Theses and Dissertations from UMD
Permanent URI for this communityhttp://hdl.handle.net/1903/2
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 give thesis/dissertation in DRUM
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
9 results
Search Results
Item Using CRISPR/Cas9 to functionally dissect Blimp1, a newly identified pair-rule gene in the hemipteran Oncopeltus fasciatus(2024) Reding, Katie; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genetic screens in the fruit fly Drosophila melanogaster identified a class of mutants displaying half as many segments as seen in wild type fly larvae (Nüsslein-Volhard and Wieschaus 1980). Careful examination of the larval cuticle revealed that one out of every two segments were deleted across the anterior-posterior (AP) axis, an unexpected phenotype suggesting that segmentation in Drosophila follows a ‘pair rule’. Thanks in part to the many genetic tools available for this model species, we now have a clear picture of how the AP axis of the Drosophila embryo is polarized and subsequently divided into distinct segments, and how the pair-rule genes (PRGs) define alternate segments during this process. Since all insects share a similar body plan, it is reasonable to expect that the processes regulating establishment of this body plan would be conserved. However, studies of the Drosophila segmentation gene orthologs in non-model insects suggest that this is not always the case. While the use of model organisms enables an unmatched depth of understanding of the mechanisms underlying development, it comes at the expense of understanding the diversity of these mechanisms across taxa. The milkweed bug Oncopeltus fasciatus (Ofas) (Hemiptera) is a particularly useful insect to study in this regard, as none of the orthologs of the Drosophila PRGs have clear PR-function in this species (Liu and Kaufman 2005b; Auman and Chipman 2018; Reding et al. 2019), while the gene E75A, which has no role in segmentation in Drosophila, is expressed in a PR pattern and its knockdown yields PR segmentation defects (Erezyilmaz et al. 2009). These results suggested that PR-regulation of segmentation in Oncopeltus might require a different set of factors than those discovered in Drosophila. To identify other non-canonical PRGs in this species, I conducted an expression pattern-based screen of transcription factor-encoding genes that are co-expressed with E75A during embryogenesis, and have identified the gene Blimp1 as an Oncopeltus PRG. Like the Drosophila PR mutants, Ofas-Blimp1 mutants display loss of alternate segments across the AP axis. No roles of Blimp1 in insect segmentation had been identified prior to this finding. This result suggests that while insect segmentation may be constrained to follow a pair rule, the genes responsible for regulating PR-segmentation are evolutionarily labile. Further, a major barrier to studying gene function in non-models is the lack of genetic tools such as visible markers and established methods for gene editing. Here I will describe deployment of CRISPR/Cas9 technology in Oncopeltus for targeted mutagenesis. While mutation of the ABC transporter-encoding gene white proved to be recessive lethal, I was able to generate a viable visible marker line by disrupting the X-linked gene Ofas-vermilion (v). Of-v is required for production of dark brown eye pigments, thus Ofas-v mutants have bright red eyes, easily discernible from the black eyes of wild type bugs. I show that a co-CRISPR approach using Of-v as a marker of germline mutation is a helpful strategy to identify mutations of interest at unlinked loci, enabling many future genetic manipulations in this species.Item EVOLUTION OF THE CRISPR IMMUNE SYSTEM FROM ECOLOGICAL TO MOLECULAR SCALES(2024) Xiao, Wei; Johnson, Philip LF; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacteria and archaea inhabit environments that constantly face viral infections and other external genetic threats. They have evolved an arsenal of defense strategies to protect themselves. My research delves into the CRISPR immune system, the only known adaptive immune system of prokaryotes. My work explores three different dimensions of the CRISPR immune system, ranging from ecological to molecular scales.From an evolutionary perspective, CRISPR is widely distributed across the prokaryotic tree, underscoring its immune effectiveness. However, the CRISPR distribution is uneven and some lineages are devoid of CRISPR. Here, I identify two ecological drivers of the CRISPR immune system. By analyzing both 16S rRNA data and metagenomic data, I find the CRISPR system is favored in less abundant prokaryotes in the saltwater environment and higher diverse prokaryote communities in the human oral environment. On the molecular level, the CRISPR system selects and cleaves its “favorite” DNA segments (also known as “spacers”) from invading viral genomes to form immune memories. I explore how the spacer sequence composition affects its acquisition rate by the CRISPR system. I develop a convolutional neural network model to predict the spacer acquisition rate based on the spacer sequence composition in natural microbial communities. The model interpretation reveals that the PAM-proximal end of the spacer is more important in predicting the spacer abundance, which is consistent with previous findings from controlled experimental studies. Combining these scales, CRISPR repeat sequences coevolve with the rest of the genome. Thus, I explore the potential of utilizing CRISPR repeat sequences for taxonomy profiling. I find a strong relationship between unique repeat sequences and taxonomy in both the RefSeq database and a human metagenomic dataset. Then I show high accuracy when utilizing repeat sequences in taxonomy annotation of human metagenomic contigs. This novel