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.

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    A Comparative Analysis of the Binding Affinity of HIV-1 Reverse Transcriptase to DNA vs. RNA Substrates
    (2010) Olimpo, Jeffrey T.; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA-DNA versus DNA-DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3´ recessed DNA base) are required for stable binding to RNA-DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA-DNA. Replacing 2´- hydroxyls on pivotal RNA bases with 2´-O-methyls did not affect stability, indicating that interactions between hydroxyls and RT amino acids do not stabilize binding. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA-DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA-DNA and DNA-DNA, respectively, allow the former to conform more easily to HIV-RT's binding cleft, leading to more stable binding. Biologically, this may aid in degradation of RNA fragments that remain after DNA synthesis.
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    Genetics of Avian Paramyxovirus serotype 2
    (2010) Subbiah, Madhuri; Samal, Siba K.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Avian Paramyxovirus (APMV) serotype 2 is one of the nine serotypes of APMV that infect a variety of bird species around the world. In chickens and turkeys, APMV-2 causes respiratory illness and drop in egg production. To understand the molecular characteristics of APMV-2, the complete genome sequences of prototype strain Yucaipa and strains Bangor, England and Kenya were determined. The genome lengths of APMV-2 strains Yucaipa, Bangor, England and Kenya are 14904, 15024, 14904, 14916 nucleotides (nt), respectively. Each genome consists of six non-overlapping genes in the order 3'N-P/V/W-M-F-HN-L5' similar to most of APMVs. Sequence comparison of APMV-2 strains England and Kenya with prototype strain Yucaipa show 94-98% nt and 90-100% aggregate amino acid (aa) identities. However, strain Bangor shares low level of nt and predicted aa sequence identities with the other three strains. The phylogenetic and serological analyses of all four strains indicated the existence of two subgroups: strains Yucaipa, England and Kenya represented one subgroup and strain Bangor represented the other subgroup. All four strains were found to be avirulent for chickens by mean death time and intracerebral pathogenicity test. To further study the molecular biology and pathogenicity of APMV-2, a reverse genetics system for strain Yucaipa was established in which infectious recombinant APMV-2 was recovered from a cloned APMV-2 antigenomic cDNA. The recovered recombinant virus showed in vitro growth characteristics and in vivo pathogenicity similar to wild type virus. Recombinant APMV-2 expressing enhanced green fluorescent protein was also recovered, suggesting its potential use as a vaccine vector. Furthermore, generation and characterization of mutant viruses by replacing the fusion protein (F) cleavage site of APMV-2 with those of APMV serotypes 1 to 9 demonstrated that the amino acid composition at F protein cleavage site does not affect the pathogenicity of APMV-2. Overall, the study conducted here has several downstream applications. The complete genome sequence of APMV-2 is useful in designing diagnostic reagents and in epidemiological studies. The reverse genetics system for APMV-2 would be of considerable utility for introducing defined mutations into the genome of this virus and develop vaccine vector for animal and human pathogens.
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    MOLECULAR BASIS OF VIRULENCE IN INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (IHNV) USING A REVERSE GENETICS APPROACH
    (2009) Ammayappan, Arun; Vakharia, Vikram N; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Infectious hematopoietic necrosis virus (IHNV) is a pathogen of major economic importance to the aquaculture industry. The long-term goal of our work is to develop a safe and effective recombinant IHNV vaccine and possibly use IHNV as a virus vector to express foreign genes. To achieve this goal, the complete genome of IHNV 220-90 virulent strain was sequenced and characterized. Subsequently, a full-length cDNA clone of IHNV was generated by constructing the full length cDNA clone, between the cytomegalovirus (CMV) promoter and the autocatalytic hammerhead and hepatitis delta virus ribozymes. Transfection of a full-length plasmid, along with the supporting plasmids resulted in the recovery of infectious rIHNV-220-90. Characterization of the rIHNV-220-90 showed that its growth characteristics in tissue culture were comparable to those of the parental virus. The possible role of IHNV proteins in virulence was explored to some extent. For this, the entire genome of attenuated virus (IHNV-61) was sequenced and compared with its virulent strain. The comparative sequencing analysis studies revealed that majority of differences were located in the glycoprotein gene. The M and G genes, and the trailer region between virulent and attenuated viruses were exchanged; recombinant chimeric viruses were recovered and studied for their pathogenicity in rainbow trout. The results obtained from in vivo studies indicate that the glycoprotein plays a major role in IHNV virulence in fish, whereas the M gene and trailer region play a negligible role in virulence of IHNV. The potential of rIHNV to serve as a viral vector was explored by expressing the VP2 protein of IPNV and hemagglutinin-estrase (HE) protein of ISAV. The recovered rIHNV-VP2 and rIHNV-HE viruses stably expressed the VP2 and HE proteins respectively for at least five serial passages and showed characteristics comparable to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively to examine the molecular determinants of virulence, pathogenesis, and new approaches for vaccine development.
