Theses and Dissertations from UMD
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Item CONFINED PHOTOTHERMAL HEATING OF NANOPARTICLE DISPLAYED BIOMATERIALS(2021) Hastman, David A; Medintz, Igor L; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Controlling the temperature of biological systems has long been utilized as a tool for regulating their subsequent biological activity. Recently, photothermal heating of gold nanoparticles (AuNPs) has emerged as an efficient and remote method to heat proximal biological materials. Moreover, this technique has tremendous potential for controlling biological systems at the subcellular level, as specific components within the system can be heated while the larger system remains unaffected. The small size, biocompatiblilty, and optical properties of AuNPs make them attractive nanoscale heat sources for controlling biological systems. While the utility of photothermal heating has significantly advanced through the optimization of AuNP size, shape, and composition, the choice of incident light source utilized has largely been unexplored. One of the more interesting excitation sources is a femtosecond (fs) pulsed laser, as the subsequent temperature increase lasts for only a few nanoseconds and is confined to the nanoscale. However, it is not yet clear how biological materials respond to these short-lived and ultra-confined nanoscale spaciotemporal temperature increases. In this dissertation, we utilize fs laser pulse excitation to locally heat biological materials displayed on the surface of AuNPs in order to understand the corresponding heating profiles and, in turn, interpret how this can be used to modulate biological activity. Due to its unique temperature sensitive hybridization properties, we exploit double-stranded deoxyribonucleic acid (dsDNA) as our prototypical biological material and demonstrate precise control over the rate of dsDNA denaturation by controlling the laser pulse radiant exposure, dsDNA melting temperature, bulk solution temperature, and the distance between the dsDNA and AuNP surface. The rate of dsDNA denaturation was well fit by a modified DNA dissociation equation from which a “sensed” temperature value could be obtained. Evaluating this sensed temperature in the context of the theoretical temperature profile revealed that the ultra-high temperatures near the AuNP surface play a significant role in denaturation. Additionally, we evaluate this technique as a potential means to enhance enzyme activity and report that enhancement is governed by the laser repetition rate, pulse width, and the enzyme’s inherent turnover number. Overall, we demonstrate that the confined and nanosecond duration temperature increase achievable around AuNPs with fs laser pulse excitation can be used to precisely control biological function and establish important design considerations for coupling this technique to more complex biological systems.Item Dihydropyrrole Formation During Sibiromycin Biosynthesis(2015) Saha, Shalini; Rokita, Steven E.; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A description of pyrrolo[1,4]benzodiazepine (PBD) biosynthesis in actinomycetes is a prerequisite for engineering production of analogs with enhanced antitumor activity. Several proteins expected to synthesize the PBD’s dihydropyrrole moiety were heterologously expressed, purified and assayed for activity. UV-visible spectroscopy revealed that predicted dioxygenases SibV and homolog Orf12 associated with PBDs sibiromycin and anthramycin, respectively, catalyze the regiospecific 2,3-extradiol dioxygenation of L-3,4-dihydroxyphenylalanine (L-DOPA) to form L-2,3-secodopa (λmax = 368 nm). 1H NMR spectroscopy indicated that L-2,3-secodopa then spontaneously cyclizes into the α-keto acid tautomer of 4-(2-oxo-3-butenoic-acid)-4,5-dehydro-L-proline 1.1 (λmax = 414 nm). Thus, the dioxygenases establish the scaffold of the dihydropyrrole moiety. Both the quaternary structure and product formed by dioxygenases are conserved in dihydropyrrole biosynthesis within both PBD and non-PBD pathways. Stability studies suggest that 1.1 is relatively labile and is likely consumed rapidly by subsequent biosynthetic steps. Hydrolysis and methylation steps were proposed to modify the dihydropyrrole scaffold in PBDs. The predicted proteins SibS and homolog TomK associated with sibiromycin and PBD tomaymycin biosynthesis, respectively, were assayed for hydrolysis activity. The predicted protein SibZ associated with sibiromycin biosynthesis was assayed for methyltransferase activity. The proposed SibZ substrate 4-vinyl-4,5-dehydro-L-proline 1.2 was synthesized. For these three proteins, no catalytic activity was observed with their proposed substrates or substrate precursors under a range of conditions. However, SibS binds 1.1 (KD = 64 ± 2 µM) suggesting it participates in dihydropyrrole biosynthesis. HPLC-MS indicated that SibS catalyzes the depurination of S-adenosylmethionine although it is unlikely this reaction is involved in sibiromycin biosynthesis. These findings suggest that the pathway requires revision. The adenylation and thiolation didomain of the predicted non-ribosomal peptide synthetase SibD associated with sibiromycin biosynthesis was expressed and purified. To test if SibD incorporates the dihydropyrrole moiety into sibiromycin, its proposed substrate 4-propenyl-4,5-dehydro-L-proline 1.5 was synthesized. A radioactivity exchange assay and peptide analysis by MS revealed that SibD does not adenylate and thiolate 1.5 or its precursors L-DOPA, 1.1 or 1.2. However, L-threonine and the metabolic precursor L-tyrosine are substrates for these reactions. SibB promotes adenylation catalyzed by SibD and represents one of two proteins, distinct from MbtH-like proteins, capable of promoting adenylation.Item Enzymatic Activity Preservation through Entrapment within Degradable Hydrogel Networks(2012) Mariani, Angela Marie; Kofinas, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation aimed to design and develop a "biogel;" a reproducible, abiotic, and biocompatible polymer hydrogel matrix, that prolongs enzymatic stability allowing for rapid production of biomolecules. The researched entrapment method preserves enzyme activity within an amicable environment while resisting activity reduction in the presence of increased pH environmental challenges. These biogels can be used in a number of applications including repeated production of small molecules and in biosensors. Five main objectives were accomplished: 1) Biogels capable of maintaining enzymatic functionality post-entrapment procedures were fabricated; 2) Biogel activity dependence on crosslinker type and crosslink density was determined; 3) Biogel composition effects on sustained activity after storage were compared; 4) Biogel activity dependence on charged monomer moieties was evaluated, and 5) Combined optimization knowledge gained from the first four objectives was utilized to determine the protection of enzymes within hydrogels when challenged with an increased pH above 8. Biogels were fabricated by entrapping beta-galactosidase (lactase) enzyme within acrylamide (ACR) gels crosslinked with poly(ethylene glycol) diacrylate (PEGDA, degradable through hydrolysis) or N,N'-methylenebisacrylamide (BIS, non-degradable). Initial hydrogel entrapment reduced activity to 40% in ACR/PEGDA gels, compared to a 75% reduction in initial activity of ACR/BIS biogels. Once entrapped, these enzymes resist activity reduction in the presence of environmental challenges, such as altering the pH from 7 to above 8. When biogels were challenged at a pH of 8, activity retention positively correlated to PEGDA crosslinker density; increasing from 48% to 91% retention in 30 to 40 mole % PEGDA biogels as compared to solution based control which retained only 23%. Retention of activity when perturbed from pH 7 is advantageous for biogel applications including the repeated production of desired small molecules and biosensors. Biogels with positive or negative monomer moiety functionalities were also investigated to increase enzyme-matrix interactions and enzyme stability. The researched entrapment method illustrates the potential to sterically hinder and diffusively impede enzymes from performing their function, potentially enabling the reactivation of the enzyme at a site and time dictated by the user by degrading the crosslinks of the network.