method not only aids in annotating CRISPR arrays but also introduces a novel tool for metagenomic sequence annotation.Item FEEDBACK-CONTROLLED BIOELECTRONIC HYBRID SYSTEM ENABLED BY ELECTROGENETIC CRISPR(2023) Wang, Sally Patricia; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)With the rise of concepts like the “internet of things” and the advances in electronic technologies, our lives have now been occupied with smart devices that easily communicate with one another. These devices, however, lack the ability to freely exchange information with the world of biology, since electronics and biology possess very different communication modalities. Recently, the field of “electrogenetics” was introduced by enlisting redox mediators like hydrogen peroxide as a novel signaling medium to facilitate the connection between electronics with biology. In this dissertation, we expanded the electrogenetic framework and established a complete network of Bio-Nano Things, which collectively allowed automated, algorithm-based feedback control of electrogenetic CRISPR activity. First, we engineered the abiotic/biotic interface in order to improve information transfer between electronics and biological systems. Inspired by nature, we created an “artificial biofilm” that immobilized living cells on the surface of the electrode by electrochemically assembling bacteria and thiolated polyethylene glycol (PEG-SH) to form a thin film. We then endowed the PEG-SH hydrogel with redox capabilities via conjugation to generate an interactive material that can autonomously synthesize hydrogen peroxide to initiate communication with a bacterial population. Additionally, a polycysteine-tagged Streptococcal protein G was introduced for PEG-SH hydrogel surface decoration to enable the recognition of cells and other biological molecules. Next, we developed oxyRS-based electrogenetic CRISPR to broaden the bandwidth of electrochemical signaling, allowing multiplexed transcriptional regulation on various genetic targets. These include two crucial quorum sensing genes that controlled the relay of electrochemical signals to a broader yet selective audience of microbial populations through quorum sensing communication. We then integrated the engineered interface with eCRISPR-mediated transcriptional regulation to present “Biospark”, a full electrogenetic system including custom-made hardware and software, for algorithm-governed automated control of gene expression. Finally, we demonstrated a network of Bio-Nano Things by connecting the Biospark system with another custom bio-electrochemical device and even users to achieve remote feedback control of eCRISPR activity and more importantly, multidirectional communication between living systems regardless of physical distance. Together, we believe this work represents a huge leap toward making “smarter” devices and networks that can seamlessly guide biological processes with electronic input and can spawn various applications in the fields of biotechnology.Item Quantifying the relative contribution and furthering qualitative understanding of ftz cis-regulatory elements in Drosophila melanogaster(2022) Fischer, Matthew Douglas; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Embryonic development is coordinated by interactions within gene regulatory networks. This process is orchestrated at the level of transcription through the regulatory properties of enhancers, which direct spatiotemporal expression patterns when bound by specific trans-acting factors. Though enhancers can act upon promoters located at great distances irrespective of orientation, the contributions from these cis-regulatory elements (CREs) are limited by insulators and/or tethering elements that organize chromatin architecture. Much research has been conducted towards understanding the coordination of the segmentation genes that pattern the basic body plan of the fruit fly, Drosophila melanogaster, during embryogenesis. The pair-rule genes (PRGs) of this pathway, such as fushi tarazu (ftz), are expressed in seven alternating stripes across the embryo. These PRGs are required for the development of body segments, and the mis-regulation of a single transcriptional domain can result in the loss of a segment. Here, I have investigated the ftz CREs to more precisely determine their sufficiency to direct expression within ftz stripe domains and their necessity for doing so in the native context of the gene. To investigate the sufficiency, I have generated 36 standardized reporter transgenes from 18 CREs, tested in both forward and reverse orientations. All CREs examined have been inserted into the same XbaI site of the reporter plasmid, and the transgenes have been inserted into the same genomic region. Through in situ hybridization experiments, I have determined that the qualitative patterns conferred by every CRE is orientation-dependent, and I have identified two putative insulators and/or tethering elements, proposed to explain this observation. To investigate their necessity, I targeted four genomic regulatory regions for precise deletion using the CRISPR/Cas9 system to generate seven deletion mutants. Though deletions were expected to cause lethality, most of the mutants are homozygous viable and fertile; only a mutant simultaneously removing two seven-stripe CREs was homozygous lethal. Quantitative gene expression analysis by fluorescent in situ hybridization chain reaction revealed that there is a critical threshold of ftz abundance required in each stripe for segmentation to proceed. In conclusion, I have determined that the ftz CREs are redundant and function together in a non-additive manner.Item INVESTIGATION INTO THE ROLE OF UVR8 IN BALANCING GROWTH AND ACCLIMATION TO UV-B RADIATION IN NATURAL AND TRANSGENIC POPULUS VARIANTS(2021) Wong, Tiffany