Cell Biology & Molecular Genetics Theses and Dissertations

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THE 3' UTR OF TURNIP CRINKLE VIRUS INTERACTS LOCALLY AND DISTALLY TO REGULATE TRANSCRIPTION AND TRANSLATION OF THE VIRUS
(2012) Young, Megan Yoke Len; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Turnip crinkle virus (TCV) is a 4054 b positive-strand RNA virus of the genus Carmovirus in the Family Tombusviridae. Upon entry into cells, TCV is translated using host translational machinery to produce its RNA-dependent RNA polymerase (RdRp). The RNA is proposed to undergo a conformational rearrangement, mediated by recruitment of the RdRp to the 3' ends of the viral RNA, which represses translation and promotes negative-strand synthesis. A second RNA switch is proposed to occur that inhibits minus-strand synthesis and promotes recruitment of the RdRp to the 3' ends of negative-strands for the asymmetrical production of positive-strands. Within the 3' UTR of TCV is a tRNA-shaped structure (TSS) that is capable of binding ribosomes and overlaps with structures necessary for translational enhancement. The RdRp has been shown to bind within this region and result in a widespread conformational shift. The binding of RdRp to the 3' end of the virus is very sensitive to perturbations of sequence or structure, with many mutations resulting in non-specific binding of the RdRp. The elements within the 3' UTR have been shown to be very interactive with alterations affecting the structure of regions hundreds of bases away. A second-site mutation study indicated that regions upstream of the 3' UTR may also be interacting with the 3' UTR. Some second-site mutations located in this upstream region were found to increase accumulation in protoplasts and additional studies are under way to explain this phenomenon. The 3' viral contribution in a luciferase reporter construct was increased to incorporate the second-site mutations. While the second-site mutations had little effect on translation, it was surprising to find that extension of the viral 3' sequence enhanced translation. Translational enhancement was mapped to just an additional twenty bases and further study revealed that a hairpin (H3) is important for viral translation and accumulation and may also be interacting with the 3' UTR.
Actin-binding protein 1 and Dynamin-2 modulate attenuation of B-cell signaling and regulate B-cell function
(2014) Seeley-Fallen, Margaret Kathryn; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
B-cells mediate humoral immune responses with production of an amazingly diverse repertoire of antigen specific antibodies. Antigen binding to the B-cell receptor (BCR) induces coordinated BCR signaling and BCR-mediated antigen internalization, processing, and presentation to helper T-cells, necessary for generation of humoral memory. Prolonged or uncontrolled BCR signaling has been linked to development of autoimmunity; therefore controlled attenuation of B-cell signaling is required for maintenance of B-cell tolerance. This study investigates the role of an actin adaptor protein, actin-binding protein 1 (Abp1/HIP-55/SH3P7), in BCR-mediated signal transduction and subsequent B-cell function. I found that in both Abp1-/- and Abp1-/--bone marrow chimeric mice, in which only B-cells lack Abp1 expression, the number of spontaneous germinal center and marginal zone B-cells, and levels of autoantibody are significantly increased. Serum levels of T-independent antibody and total IgM and IgG antibodies are elevated in Abp1-/- mice, whereas T-dependent IgG responses are reduced and fail to undergo affinity maturation. Upon binding membrane-associated antigen, surface BCR clustering is enhanced and B-cell contraction delayed in Abp1-/- B cells, concurrent with slow but persistent increases in F-actin at BCR signalosomes. Furthermore, BCR signaling is enhanced in Abp1-/- B cells, including Ca2+ flux and phosphorylation of B-cell linker protein (BLNK), mitogen-activated protein kinase kinase MEK1/2, and extracellular signal-regulated kinase (ERK), coinciding with reductions in recruitment of the inhibitory signaling molecules, hematopoietic progenitor kinase 1 (HPK1) and SH2-containing inositol 5-phosphatase (SHIP-1) to BCR signalosomes. Our previous studies demonstrated a role for Abp1 in BCR internalization by linking the actin cytoskeleton to the endocytic machinery protein dynamin2. This study shows that dyanmin2 is recruited to the BCR upon antigen binding, and its recruitment is dependent on its proline rich domain (PRD). BCR internalization and trafficking to late endosomes requires the PRD, GTPase function, and tyrosine phosphorylation sites of dynamin2. B-cells expressing a GTPase dead mutant of dynamin2 exhibit enhanced BCR clustering and signaling. These results indicate Abp1 negatively regulates BCR signaling by coupling actin remodeling to B-cell contraction, activation of inhibitory signaling molecules, and the endocytic machinery protein dynamin2, revealing a novel regulatory mechanism for peripheral B-cell development and antibody response.