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    Biomedical Innovation and the Politics of Scientific Knowledge: A case study of Gardasil
    (2008) Clark, Aleia Yvonne; Mamo, Laura; Sociology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Vaccine development represents a special case where historically, public health priorities are central. Trends of privatization have increased the role played by pharmaceutical and biotech companies in developing new biomedical technologies. As the innovative science behind new medical technologies moves into pharmaceutical laboratories and biotech companies, the "logics of action" that pattern knowledge production shift. This project explores how different logics of action based on commercial investment and public good shaped the development of Gardasil, a new vaccine to prevent cervical cancer. The study found that both the logics of public good and commercial profit significantly shaped the final product. The study also found that variations in the definition of public good allowed for the settlement of tensions between good and profit. The findings have implications for the future of vaccine development, as well as for the analysis of biomedical innovation in our contemporary political economy.
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    Nucleocapsid protein modulates the specificity of plus strand priming and recombination patterns in Human Immunodeficiency Virus
    (2008-11-30) Jacob, Deena Thankam; DeStefano, Jeffrey J; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Replication in HIV (human immunodeficiency virus) occurs through reverse transcription in which the genomic single stranded RNA is copied into double stranded DNA. This process involves two priming events namely those of the minus and plus strand DNAs. The tRNA primer required to initiate the minus strand is carried by the virus into the host cell, while the plus strand primer is generated from a region of the genomic RNA called the polypurine tract (PPT). Results in this dissertation indicate a new role for HIV nucleocapsid protein (NC) in modulating the specificity of plus strand priming. For HIV, the central and 3′ (PPTs) are the major sites of plus strand initiation and other primers are rarely used. Using reconstituted in vitro assays, results showed that NC greatly reduced the efficiency of extension of non-PPT RNA primers, but not PPT. Extension assays in presence of mutant NCs show that the helix destabilization activity of NC and its ability to block the association of RT to non-PPT primers are responsible for the preferential extension of PPT in presence of NC. The effect of varying NC and Mg2+ concentrations on recombination during reverse transcription was also analyzed in this thesis. NC strongly influences the efficiency of recombination as well as the location where crossovers occurred. In contrast Mg2+ had a smaller effect on crossover locations. Both NC and Mg2+ influenced the level of pausing by RT during synthesis on RNA templates although NC's effect was more profound. At high NC concentrations, pausing was nearly eliminated even in locations with high predicted secondary structure. The results suggest that RT pausing may be limited during virus replication.
