UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
17 results
Search Results
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 The role of sequence in the structure of self-assembling 3D DNA crystals(2015) Saoji, Maithili; Paukstelis, Dr. Paul J; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA is a widely used biopolymer for the construction of nanoscale objects due to its programmability and structural predictability. DNA oligonucleotides can, however, exhibit a great deal of local structural diversity. DNA conformation is strongly linked to both environmental conditions and the nucleobase identities inherent in the oligonucleotide sequence, but the exact relationship between sequence and local structure is not completely understood. We previously determined the X-ray crystal structure of a DNA 13-mer that forms a continuously hydrogen bonded three-dimensional lattice through Watson-Crick and non-canonical base pairs. In the current work I examined how the sequence of the Watson-Crick duplex region influenced crystallization of this 13-mer. I screened all possible self-complementary sequences in the hexameric duplex region and found 21 oligonucleotides that crystallized. Sequence analysis showed that one specific Watson-Crick base pair influenced the crystallization propensity and the speed of crystal self-assembly. I determined X-ray crystal structures for 13 of these oligonucleotides and found sequence-specific structural changes suggesting that this base pair may serve as a structural anchor during crystal assembly. I explored the crystal self-assembly and nucleation process and demonstrated that crystals grown from mixtures of two different oligonucleotide sequences contained both the oligonucleotides. These results suggested that crystal self-assembly is nucleated by the formation of Watson-Crick duplexes. Finally, I also examined how a single nucleotide addition to the DNA 13-mer leads to a significantly different overall structure under identical crystallization conditions. The 14-mer crystal structures described here showed that all of the predicted Watson-Crick base pairs were present, but the major difference as compared to the parent 13-mer structure was a significant rearrangement of non-canonical base pairs. This included the formation of a sheared A-G base pair, a junction of strands formed from base triple interactions, and tertiary interactions that generated structural features similar to tandem sheared G-A base pairs. The adoption of this alternate non-canonical structure was dependent in part on the sequence of the Watson-Crick duplex region. These results provided important new insights into the sequence/structure relationship of short DNA oligonucleotides and demonstrated a unique interplay between Watson-Crick and non-canonical base pairs that are responsible for crystallization fate.Item BIOLOGICAL CHARACTERIZATION OF TWO PUTATIVE DNA METABOLISM ENZYMES IN DEINOCOCCUS RADIODURANS(2014) Mueller, Charles; Julin, Douglas; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)HerA proteins are members of the FtsK-HerA superfamily of P-loop ATPases. FtsK is a bacterial protein that translocates double-stranded DNA during cell division. In archaea, herA is an essential gene that encodes an enzyme believed to be important for recombinational DNA repair. It is typically found in an operon with a gene that codes for a nuclease, nurA. Homologs of herA and nurA are found in a few bacterial genomes. In most cases, these bacteria lack an ftsK homolog. The functions of NurA and HerA in bacteria are not known. We chose to investigate the roles of NurA and HerA in Deinococcus radiodurans, typically studied for its extreme resistance to double-strand DNA breaks. The D. radiodurans genome has homologs of nurA and herA in an operon, and it also has an ftsK gene. We made strains with deletions of either herA or nurA and characterized their sensitivity to DNA damaging agents and basic growth properties. The results indicate that neither gene is essential in D. radiodurans, and deletions of the genes do not cause significant sensitivity to DNA damaging agents. The herA deletion strain displayed a distinct phenotype consisting of slower growth and larger cell types. The herA phenotype in D. radiodurans is similar to that of mutation of ftsK homologs in Escherichia coli and Bacillus subtilis. The results suggest that HerA has an FtsK-like function in cell division, rather than acting in DNA repair, in D. radiodurans.Item Jamming effects in glasses and