The activation of memory B cells to generate high affinity antibody responses in vitro and in vivo
(2011) Richard, Katharina; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Immunological memory is the hallmark of the adaptive immune system. The humoral branch of the immunological memory is mediated by memory B-cells (mB). Memory B cells are marked by longevity, expression of antibodies with high affinity, and ability to generate robust antibody responses upon reencountering pathogens. However, requirements for the activation of mB cells and the induction of humoral memory responses are not well understood. This thesis examines the role of Toll-like receptors (TLRs) in mB activation using an immunized mouse model. TLRs are a family of receptors that recognize common molecular patterns of microbial pathogens and stimulate innate immune responses. Our study found that mouse mB expressed TLR9 and 4, and responded to their agonists in vitro by differentiating into high affinity IgG secreting plasma cells. However, TLR agonists alone were not sufficient to activate memory B cells in vivo. Antigen was required for the clonal expansion of antigen-specific memory B cells, the differentiation of mB cells to high affinity IgG secreting plasma cells, and the recall of high affinity antibody responses. The Ag- specific B cells that had not yet undergone isotype switching showed a relatively higher expression of TLR4 than memory B cells, which was reflected in a heightened response to its agonist, but in both cases of TLR4 and 9 yielded mostly low affinity IgM secreting plasma cells. When immunized together with the antigen, TLR agonists not only boosted the antigen-specific titers, but also increased affinity and isotype switching of the immunoglobulin. Thus, while TLR agonists alone are unable to activate mB in vivo, they can enhance humoral memory responses induced by the antigen.
Activation of NLRP3 inflammasomes during complement-mediated phagocytosis by macrophages
(2015) Suresh, Rahul; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The Complement System is an important component of innate immunity, whose activation has been associated with acute inflammation. Activation of complement leads to the generation of a Membrane Attack Complex (MAC) composed of C5b-C9 proteins. The function of the MAC has been primarily associated with target cell death through the polymerization and insertion of C9 on the surface of complement activating particles. Here, I provide evidence that MAC assembled on the surface of complement activating particles, can be transferred to host macrophages during the process of phagocytosis. This “bystander activation” onto macrophages causes potassium efflux and ROS production, which induces the assembly of the NLRP3 inflammasome, which in turn results in the activation of caspase-1 and the processing and secretion of IL-1β and IL-18 to regulate both innate and adaptive immunity. Inflammasome activation is not induced when macrophages phagocytize unopsonized particles or particles opsonized with serum deficient in one of the terminal complement components. The secretion of IL-1β and IL-18 by macrophages is dependent on NLRP3, ASC, and caspase-1, as macrophages deficient in any one of these components fail to secrete these ii cytokines following complement-mediated phagocytosis. The phagocytosis of complement-opsonized particles increases leukocyte recruitment and promoted Th17 biasing. Leishmania major, an intracellular pathogen, follows a similar pattern, when infecting macrophages: Phagocytosis of the pathogen was accompanied by the activation of complement pathway resulting in “bystander activation” and NLRP3 inflammasome assembly. When macrophages are primed with IFN and IL-1β together prior infection, the killing of parasites is enhanced demonstrating that IL-1β can work in concert with IFNγ to activate macrophages and thus reduce the intracellular burden of the pathogen. This study demonstrates that the phagocytosis of complement-opsonized particles can induce inflammasome activation by a novel mechanism involving MAC-mediated “bystander activation” of host macrophages. This work provides another mechanism whereby complement activation can lead to acute inflammation and identifies a previously undescribed function of the MAC to activate inflammasomes on macrophages.
