prediction Program (RAMMPP) in the summer of 2002. Comparison of modeled and observed CO total columns were generally in agreement within 5-10%. There is little evidence that the CO emissions inventory is grossly overestimated. CMAQ predicts the same vertical profile shape for all of the observations, i.e. CO is well mixed throughout the boundary layer. However, the majority of observations have poorly mixed air below 500 m, and well mixed air above. CMAQ appears to be transporting CO away from the surface more quickly than what is observed. Turbulent mixing in the model is represented with K-theory. A minimum Kz that scales with fractional urban land use is imposed in order to account for subgrid scale obstacles in urban areas and the urban heat island effect. Micrometeorological observations suggest that the minimum Kz is somewhat high. A sensitivity case where the minimum Kz was reduced from 0.5 m2/s to 0.1 m2/s was carried out. Model performance of surface ozone observations at night increased significantly. The model better captures the observed ozone minimum with slower mixing, and increases ozone concentrations in the residual layer. Model performance of CO and ozone morning vertical profiles improves, but the effect is not large enough to bring the model and measurements into agreement. Comparison of modeled CO and O3 vertical profiles shows that turbulent mixing (as represented by eddy diffusivity) appears to be too fast, while convective mixing may be too slow.
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    NANOSTRUCTURED THIN FILM POLYMER ELECTROLYTES FOR FLEXIBLE BATTERY APPLICATIONS
    (2009) Ghosh, Ayan; Kofinas, Peter; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    In recent years, the interest in polymeric batteries has increased dramatically. With the advent of lithium ion batteries being used in cell phones and laptop computers, the search for an all solid state battery has continued. Current configurations have a liquid or gel electrolyte along with a separator between the anode and cathode. This leads to problems with electrolyte loss and decreased performance over time. The highly reactive nature of these electrolytes necessitates the use of protective enclosures which add to the size and bulk of the battery. Polymer electrolytes are more compliant than conventional inorganic glass or ceramic electrolytes. The goal of this work was to design and investigate novel nanoscale polymer electrolyte flexible thin films based on the self-assembly of block copolymers. Block copolymers were synthesized, consisting of a larger PEO block and a smaller block consisting of random copolymer of methyl methacrylate (MMA) and the lithium salt of methacrylic acid (MAALi). The diblock copolymer [PEO-b-(PMMA-ran-PMAALi)] with added lithium bis(oxalato)borate, LiBC4O8 (LiBOB) salt (in the molar ratio ethylene oxide:LiBOB = 3:1) was used to form flexible translucent films which exhibited nearly two orders of magnitude greater conductivity than that shown by traditional high molecular weight PEO homopolymer electrolytes, in the absence of ceramic fillers and similar additives. The presence of the smaller second block and the plasticizing effect of the bulky lithium salt were shown to effectively reduce the crystallinity of the solid electrolyte, resulting in improved ion transporting behavior. The tailored solid self-assembled diblock copolymer electrolyte matrix also exhibits an exceptionally high lithium-ion transference number of 0.9, compared to a value between 0.2 and 0.5, shown by typical polymer-lithium salt materials. The electrolyte material also has a wide electrochemical stability window and excellent interfacial behavior with lithium metal electrode. The combination of these properties make electrolyte membranes composed of the diblock copolymer PEO-b-(PMMA-ran-PMAALi) and LiBOB salt, viable electrolyte candidates for flexible lithium ion based energy conversion/storage devices.
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    Evaluation of the transcription of small RNA SgrS and glucose transporter mRNA ptsG in E. coli B and E. coli K cultures under high glucose conditions
    (2009) Ng, Weng Ian; Wang, Nam Sun; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Escherichia coli is commonly used as the production system for recombinant proteins. However, acetate accumulation in fermentation affects cell growth and protein yield. Recent studies have showed that the small RNA SgrS regulates the major glucose transporter mRNA ptsG in a post–transcriptional manner when the metabolic intermediate glucose–6–phosphate is accumulated intracellularly in E. coli K. Here, comparative analysis of the transcription of SgrS and ptsG is performed between E. coli B and E. coli K cultures in both shake flasks and bioreactor. Both strains expressed SgrS when grown on the non–metabolizable glucose analog α–methyl–glucoside. However, under high glucose conditions, only E. coli B showed significant expression of SgrS. This behavior is unaffected by oxygen supply and pH control. E. coli B produced less acetate on glucose than E. coli K in the bioreactor settings. This provides evidence of a possible connection between SgrS and acetate production in aerobic fermentation of E. coli.