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    Host Molecular Responses in Chickens Infected with an Avian Influenza Virus
    (2008-11-20) Ramirez-Nieto, Gloria Consuelo; Perez, Daniel R.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Avian influenza virus has a segmented RNA genome that allows the virus to evolve continuously and generate new strains. Wild birds serve as natural reservoirs of avian influenza virus and provide a potential source for emergence of new viruses, which traverse host barriers and infect new avian or mammalian species. The mechanisms involved in this process are not completely understood. Our main goal is to understand host-pathogen interactions involved in avian influenza pathogenicity. As part of our approach we studied the effect of pre-exposure of chickens to IBDV (infectious bursal disease virus) on host susceptibility to infection, disease progression, and host molecular responses to infection with a mallard H5N2 low pathogenic avian influenza (LPAI) virus. We found that prior exposure of chickens to IBDV led to increased susceptibility to infection with the mallard H5N2 LPAI virus compared to normal chickens. This increased susceptibility allowed us to further adapt the virus to chickens. After 22 passages (P22) in IBDV-pre-exposed chickens, the LPAI virus replicated substantially better than the wild-type (WT) mallard virus in both IBDV-exposed and normal chickens. Interestingly, the P22 virus showed similar levels of replication in the respiratory and intestinal tracts of both groups, although it caused exacerbated signs of disease and severe lesions in the IBDV-pre-exposed group. We suggest that prior IBDV exposure provides a port of entry for avian influenza in an otherwise resistant chicken population. Furthermore, adaptation of avian influenza (AI) in IBDV-exposed chickens may allow for the selection of AI virus strains with expanded tissue tropism. We also studied the effects of host response to H5N2 AI in normal and IBDV-infected birds using high-throughput gene expression analysis. We demonstrated that IBDV-exposed chickens showed less than optimal humoral responses to LPAI infection as well as alterations in local molecular pathways that eventually led to exacerbated disease and death. At the molecular level we found amino acid substitutions in the surface glycoprotein hemagglutinin (HA). Those changes suggest selection for a virus that binds to and replicates more efficiently in chickens. Taken together our results suggest that IBDV-pre-exposure may play a role in exacerbating AI-induced pathogenicity.
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    Molecular markers of interspecies transmission of H2N2 and H9N2 avian influenza A viruses
    (2008-11-19) Sorrell, Erin Maureen; Perez, Daniel R; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Most avian influenza viruses do not replicate or transmit efficiently in mammals. The events that lead to interspecies transmission and host adaptation are unknown. Part one of this project set out to establish quail as an intermediate host of influenza. Our results indicate that adapting a mallard H2N2 virus in quail leads to expanded host range in chickens. The molecular changes, which occur during the adaptation in quail, are crucial for viral replication and transmission in chickens. Further adaptation of this quail-adapted virus in chickens leads to a 27 amino acid-deletion in the stalk region of the NA, changing the tissue tropism and temperature phenotype of the virus. H9N2 influenza viruses have created in poultry an endemic situation in much of Asia, Europe and the Middle East. This subtype, albeit low pathogenic, carries with it human receptor specificity and the ability to infect humans without prior adaptation. The generation of an influenza pandemic requires interspecies transmission of a novel strain, which can adapt to its new host through either reassortment or point mutations. Given that two previous pandemics were the result of reassortment between low pathogenic avian viruses and human subtypes of that period, and given the endemic situation of avian H9N2 viruses in Eurasia, for part two of this project, we wanted to determine if adaptation of an avian-human H9N2 reassortant in ferrets could support mammalian respiratory droplet transmission. Here we show for the first time that a reassortant virus carrying the HA and NA of an avian H9N2 virus can transmit in respiratory droplets. This is the first report of respiratory droplet transmission of H9N2 influenza, which carries profound implications for pandemic preparedness. The amino acid changes on the HA might identify critical, adaptive mutations necessary for respiratory transmission in subsequent pandemic avian influenza strains. Using reverse genetics we identified key combinations of this adapted reassortant that support respiratory droplet transmission.
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    Inhibition of PRRSV Replication by Combination of Antisense Morpholino Oligomers
    (2008-08-22) Han, Xue; Zhang, Yanjin; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Porcine reproductive and respiratory syndrome (PRRS) has caused heavily economic losses in the swine industry worldwide and current strategies to control PRRS are inadequate. Previous studies have shown that antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) can be effective antivirals against PRRS virus (PRRSV). PPMO are structurally similar to DNA with modified backbone and resistant to nuclease. In this study, we characterized the combined effect of PPMO. Two pairs of PPMO combinations were identified to show enhanced inhibitory effect on PRRSV replication in cell culture. The PPMO combination also inhibited replication of heterologous PRRSV strains within the same genotype. Treatment of the cells with the combination reduced PRRSV RNA and protein levels. In transiently transfected cells, the PPMO combination suppressed target mRNA expression, indicating that the suppression was due to their antisense effect. These results suggest potential application of these PPMO combinations for the control of PRRSV infection and spread.