Marie; Eisenstein, Edward; Sullivan, Joseph; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Research on woody plants offers promise for the development of next-generation biofuel feedstocks with reduced lignin recalcitrance and enhanced saccharification for ethanol production. Natural variants of Populus trichocarpa with diverse lignin content and saccharification differences, and transgenic Populus deltoides constructed for reduced lignin levels for improved cellulose extraction, offer clues to enhance biofuel production but with a tradeoff to overall fitness and biomass. One concern of engineering lignin relates to the protection of plants against environmental stress such as UV-B radiation. Secondary metabolite biosynthesis initiated by UV-B, particularly phenylpropanoids (lignin precursors) and flavonoids, plays an important role in managing and protection of UV stress. Genetic modifications affecting the production of these compounds may have significant physiological consequences. Thus, the goal of this research was to develop a model for biosynthetic compensation of low-lignin Populus to UV-B stress. The effect of UV-B on Populus was evaluated by spectroscopic and metabolomic measurements on leaves. UV-B promoted shifts in physiological and metabolomic responses of natural and transgenic Populus with varying levels of lignin were complex, reflecting compensation from variety of biosynthetic alterations. Therefore, the impact of modulating the expression of the photoreceptor, UVR8, in regulating the response of Populus to UV-B was pursued. Modulation of UVR8 expression in Populus hybrid was achieved by constructing transgenic plants using CRISPR and RNAi, in wild-type, and an RNAi-constructed cinnamyl alcohol dehydrogenase knockdown line. UV-B response of UVR8 modulated Populus indicated that flavonoids were upregulated in UVR8 overexpression lines, and that in a CAD knockdown background, these effects were slightly enhanced. Salicylates were upregulated in UVR8 knockout poplars, suggesting metabolic flux in the pathway, but little difference was seen relative to wild-type plants in CAD lines, and UV-B treatment had little effect. An interesting and unexpected finding was that UVR8 modulated Populus exhibited more rapid growth than wild-type plants. The findings underscore the key role of UVR8 in synchronizing protective metabolic responses to UV-B and suggest an additional function of the photoreceptor in regulating growth and development of Populus through shifts in the chemical equilibria of UVR8 monomers and dimers and interactions with other regulatory factors.Item Investigating the Distribution of CRISPR Adaptive Immune Systems Among Prokaryotes(2019) Weissman, Jake; Johnson, Philip L.F.; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Just as larger organisms face the constant threat of infection by pathogens, so too do bacteria and archaea. In response, prokaryotes employ a diverse set of strategies to simultaneously cope with their viral and physical environments. Here I explore the ecology and evolution of the CRISPR adaptive immune system, a powerful form of protection against viruses that is the only known example of adaptive immunity in prokaryotes. CRISPR systems are widespread across diverse bacterial and archaeal lineages, suggesting that CRISPR effectively defends against viruses in a broad array of environments. Nevertheless, this defense system is nearly absent in many bacterial groups, and in many environments. I focus on understanding these patterns in CRISPR incidence and the ecological drivers behind them. First, I identify the ecological conditions that favor the adoption of a CRISPR-based defense strategy. I develop a phylogenetically-conscious machine learning approach to build a predictive model of CRISPR incidence using data on over 100 phenotypic traits across over 2600 species and discovered a strong but hitherto-unknown negative interaction between CRISPR and aerobicity. I then consider the multiplicity of CRISPR arrays on a genome, testing whether or not selection favors redundancy in immunity. I use a comparative genomics approach, looking across all prokaryotes to demonstrate that on average, organisms are under selection to maintain more than one CRISPR array. I then explain this surprising result with a theoretical model demonstrating that a trade-off between memory span and learning speed could select for paired “long-term memory” and “short-term memory” CRISPR arrays. Finally, I provide a theoretical examination of the phenomenon of immune loss, specifically in the context of CRISPR immunity. In doing so, I propose an additional mechanism to answer the perennial question: “How do bacteria and bacteriophage coexist stably over long time-spans?” I show that the regular loss of immunity by the bacterial host can produce host-phage coexistence more reliably than other mechanisms, pairing a general model of immunity with an experimental and theoretical case study of CRISPR-based immunity.Item Novel Models for Studying Trophoblast Development and Placental Pathologies(2019) Pence, Laramie; Telugu, Bhanu; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Placental development begins in the mammalian blastocyst, when the first lineage specification event commits one cell population to making extraembryonic tissues, including the placenta, and commits another cell population to making the embryo proper. The mouse is an excellent animal model to study these early events and how the resulting placenta organ supports normal fetal development and a healthy pregnancy in the mother. The studies included in this Dissertation use the mouse to understand the role of long non-coding RNAs during early placental development, and to create a lineage biasing model that takes advantage of what is known about