biopolymers(2014) Kang, Hongsuk; Thirumalai, Devarajan; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this dissertation, jamming effects in highly packed systems are studied in two specific materials: glasses and biopolymers in cellular environments. Suspensions consisting of highly charged colloids, which are well-known glass-forming systems, are investigated using molecular dynamics simulations in order to test Random First Order Transition (RFOT) theory. I found that there is a critical volume fraction at which ergodic-to-nonergodic transitions for three dynamic observables take place in accordance with RFOT. Based on numerical observations, it is also proposed that the dynamic heterogeneity can be attributed to the violation of law of large numbers. In addition, the bond orientational order of colloidal suspensions and soft-spheres is discussed in the context of liquid-glass transitions. The response of biopolymers to a crowded environment is another interesting issue because 20-40% volume of a cell is occupied by various cellular components such as ribosomes and proteins in vivo. In this work, using low-friction langevin dynamics simulations with explicit crowding particles, I examined the conformational change of biopolymers in the presence of crowders of various sizes and shapes. The simulation results reveal that cylindrical crowders induce much greater compaction of the polymers than spherical ones at low volume fractions and the stronger crowding effects disappear at higher volume fractions due to local nematic ordering of cylindrical particles. The reduction in the size of polymer is even more dramatic in a mixture of spherical and cylindrical shapes because of cooperative crowding effects explained by the phase separation of spheres and rodlike particles. Finally, the crowding effects of cellular components on bacterial chromosomes are estimated using a mixture of spherical crowders with the composition found in bacterial cytoplasms.Item Effects of chronic exercise on global DNA methylation and epigenetic factors in sperm and testes of mice.(2012) Marini, Michael Paul; Roth, Stephen M; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Epigenetic alterations of DNA affect DNA transcription and translation. These alterations occur frequently, however environmental exposures induce epigenetic changes to DNA that would otherwise remain in autoregulatory stasis. This study aimed to look at exercise as a possible environmental factor causing epigenetic change. The study also assessed global DNA methylation in sperm, which may transmit such epigenetic changes via the paternal germ line. Measurements were compared between groups of mice that engaged in chronic exercise or remained sedentary. This study also examined enzymes causing methylation shifts in sperm by comparing levels of mRNA expression of genes responsible for new DNA methylation - DNMT3A, DNMT3B and DNMT3L - in testes. These results were compared between exercise and sedentary cohorts, and in progeny to assess heritability of epigenetic change. The results showed a significant difference in global methylation in the sperm between exercise and sedentary cohorts and a concomitant increase in gene expression in multiple DNMT3 genes.Item DESIGN AND DEVELOPMENT OF THREE-DIMENSIONAL DNA CRYSTALS UTILIZING CGAA PARALLEL BASE PAIRED MOTIFS(2013) Muser, Stephanie Elizabeth; Paukstelis, Paul; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Three-dimensional (3D) DNA crystals hold great potential for various applications such as the development of molecular scaffolds for use in protein structure determination by x-ray crystallography. The programmability and predictability of DNA make it a powerful tool for self-assembly but it is hindered by the linearity of the duplex structure. Predictable noncanonical base pairs and motifs have the potential to connect linear double-helical DNA segments into complex 3D structures. The sequence d(GCGAAAGCT) has been observed to form 3D crystals containing both noncanonical parallel pairs and canonical Watson-Crick pairs. This provided a template structure that we used in expanding the design and development of 3D DNA crystals along with exploring the use of predictable noncanonical motifs. The structures we determined contained all but one or two of the designed secondary structure interactions, depending on pH.Item RNA-SEQUENCING ANALYSIS: READ ALIGNMENT AND DISCOVERY AND RECONSTRUCTION OF FUSION TRANSCRIPTS(2013) Kim, Daehwan; Salzberg, Steven L; Computer Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)RNA-sequencing