Active and Passive Immunization Strategies for Protection of Mice and Monkeys Against Orthopoxvirus Infection
(2006-10-18) Fogg, Christiana; Simon, Anne; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The Poxviridae are large DNA viruses that replicate in the cytoplasm of vertebrates or invertebrates. The genus Orthopoxvirus includes variola virus, the cause of smallpox, and vaccinia virus (VACV), the prototypal family member used in the licensed smallpox vaccine. Interest in the development of an alternative smallpox vaccine emerged because of complications associated with recent vaccination efforts and the growing number of people excluded from vaccination. Antibody therapies are also of interest for Orthopoxvirus infection treatment instead of vaccinia immune globulin from human donors. Essential to these efforts are studies that elucidate aspects of the immune response required for protection against disease. Two infectious forms of virus exist, intracellular mature virus (IMV), which mediates spread between hosts, and extracellular virus (EV), which is required for efficient spread within a host. IMV and EV each possess an outer membrane with viral proteins targeted by the adaptive immune response. I have used soluble baculovirus-expressed forms of VACV proteins from the IMV and EV in order to understand the role of immunity to these particles during infection. Subcutaneous immunization of mice multiple times with the EV proteins A33 and B5 and the IMV protein L1 either individually or in combinations induced specific antibody responses and protected against weight loss and death caused by virus infection, especially following immunization with A33+B5+L1 or A33+L1. Similar patterns of protection were observed by passive immunization of mice with polyclonal or monoclonal antibodies against A33, B5, or L1 prior to or after intranasal challenge. A27 was investigated as an alternative IMV protein to L1, but proved less effective alone or in combination with A33. Potent and more rapid immune responses to the A33 and L1 proteins were stimulated by the use of the adjuvants QS-21, or alum mixed with CpG oligodeoxynucleotides. Protection against a lethal challenge was observed in a small study with monkeys that were immunized with A33, B5, and L1 and challenged with monkeypox. My data indicate protection against orthopoxviruses is seen in animal models so long as a good antibody response is made to both the IMV and EV forms.
The agarase system of saccharophagus degradans strain 2-40 : analysis of the agarase system and protein localization
(2005-12-05) Ekborg, Nathan Alexander; Weiner, Ronald; Hutcheson, Steven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Saccharophagus degradans (formerly "Microbulbifer degradans") strain 2-40 is a Gram-negative marine bacterium isolated from the Chesapeake Bay. Analysis of 16s rDNA sequence indicated that S. degradans is related to a group of marine proteobacteria adept at degrading complex polysaccharides (CPs). S. degradans can depolymerize at least ten CPs including agarose. Agarose, an algal galactan, is degraded by few organisms. The agarase system of S. degradans was shown to be composed of five enzymes AgaA, AgaB, AgaC, AgaD and AgaE. These proteins contain glycoside hydrolase domains GH16, GH50 and GH86. S. degradans is the only organism known to collectively encode agarases with at least one of these domains. Unusual for agarases, AgaB and AgaE also contain multiple type-six carbohydrate binding modules. Furthermore, AgaE contains four thrombospondin type-three repeats whose function in prokaryotic proteins were unknown. The predicted agarases were characterized using a variety of methods including genomics, biochemical assays, proteomics and a newly described mutagenic technique. Agar degradation by S. degradans includes two depolymerases, AgaB and AgaC, a B-agarase II (AgaE) and a possible a-neoagarobiose hydrolase (AgaA). AgaB was found to be freely secreted while AgaC and AgaE were surface associated. AgaC is a predicted lipoprotein while AgaE did not have domains characteristic of surface localization. The Tsp-3 repeats, which are similar to repeats found on other cell surface enzymes, are the proposed cell surface anchoring sequences of AgaE.
ALTERED PORCINE ALVEOLAR MACROPHAGE PHENOTYPE BY ALL-TRANS RETINOIC ACID AND ASCARIS SUUM INFECTION
(2011) Perry, Trinity Lynn; Mosser, David M; Urban, Joseph F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Ascaris suum is known to cause ascariasis of both pigs and humans. Ascariasis is associated with a reduced control of intracellular pathogens and decreased vaccine efficacy. All-trans retinoic acid (ATRA), an active metabolite of Vitamin A (VA), is known to mediate a diverse array of physiological and biological processes including immune and inflammatory responses. The phenotype of alveolar macrophages (AM), the most abundant cell in the lung, has not been critically examined during chronic infection with A. suum nor has the effect of administration of dietary ATRA alone or during infection. This combination of treatments is likely common in underdeveloped regions where infection with Ascaris is prevalent and public health initiatives to control nutrient deficiencies often include VA supplementation. In this study we showed that a low dose and repeated "trickle" infection with A. suum alone and with ATRA administration altered the AM cell surface antigen phenotype.