the first lineage specification event in mammals. Using expression analysis and the CRISPR/Cas9 system to create a knockout mouse strain, a placental-specific lncRNA was discovered and shown to be expressed in derivatives of the ectoplacental cone. Additionally, using the line age bias model to cause biased ablation of Hif1α in the placenta has revealed a role for fetal vs. placental contribution of resulting phenotypes.Item Diversity, dynamics, and dissemination of microbial communities in reclaimed and untreated surface waters used for agricultural irrigation(2019) Chopyk, Jessica; Sapkota, Amy R; Public and Community Health; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)High quality freshwater is a vital resource for sustaining agriculture and feeding a growing global population. Yet, due to increasing declines in groundwater, key food production regions across the world face uncertainty with regard to water availability. Nontraditional irrigation water sources, such as reclaimed water (advanced treated municipal wastewater) and untreated surface water (e.g. creeks, ponds, and brackish rivers), may contribute to sustainable solutions to conserve groundwater supplies. However, the microbial community composition and dynamics within these water sources are typically poorly characterized and comparative analysis of their microbial communities are rare. Using high-throughput, cultivation-independent sequencing methodologies, this dissertation research focused on three aims: 1) exploring the functional and taxonomic features of bacteria in nontraditional irrigation water sources; 2) assessing the bacterial and viral communities of agricultural pond water in relation to seasonality; and 3) describing the dynamics, composition, and potential dissemination of irrigation water microbiota from a freshwater creek to an irrigated field. The first aim was addressed through a broad investigation of bacteria within agricultural ponds, freshwater creeks, brackish rivers, and reclamation facilities. Through metagenomic-based analyses, features of the bacterial community, such as antimicrobial resistance genes (ARGs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays, were found to vary by sampling date and specific site. For the second aim, agricultural pond water was sampled over two time periods and found to harbor diverse bacteria and bacteriophage species, the abundance and composition of which were influenced by factors characteristic of the pond’s topography and seasonality. For the final aim, samples from a creek used actively for agricultural irrigation, as well as samples of pre- and post-irrigated soil, were analyzed. ARGs and virulence factors were identified in the water and soil samples, with the majority being specific to their respective environment. Moreover, analyses of CRISPR arrays from the creek samples indicated the persistence of certain bacterial lineages, as well as specific interactions between creek bacteriophage and their hosts. Overall, this research improves scientific knowledge of bacterial and viral composition, dynamics, and interactions that can be utilized to assess the suitability and safety of nontraditional irrigation water sources.Item ALTERNATIVE APPROACHES IN MOLECULAR CHARACTERIZATION OF FOODBORNE PATHOGENS: SHIGA TOXIN-PRODUCING Escherichia coli AND Salmonella SEROTYPES(2014) Toro Ibaceta, Magaly Alejandra; Meng, Jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Shiga toxin-producing E. coli (STEC) and Salmonella enterica subspecies enterica (S. enterica) are two major foodborne pathogens. They cause almost 1.5 million of cases of disease each year in the US. Due to their public health impact, development of new methods for their detection and identification are top priority. This research focused on identifying alternative molecular methods and markers for the identification of STEC and Salmonella. First, a suspension array was developed to simultaneously identify the seven most prevalent STEC (O26, O45, O103, O111, O121, O145, and O157) in the US. The panel targeted genes wzx or wzy and Shigatoxin genes. Testing and optimization employed four to eleven isolates of each serotype in the panel. STEC fluorescence values were 30 to >270 times greater than those of negative controls, demonstrating the method's effectiveness for the molecular serotyping of STEC. STEC strains (n=194) of 43 serotypes were examined for clustered regularly interspaced short palindromic repeats (CRISPR) arrays to study relatedness among serotypes. A subset of strains (n=81) was analyzed for cas and virulence genes to determine a possible relationship. CRISPR spacer content correlated well with serotypes, although some strains with different serogroup but the same H type shared identical arrays (O26:H11, O103:H11, and O111:H11). cas and virulence genes were not associated, but strains with greater probability of causing outbreaks and disease showed fewer spacers than those less likely to cause them (p<0.05). Therefore, CRISPR array content correlated well with STEC serotype, and CRISPR-cas systems were inversely related to strain virulence potential. Finally, the CRISPR arrays of 221 S. enterica of 53 serotypes were analyzed to define their relationship. CRISPR-cas systems of 50 S. enterica serotype Bareilly (S. Bareilly) were analyzed to resolve intra-serotype variations. CRISPR arrays correlated well with serotypes, although some serotypes displayed more than one type of array (e.g. S. Bareilly). Additionally, CRISPR-cas system elements reflected S. Bareilly phylogeny, but the array content was not linked to food vehicle or isolate's geographical origin. In conclusion, CRISPR array are useful for designing molecular serotyping assays, but a range of strains should be included to account for variation in S. enterica.