technologies, which sequence the RNA molecules being transcribed in cells, allow us to explore the process of transcription in exquisite detail. One of the primary goals of RNA sequencing analysis is to reconstruct the full set of transcripts (isoforms) of genes that were present in the original cells. In addition to the transcript structures, experimenters need to estimate the expression levels for all transcripts. The first step in the analysis process is to map the RNA-seq reads against the reference genome, which provides the location from which the reads originated. In contrast to DNA sequence alignment, RNA-seq mapping algorithms have two additional challenges. First, any RNA-seq alignment program must be able to handle gapped alignment (or spliced alignment) with very large gaps due to introns, typically from 50-100,000 bases in mammalian genomes. Second, the presence of processed pseudogenes from which introns have been removed may cause many exon-spanning reads to map incorrectly. In order to cope with these problems effectively, I have developed new alignment algorithms and implemented them in TopHat2, a second version of TopHat (one of the first spliced aligners for RNA-seq reads). The new TopHat2 program can align reads of various lengths produced by the latest sequencing technologies, while allowing for variable-length insertions and deletions with respect to the reference genome. TopHat2 combines the ability to discover novel splice sites with direct mapping to known transcripts, producing more sensitive and accurate alignments, even for highly repetitive genomes or in the presence of processed pseudogenes. These new capabilities will contribute to improvements in the quality of downstream analysis. In addition to its splice junction mapping algorithm, I have developed novel algorithms to align reads across fusion break points, which result from the breakage and re-joining of two different chromosomes, or from rearrangements within a chromosome. Based on this new fusion alignment algorithm, I have developed TransFUSE, one of the first systems for reconstruction and quantification of full- length fusion gene transcripts. TransFUSE can be run with or without known gene annotations, and it can discover novel fusion transcripts that are transcribed from known or unknown genes.Item Trapping Labile Adducts Formed Between an ortho-Quinone Methide and DNA(2012) McCrane, Michael Patrick; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exogenously generated electrophiles are capable of alkylating DNA. If not repaired, the resulting DNA adducts can lead to mutations and either cancer or cell death. Electrophilic ortho-quinone methides (o-QM) are reactive intermediates that alkylate DNA and are generated during xenobiotic metabolism of a variety of compounds including environmental toxins and therapeutic agents. Identifying the full alkylation profile of o-QM towards DNA would allow for the genotoxicity of o-QM precursors to be better understood. From model studies based on nucleosides, o-QMs react most readily, but reversibly with the strong nucleophiles 2'-deoxycytidine (dC) N3, 2'-deoxyguanosine (dG) N7, and 2'-deoxyadenosine (dA) N1 and less efficiently, but irreversibly with the weak nucleophiles dG N1, dG N2, and dA N6. The reverse reactions complicate analysis of their products in DNA, which requires enzymatic digestion and chromatographic separation. Selective oxidation by bis[(trifluoroacetoxy)iodo]benzene (BTI) can transform the reversible o-QM-DNA adducts into irreversible derivatives capable of surviving such analysis. To facilitate this analysis, a series of oxidized o-QM-dN adducts were synthesized as analytical standards. Initial oxidative trapping studies with an unsubstituted o-QM and dC demonstrated the necessity of an alkyl substituent para to the phenolic oxygen to block over-oxidation. A novel o-QM included a methyl group para to the phenolic oxygen that successfully blocked the over-oxidation allowing for generation of a stable MeQM-dC N3 oxidized product. Further oxidative trapping studies with MeQM and dG resulted in the formation of three stable MeQM-dG oxidized products (guanine N7, dG N1, and dG N2). Initial studies with duplex DNA optimized the enzymatic digestion and confirmed that the assay conditions were compatible with oxidative trapping. The low yielding MeQM alkylation of duplex DNA needs to be scaled up prior to the oxidative trapping studies. Alternative studies quantified the release of MeQM from DNA with the use of 2-mercaptoethanol as a nucleophilic