ALTERING THE AI-2 MEDIATED QUORUM SENSING CIRCUITRY TO QUENCH BACTERIAL COMMUNICATION NETWORKS
(2011) Roy, Varnika; Bentley, William E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The emergence of antibiotic resistant bacteria poses a global threat to human health and has been classified as a clinical super-challenge of the 21st century. This has necessitated research on new antimicrobials that inhibit bacterial virulence by mechanisms other than those that target bacterial growth or viability. Such approaches have been reported to pose less evolutionary pressure on bacteria to evolve and become resistant to antibiotics. Bacterial cell-cell communication, termed quorum sensing (QS), is mediated by signatures of small molecules. QS via these small molecules has been linked to numerous undesirable bacterial phenotypes such as biofilm formation, onset of pathogenicity, triggering of virulence genes etc. The small signaling molecules represent targets for intercepting bacterial communication (and their resultant undesirable phenotypes). We have devised two strategies that interrupt bacterial communication in multispecies bacterial cultures by targeting the interspecies signaling molecule autoinducer-2 (AI-2), which is produced or recognized by over 70 species of bacteria. Our first approach is to bring the native intracellular AI-2 signal processing mechanisms to the extracellular surroundings to quench the QS response of bacteria. Specifically we deliver the Escherichia coli AI-2 kinase, LsrK, to E. coli populations ex vivo and phosphorylate and degrade the extracellular AI-2. This significantly attenuates the native QS response in E. coli. Similar results are obtained in a tri-species synthetic ecosystem comprising E. coli, Salmonella typhimurium and Vibrio harveyi. In our second quenching strategy, we explore a panel of small synthetic molecules that are analogs of AI-2 (C1-alkyl analogs). The analogs are observed to cause species-specific and cross-species quorum quenching in the tri-species synthetic ecosystems of the aforementioned strains. Some of the AI-2 analogs quench pyocyanin (toxin production) in the opportunistic pathogen Pseudomonas aeruginosa. Based on these observations, I used analog cocktails to quench QS en masse in assembled synthetic ecosystems. Finally, I tested the efficiency of the analogs in quenching pathogenic phenotypes such as biofilm formation in E. coli. The analogs inhibit biofilm formation and act in concert with antibiotics to reduce biofilm formation even further. Our results suggest entirely new modalities for interrupting or tailoring the networks of communication among bacteria and identifying drug targets to develop the next generation of antimicrobials based on QS.
Alternate Conformations Regulate Ribosomal Recoding in a Positive-sense RNA Virus
(2016) Kuhlmann, Micki Michelle; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Positive-sense RNA viruses are important animal, plant, insect and bacteria pathogens and constitute the largest group of RNA viruses. Due to the relatively small size of their genomes, these viruses have evolved a variety of non-canonical translation mechanisms to optimize coding capacity expanding their proteome diversity. One such strategy is codon redefinition or recoding. First described in viruses, recoding is a programmed translation event in which codon alterations are context dependent. Recoding takes place in a subset of messenger RNA (mRNAs) with some products reflecting new, and some reflecting standard, meanings. The ratio between the two is both critical and highly regulated. While a variety of recoding mechanisms have been documented, (ribosome shunting, stop-carry on, termination-reinitiation, and translational bypassing), the two most extensively employed by RNA viruses are Programmed Ribosomal Frameshifting (PRF) and Programmed Ribosomal Readthrough (PRT). While both PRT and PRF subvert normal decoding for expression of C-terminal extension products, the former involves an alteration of reading frame, and the latter requires decoding of a non-sense codon. Both processes occur at a low but defined frequency, and both require Recoding Stimulatory Elements (RSE) for regulation and optimum functionality. These stimulatory signals can be embedded in the RNA in the form of sequence or secondary structure, or trans-acting factors outside the mRNA such as proteins or micro RNAs (miRNA). Despite 40+ years of study, the precise mechanisms by which viral RSE mediate ribosome recoding for the synthesis of their proteins, or how the ratio of these products is maintained, is poorly defined. This study reveals that in addition to a long distance RNA:RNA interaction, three alternate conformations and a phylogenetically conserved pseudoknot regulate PRT in the carmovirus Turnip crinkle virus (TCV).