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    RNA packaging and gene delivery using Tobacco mosaic virus pseudo virions
    (2008-04-28) Hung, Chi-Wei; Bentley, William; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    RNA interference (RNAi) has emerged as a powerful tool for the study of gene function and post-transcriptional regulation. However, the lack of a proper delivery system for RNAi is a major problem for its application as a therapeutic agent. In this study, Tobacco mosaic virus (TMV) is utilized as an RNAi carrier for gene delivery into mammalian and insect cells. The self assembly and disassembly of TMV is investigated to create chimeric viruses for gene delivery. The origin of assembly sequence (OAS) within the TMV RNA initiates its association with coat protein through a unique hairpin structure. Studies in this dissertation show that by incorporating TMV OAS into an RNA of interest, the RNA can assemble into "pseudo-virions" by the virus coat protein. The length of the pseudo-virions changed in proportion with the size of the RNA. To deliver the RNA to the targeted cells, virions are further surface-modified with synthetic cell-penetrating peptides to facilitate cell endocytosis. Two genes were selected as targets: 1) EGFP as a visual marker and 2) Cyclin E for control of cell cycle. EGFP is expressed in a transient expression experiment using a plasmid vector, pEFGP-N1. Cyclin E is regulated endogenously in High FiveTM cells, and its translation is targeted using the pseudo virions. Pseudo-virions targeting EGFP RNA (antisense EGFP) are able to suppress transient EGFP production by 61% whereas pseudo virions targeting cyclin E (antisense cycE) are capable of arresting cells at G1 phase. This RNA packaging system protects packaged RNA and provides a means of delivering RNAi constructs into various host cells.
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    Functions of the Tobacco mosaic virus helicase domain: regulating formation of the virus replication complex and altering the activity of a host-encoded transcription factor
    (2008-04-23) Wang, Xiao; Culver, James N; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Tobacco mosaic virus (TMV)-encoded 126-kDa and 183-kDa replicases are multidomain and multifunctional proteins. The helicase domain shared by both replicases has been shown to perform multiple tasks during the virus life cycle. In vitro structural and functional analyses demonstrated that monomers and dimers of the TMV helicase domain were the active forms for ATP hydrolysis. However, self-interaction of the helicase polypeptides resulted in the formation of higher-order structures that likely serve as structural scaffolds for the assembly of virus replication complexes (VRCs). Mutagenesis studies of the TMV helicase motifs showed that conserved amino acid residues played important roles in protein ATPase and/or RNA binding activities. A close correlation between ATPase activity of the helicase domain and assembly of wild-type VRC-like vesicles by the 126-kDa replicase further suggests that ATPase activity of the TMV helicase domain may modulate proper VRC assembly. In addition to helicase self-interaction, a novel virus-host interaction involving ATAF2, a NAC domain transcription factor was identified. Members within the NAC domain family are involved in plant developmental processes and stress/defense responses. In this study, transgenic plants overexpressing ATAF2 showed a strong developmental phenotype. Inoculation of TMV in these transgenic plants resulted in reduced virus accumulations. Additionally, transcriptional induction of ATAF2 occurred in response to TMV infection and salicylic acid treatment. Combined, these results suggest that ATAF2 is involved in a host defense response. One interesting finding was that in susceptible hosts, virus-directed induction of ATAF2 and PR1, a well-defined pathogenesis-related (PR) marker gene for host defense system, occurred only in locally-infected but not in systemically-infected tissues. Dynamic changes in the expression of host defense genes suggest that viruses have evolved certain mechanisms to actively modulate host gene expression. Interaction between the TMV helicase domain and ATAF2 may provide one way to suppress the ATAF2-mediated host defense signaling pathway. Combined these studies investigated the importance of the TMV helicase domain in VRC formation and in manipulating the host defense system. The results confirmed the functional versatility of the TMV helicase domain in establishing a successful virus life cycle.