trap. These studies revealed single stranded DNA as a superior carrier of MeQM than duplex DNA and, therefore, a better target DNA for the oxidative trapping studies due to increased yield of MeQM adducts. With the increased MeQM-DNA yield, the intrinsic selectivity and reactivity of MeQM towards DNA can be determined.Item MANIPULATION OF DNA TOPOLOGY USING AN ARTIFICIAL DNA-LOOPING PROTEIN(2012) Gowetski, Daniel; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA loop formation, mediated by protein binding, plays a broad range of roles in cellular function from gene regulation to genome compaction. While DNA flexibility has been well investigated, there has been controversy in assessing the flexibility of very small loops. We have engineered a pair of artificial coiled-coil DNA looping proteins (LZD73 and LZD87), with minimal inherent flexibility, to better understand the nature of DNA behavior in loops of less than 460 bp. Ring closure experiments (DNA cyclization) were used to observe induced topological changes in DNA upon binding to and looping around the engineered proteins. The length of DNA required to form a loop in our artificially rigid system was found to be substantially longer than loops formed with natural proteins in vivo. This suggests the inherent flexibility of natural looping proteins plays a substantial role in stabilizing small loop formation. Additionally, by incrementally varying the binding site separation between 435 bp and 458 bp, it was observed that the LZD proteins could predictably manipulate the DNA topology. At the lengths evaluated, the distribution of topological products correlates to the helical repeat of the double helix (10.5 bp). The dependence on binding site periodicity is an unequivocal demonstration of DNA looping and represents the first application of a rigid artificial protein in this capacity. By constructing these DNA looping proteins, we have created a platform for addressing DNA flexibility in regards to DNA looping. Future applications for this technology include a vigorous study of the lower limits of DNA length during loop formation and the use of these proteins in assembling protein:DNA nanostructures.Item DESIGN AND SYNTHESIS OF AN ELECTRON RICH QUINONE METHIDE PRECURSOR FOR SEQUENCE -DIRECTED ALKYLATION OF DNA(2012) Huang, Chengyun; Rokita, Steven E; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Quinone methides (QM) can be delivered to alklyate specific sites with a single strand DNA through target promoted alkylation. Previous experimental results indicated that alkylation by DNA-QM self-adduct was too slow for application in a biological system. A new quinone methide precursor (QMP) with enhanced reactivity is necessary to accelerate the reaction. Previous study showed that an electron donating group present in the QMP would facilitate the generation of QM from the precursor and its regeneration from the reversible alkylation adducts. Therefore, new QMPs with increased electron density were designed. An electron rich QMP2 was successfully synthesized through a benzylaldehyde derivative. As predicted, DNA-QM self-adduct formation was much faster using QMP2 than using the conventional precursor QMP1 without an electron donating group. Only 20 min was needed for QM2 to complete the conversion from DNA-QMP conjugate while QM1 needed 24 hrs to finish the same conversion. The DNA-QM2 self-adduct also exhibited faster reaction for alkylation of the target single strand. A two-day incubation was necessary to achieve its maximal yield of 20% compared to 6 days required to achieve the maximal yield of 16% for DNA-QM1. In order to target duplex DNA, QMP was coupled to triplex forming oligonucleotides (TFO) to deliver the QM to the major groove of DNA through triple helix formation. Alkylation products were observed with the DNA-QMP1 conjugate but not the DNA-QM1 self-adducts. An adjacent guanine in the sequence can increase alkylation yield from around 10% to up to 20%. QMP2 was also coupled to the TFO to generate the self-adduct DNA-QM2. Maximal duplex alkylation yield (15%) using DNA-QM2 self-adduct was achieved in 3 days if the triplex samples were incubated at room temperature. The alkylation yield increased to 20% with the DNA-QM2 self-adductwhen samples were incubated at 37 °C. The DNA-QMP2 conjugate could even be activated at 37 oC without fluoride and resulted in an alkylation yield of up to 25%. The enhanced reactivity of the electron rich QMP2 improved the duplex alkylation effectiveness and prepared it for future in vivo application.