ANALYSIS OF CONSENSUS GENOME-WIDE EXPRESSION-QTLS AND THEIR RELATIONSHIPS TO HUMAN COMPLEX TRAIT DISEASES
(2014) YU, CHEN-HSIN; Moult, John; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Genome-wide association studies of human complex disease have identified a large number of disease associated genetic loci. However, most of these risk loci do not provide direct information on the biological basis of a disease or on the underlying mechanisms. Recent genome-wide expression quantitative trait loci (eQTLs) association studies have provided information on genetic factors, especially SNPs, associated with gene expression variation. These eQTLs might contribute to phenotype diversity and disease susceptibility, but interpretation is handicapped by low reproducibility of the expression results. Our first major goal was to establish a list of consensus eQTLs by integrating publicly available data for specific human populations and cell types. We used linkage disequilibrium data from Hapmap and the 1000 Genomes Project to integrate the results of eQTL studies. Overall, we find over 4000 genes that are involved in high confidence eQTL relationships. We also assessed the possible underlying mechanisms of tissue dependent eQTLs by mapping these to known genome sites of functional elements. Results of comparison of eQTLs across studies on the same cell type versus those on different cell types suggest that tissue specific eQTLs are less common than pan-tissue eQTLs. Our second major goal was to use these results to elucidate the role eQTLs play in human common diseases. For this purpose, we matched the high confidence eQTLs to a set of 335 disease risk loci identified from the Wellcome Trust Case Control Consortium (WTCCC1) genome-wide association study and follow-up studies for seven human common diseases. Our results show that the data are consistent with approximately 50% of these disease loci arising from an underlying expression change mechanism. In many cases, the results provide a proposed expression mechanism for genes previously suggested as disease relevant, in others, new disease relevant genes are identified. A web-based database, ExSNP, was designed to provide comprehensive access to the eQTL data and results from our analysis, including original eQTLs, high-confidence eQTLs, cell type dependent eQTLs, population dependent eQTLs, disease associated eQTLs, and functionally annotated eQTLs. The website also incorporates a genome browser that allows visualization of the relative positions of eQTL SNPs to their associated genes and other neighboring genes, as well as the relationship to functional elements and disease associations.
Analysis of Frameshifting Frequencies Due to Homopolymeric Nucleotide Tracts in Neisseria gonorrhoeae
(2006-03-10) Holder, Robert Christopher; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Organisms that are sensitive to the antimicrobial agent nitrofurantoin express nitroreductases, which reduce nitrofuran containing compounds to highly reactive and damaging intermediates. A single nitroreductase, nfsB, was identified in N. gonorrhoeae FA1090. The sequence of nfsB was modified to contain polyguanine tracts of varying lengths. Analysis indicated that mutations yielding nitrofurantoin resistance occurred at a higher rate in strains containing modified nfsB genes when compared to wildtype FA1090. The frequency of mutation also increased as polyguanine tract length increased. A polyguanine tract length of 5 residues did not produce elevated mutation frequencies. The use of PCR and polyacrylamide gel electrophoresis proved to be a reliable method for quickly identifying nitrofurantoin resistant mutants that contained nfsB frameshift mutations. When nfsB fragments amplified from nitrofurantoin resistant mutants were electrophoresed, the presence of insertions or deletions in the nfsB coding sequence was easily detected.
ANTIBACTERIAL MECHANISM OF PLANT-DERIVED PHENOLICS AGAINST SALMONELLA ENTERICA SEROVAR TYPHIMURIUM
(2023) Alvarado-Martinez, Zabdiel; Biswas, Debabrata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Salmonella enterica serovar Typhimurium (ST) remain one of the main bacterial pathogens responsible for illnesses, hospitalizations, and deaths in the USA. Its ubiquitous prevalence in nature, invasive pattern and increasing antibiotic resistance make it a public health threat, warranting the discovery of novel antimicrobials that can be implemented as either treatments or as forms of control. Plant-derived compounds have been proposed as potential antimicrobials that can be used against gram-negative pathogens, with phenolic acids being of interest for their prevalence in nature and bioactivity. This research studied the effects of gallic acid (GA), protocatechuic acid (PA) and vanillic acid (VA) against ST. Findings showed these compounds to be able to inhibit bacterial growth in vitro, while also showing a reduction in the expression of key virulence genes, without inducing resistance over multiple passages. Further studies using a human epithelial cell line for studying host-pathogen interactions, showed their capability to reduce the number of ST that were able to invade the host cells. Further studies were performed in cecal fluid to test their potency in more complex environments and assess their effects on the microbiome. When in cecal fluid, compounds showed a reduced inhibitory potency compared to in vitro, but still exerted antimicrobial pressure against ST. When analyzing relative abundance of other bacteria through 16S-rRNA gene sequencing, there was an overall decrease in the Protobacteria phylum, while no significant negative effect was seen for other phyla like that of the Firmicutes and Actinobacteria. Experiments to determine the mechanism of action against ST showed these phenolic acids to permeabilize the cell plasma membrane, in addition to reducing cell wall synthesis. Scanning electron microscopy showed treated bacteria to have dents at the polar ends of the cell, while others were found in a duplet formation, suggesting further disruption of specific bacterial functions associated to cell division and structure. These findings suggest that despite their similarities, these compounds are capable of exerting different types of antimicrobial pressure against ST that could better inform their future use as control measures against ST, and their potential use case based on the desired outcome.