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    Self-Assembled Photoresponsive and Thermoresponsive Nanostructures
    (2009) Sun, Kunshan; Raghavan, Srinivasa R.; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Responsive complex fluids based on nanostructures (e.g., micelles, vesicles and nanoparticles) have received considerable attention recently. The ability of these materials to be tuned by light or heat can have many potential applications in the areas of drug delivery, coatings, sensors, or microfluidic valves and dampers. However, most current photoresponsive and thermoresponsive formulations require the synthesis of complex organic molecules, and this prevents them from being used widely for commercial applications. In this dissertation, we seek to develop new classes of photoresponsive (PR) and thermoresponsive (TR) nanostructures based on commercially available, inexpensive precursors. In the first part of this study, we report a new PR fluid based on light-activated nanoparticle assembly. Our system consists of disk-like nanoparticles of laponite along with a surfactant stabilizer (Pluronic F127) and the photoacid generator (PAG), diphenyliodonium-2-carboxylate monohydrate. Initially, the nanoparticles are sterically stabilized by the surfactant and the result is a stable, low-viscosity dispersion. Upon UV irradiation, the PAG gets photolyzed, lowering the pH by about 3 units. In turn, the stabilizing surfactant is displaced from the negatively charged faces of the nanoparticle disks while the edges of the disks become positively charged. The particles are thereby induced to assemble into a 3 dimensional "house-of-cards" network that extends through the sample volume. The net result is a light-induced sol to gel transition, i.e., from a low, water-like viscosity to an infinite viscosity and yield stress. The yield stress of the photogel is sufficiently high to support the weight of small objects. The gel can be converted back to a sol by either increasing the pH or the surfactant content. Evidence for the above mechanism is provided from a variety of techniques, including small-angle neutron scattering (SANS). In the second part of this study, we demonstrate that laponite/PF127 mixtures also show thermogelling, i.e., the fluids transform from low viscosity sols to stiff gels upon heating above a critical temperature. This phenomenon is reversible and it requires the presence of sufficient amounts of both components. At room temperature, PF127 adsorbs onto laponite disks and stabilizes them by steric repulsion. Upon heating, the PF127 layer on the disks becomes thicker, and more importantly, PF127 micelles in the bulk solution grow significantly. Evidence for the growth of micelles is presented from SANS modeling and from transmission electron microscopy (TEM). At a distinct temperature, we believe the micelles induce depletion flocculation of the laponite particles into a gel network. Interestingly, if the PF127 concentration is increased further, the thermogelling is eliminated - this is suggested to be due to the micelles providing depletion stabilization of the particles.
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    Polymer capsules as building blocks for soft, connected mesostructures
    (2009) George, Elijah; Raghavan, Srinivasa; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    We show that polymer capsules can serve as soft building blocks for creating a range of mesoscale (0.1 to 10 mm) structures. The central innovation is a new approach for connecting spherical capsules by exploiting electrostatic complexation. Using this approach, connected structures with complex shapes can be easily assembled, and more importantly, a single connected structure can be made to have a diverse array of functions. The modular approach to shape and function is very much like using Lego bricks of different colors. The connected structures can be made responsive (capable of being actuated) by magnetic fields by including magnetic capsules within them. One motivation for creating these structures is to mimic the mechanics and motility of small creatures such as the earthworm or ant - this could eventually enable the design of autonomous biomimetic robots. In addition, soft connected structures could be employed to transport cargo such as drugs or proteins in blood vessels, or to construct valves, rotors, or mixers in microfluidic or lab-on-a-chip devices.
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    A Simplified Model of Planetary Chemical Vapor Deposition Reactors
    (2009) Shahshahan, Negin; Adomaitis, Raymond A.; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    A simplified model for planetary chemical vapor deposition reactors is proposed and used to compute deposition species mole fraction and deposition rate in the reactor depletion zone. First, the modeling and optimization work performed in the literature is reviewed and their representative deposition rate profiles are extracted. Afterwards, several simplifying assumptions are applied to derive the reactor modeling equation, and the eigenfunction expansion solution is subsequently computed using a previously developed MATLAB object-oriented computational framework. The simulation result for the deposition profile is improved by modifying the inlet boundary condition, and is then compared with the previously published profiles. The MATLAB optimization toolbox is used to find the optimal deposition profile giving the best match with the published, detailed simulator profiles. Finally, an evaluation of the model consistency with the published results is given.
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    BUTANOL PRODUCTION FROM GLYCEROL BY Clostridium pasteurianum IN DEFINED CULTURE MEDIA- A PHENOTYPIC APPROACH.
    (2009) Ramos Sanchez, David Leonardo; Wang, Nam S; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The fluctuations in oil prices have stimulated the production of renewable biofuels, in particular the production of bioethanol and biodiesel. The production of biodiesel has expanded almost six fold in the past years. The ten wt% of the biodiesel process results in crude glycerol. Once a valuable product, nowadays glycerol is considered a waste and a surplus material. Its current low price makes it an attractive substrate for a fermentation process. Molecular genetics have unveiled new insights about solvent production in Clostridia. It has been recognized that endospore development and solvent formation share a regulatory mechanism. The solvent production, particularly the butanol fermentation of glycerol by Clostridium pasteurianum was studied. Taking advantage of the characteristics of the sporulation phenotype, the study of the butanol fermentation was approached. A relation between spore formation and butanol production was found in C. pasteurianum by applying molecular genetics concepts.