Antibody Responses During Infection with Leishmania spp.
(2005-11-30) Miles, Suzanne Adell; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Classically activated macrophages produce high levels of IL-12 and moderate levels of IL-10. When IgG immune complexes ligate the Fc gamma receptors on activated macrophages, they shut off IL-12 synthesis and increase IL-10 production. These macrophages are termed 'type II' activated macrophages. The anti-inflammatory responses of type II activated macrophages could be harmful to the host when an inflammatory response is critical to pathogen clearance. The clearance of intracellular pathogens, such as Leishmania spp., relies upon an inflammatory response. Inflammatory cytokines lead to classical macrophage activation, which is critical to the destruction and clearance of phagocytosed parasites. Without a robust inflammatory response, Leishmania parasites are free to replicate within macrophages, leading to disease pathology. Based on this knowledge, it was hypothesized that IgG coated Leishmania amastigotes would ligate Fc gamma receptors on host macrophages and induce the production of IL-10. Therefore, the role of IgG in mediating host defense to Leishmania was examined. For these studies, JH mice, which lack IgG and are on the Leishmania major susceptible BALB/c background, were used. We show that IgG not only fails to provide protection against this intracellular pathogen, but it actually contributes to disease progression. JH mice were more resistant to disease than control BALB/c mice, and reconstitution with anti-L. major antisera resulted in increased disease, with larger lesions and higher numbers of parasites. Antibody administration correlated with an increase in IL-10 production in lesions, however, this IgG mediated exacerbation of disease could be reversed by simultaneously treating with anti-L. major antisera and a monoclonal antibody against the IL-10 receptor. The anti-L. major IgG profiles of resistant C57BL/6 mice and susceptible BALB/c mice were examined. BALB/c mice exhibited higher levels of IgG, in both circulating titers and on the surfaces of amastigotes, than C57BL/6 mice. The differences in the anti-L. major IgG titers between resistant and susceptible strains provide evidence of the integral role of IgG during leishmaniasis. These studies demonstrate that IgG can cause a novel form of immune enhancement due to its ability to induce IL-10 production from macrophages.
THE ANTIVIRAL ROLES OF ATG1 IN DROSOPHILA MELANOGASTER: IMMUNE RESPONSES AGAINST DROSOPHILA X VIRUS
(2014) Wang, Qian; Wu, Louisa; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
In mammals, autophagy is important for the immune response against select viruses and is responsible for delivering virus to the lysosome for degradation. In Drosophila melanogaster, the roles of autophagy genes in an antiviral immune response are not fully understood. Here we identify a novel antiviral role for Atg1 in Drosophila melanogaster upon infection with Drosophila X virus (DXV). Flies with a decreased level of Atg1 expression in the fat body developed an increased susceptibility to DXV and have a higher viral load compared to wildtype. However, silencing of other autophagy components (Atg7, Atg8) does not have the same effect. Moreover, we could find no evidence that classical autophagy is directly associated with DXV upon viral infection. This suggests that the antiviral function of Atg1 may be independent of classical autophagy. To address this, we examined the effect of Atg1 knockdown on the fly transcriptome in both DXV infected and uninfected flies. Interestingly, lipid droplet lipolysis and β-oxidation, two major processes responsible for energy production, are induced upon DXV infection. Facilitating lipolysis by knocking down lsd2, a positive regulator of lipase bmm, results in an increased host susceptibility to DXV, together with an increased viral load. In contrast, blocking lipolysis in the negative regulator lsd1 null mutant renders the host more resistant to the virus. This indicates that the increased energy production favors the virus for active replication and does not favor the elimination of virus. Surprisingly, silencing of Atg1, even in the absence of infection, also increases the rates of lipolysis and &beta-oxidation, shown by an increased expression of genes that are involved in lipid metabolism and an decreased lipid droplet size in the Atg1-silenced flies. The differences in gene expression and lipid droplet size between Atg1 RNAi flies and WT flies become more apparent as the infection progresses. In summary, we identify a novel role for Atg1 in restricting energy production and limiting DXV replication. This finding may shed light on antiviral studies against other dsRNA viruses that manipulate host energy homeostasis. Finally, our data reveal an important and unexpected role for Atg1 in innate immune antiviral responses independent of autophagy.
ARCHAEAL DNA REPLICATION PROTEINS: MEMBERS AND FUNCTIONS
(2013) Li, Zhuo; Kelman, Zvi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The mechanism of DNA replication in archaea, the third domain of life, has been studied for more than two decades using biochemical, structural and bioinformatic approaches. Historically, many of the proteins that participate in archaeal replication were identified via similarity to enzymes needed for DNA replication in bacteria and eukarya. This study uses a different approach to identify new factors that may be involved in replication. Genetic tools developed for the thermophilic archaeon Thermococcus kodakarensis were used to identify new replication factors that could not be recognized through in silico methods. First, a network of proteins that may participate in replication was identified using in vivo tagging of known replication enzymes. Following affinity purification the proteins that co-purified with the tagged enzymes were identified using mass spectrometry. This study describes the identification of a number of new putative replication factors. Next, the biochemical properties of two proteins identified in the screen were characterized. One, the product of gene TK1525, was identified via its interaction with the GINS complex. This protein was predicted to be an archaeal homologue of the bacterial RecJ nuclease. It was found that the protein is a processive, manganese-dependent, single strand DNA-specific exonuclease. The protein was designated GAN for GINS-associated nuclease. GAN forms a complex with GINS and also interacts with the archaeal-specific DNA polymerase D in vivo. Subsequent bioinformatic analysis suggested that GAN may be the archaeal homologue of the eukaryotic Cdc45 protein. The second protein characterized is the product of TK0808. This protein was identified via its interactions with proliferating cell nuclear antigen (PCNA). The protein, upon binding to PCNA, inhibits PCNA-dependent activities. The protein was therefore designated TIP for Thermococcales inhibitor of PCNA. While most proteins that interact with PCNA do so via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA, TIP neither contains the canonical PIP motif nor interacts with PCNA via the IDCL. These findings suggest a new mechanism for PCNA binding and suggest a new mechanism to regulate PCNA-dependent activities.
AUTOINDUCER-2 (AI-2) MEDIATED QUORUM SENSING IN ESCHERICHIA COLI
(2004-12-14) Wang, Liang; Bentley, William E; Hutcheson, Steven W; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Bacteria have evolved complex genetic circuits to regulate their physiological activities and behaviors in response to extracellular signals. In a process termed "quorum sensing", or density-dependent gene regulation, bacteria produce, release and respond to certain signaling molecules termed autoinducers. The bacterial autoinducer-2 (AI-2) has received intense interest recently because the gene for its synthesis, luxS, is common in a large number of Gram-negative and Gram-positive bacterial species. In this study, the luxS controlled genes were identified in Escherichia coli K12 strain under two different growth conditions using DNA microarrays. Deletion of the luxS was shown to affect expression of genes involved in AI-2 transport (the lsr operon) and methionine biosynthesis (metE), and to a lesser degree those involved in methyl transfer, iron uptake, resistance to oxidative stress, utilization of various carbon sources, and virulence. The effects of glucose on extracellular AI-2 level were investigated further. It was shown that both AI-2 synthesis and uptake in Escherichia coli are subject to catabolite repression through the cAMP-CRP complex. This complex directly stimulates transcription of the lsr (luxS regulated) operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with LsrR repressor to regulate AI-2 uptake. This study, for the first time, has shown that quorum sensing regulates specific activities in E. coli K12, and has elucidated regulatory mechanisms for AI-2 biosynthesis and transport in this organism. With a better understanding of AI-2/luxS mediated gene regulation, we may be able to develop strategies for harnessing AI-2 quorum sensing for our advantage in bioreactor studies and ultimately in control of the bacterial pathogenicity.
B Cell Memory, CD23, and Lipid Metabolism: A Preliminary Study
(2012) Wiggins, Melvin Daniel; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
We each receive vaccinations throughout our lives, which protect us from many pathogens and gives long-term protection through generating a subset of memory lymphocytes. This study explores whether CD23 (Fcε receptor) and high fat diet have roles in regulating memory B cells. CD23 in B cells was examined using CD23 transgenic mice. My data show that, after antigenic stimulation, CD23 co-aggregates with the BCR. The percentages of isotype switched B cells as well as other peripheral B cell subsets in the spleen are not altered in unimmunized CD23 transgenic mice, implicating that CD23 does not have any significant role in the generation of memory B cells. High fat diet with and without high cholesterol led to increased numbers of mature follicular B cells and decreases in transitional B cells in a NPC1L1 independent manner.The marginal zone B cells numbers are increased in the mice fed high fat/high cholesterol diets. This suggests a possible role of high fat/high cholesterol diet in regulating the peripheral development of B cells.
The behavior and evolution of Class II transposable elements in the malarial mosquito, Anopheles gambiae
(2008-02-11) Subramanian, Ramanand Arun; O'Brochta, David A; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Transposable elements are DNA sequences with a unique ability to change their genomic location. Transposable elements are fascinating because of their ability to move, and their ubiquitous presence and contribution to the evolution of all prokaryotic and eukaryotic genomes. Their mobility properties have made them extremely useful as molecular tools in the laboratory. Transposable elements have also been proposed to be useful as genetic drive agents to introduce phenotype-altering genes in natural populations of mosquitoes, to control vector-borne diseases such as malaria. Presented in this thesis are studies on the behavior and evolution of two endogenous Class II transposable elements, Herves and Topi in natural populations of Anopheles gambiae, a species seriously being considered for population modification using genetic manipulation. In Chapters 2 and 4, results from the analysis of copy number, activity, and nucleotide sequence as well as structural diversity of Herves and Topi elements, respectively in 5-6 An.gambiae populations in Africa are described. In Chapter 3, studies to identify and assess the activity of the natural variants of Herves transposase in An.gambiae are described. The results from these studies show that both Herves and Topi elements have long histories in An.gambiae with Topi present in An.gambiae earlier than Herves. Herves, but not Topi, is still active in natural populations of An.gambiae with more than one active form of Herves transposase responsible for its activity. Both the elements, despite their long history in An.gambiae, have a very high percentage of individuals with complete forms of the element. This observation is an unusual feature of these elements, which would not be predicted for elements with such a long history. The presence of complete and active forms of Herves and Topi, elements with long histories in An.gambiae, argues against the possibility of rapid accumulation of deleted forms of transposable elements as a general feature of their evolution. Better understanding of the behavior and evolution of Class II transposable elements in An.gambiae shows that Class II transposable elements still hold promise as a genetic drive agent for this particular species.
Binding Interactions in the Bacterial Chemotaxis Signal Transduction Pathway
(2008-12-08) Eaton, Anna Kolesar; Stewart, Richard C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The investigation of signal transduction pathways is critical to the basic understanding of cellular processes as these pathways function to regulate diverse processes in both eukaryotes and prokaryotes. This dissertation focuses on understanding some of the biochemical events that take place in the chemotaxis signal transduction pathway of bacteria. In this system, cell-surface receptor proteins regulate a histidine protein kinase, CheA, that autophosphorylates and then transfers its phosphate to an effector protein, CheY. Phospho-CheY, in turn, influences the direction of flagellar rotation. This sequence of biochemical events establishes a chain of communication that ultimately allows the chemotaxis receptor proteins to regulate the swimming pattern of the bacterial cell when it encounters gradients of attractant and repellent chemicals in its environment. The three projects presented in this dissertation sought to fill basic gaps in our current understanding of CheA and CheY function. In the first project, I examined the nucleotide binding reaction of CheA using the fluorescent nucleotide analogue, TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate]. TNP-ATP is an effective inhibitor for CheA. By monitoring the fluorescence of TNP-ATP when it bound to CheA, I examined the affinity of the binding interaction and discovered that the two ATP binding sites of each CheA dimer exhibited negative cooperativity in their interactions with TNP-ATP. This is the first evidence of cooperativity in the histidine protein kinase superfamily. In the second project, I focused on elucidating the binding mechanism that underlies formation of the CheA:TNP-ATP complex. My results indicated a three-step mechanism, including rapid formation of a low-affinity complex, followed by two steps during which conformational changes give rise to the final high-affinity complex. This same basic mechanism applied to CheA from Escherichia coli and from Thermotoga maritima. In the third project, I turned my attention to studying the CheY phosphorylation and binding reactions using fluorescently labeled versions of CheY. The results of this final study indicated that CheY proteins labeled with the fluorophore Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] could be useful tools for investigating CheY biochemistry. However my results also brought to light some of the limitations and difficulties of this approach.
BIOINFORMATIC ANALYSIS OF THE FUNCTIONAL AND STRUCTURAL IMPLICATIONS OF ALTERNATIVE SPLICING.
(2007-01-23) Melamud, Eugene; Moult, John; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
In higher Eukaryotes, upon transcription of a gene, a complex set of reactions take place to remove fragments of a sequence (introns) from transcribed RNA. A large macro-molecular machine (the spliceosome) recognizes the ends of introns, brings ends into close proximity and catalyzes the splicing reaction. The selection of the location of the ends of introns (splice sites) determines the final message produced at the end of the process. In some cases, an alternative set of splice sites are chosen, and as a consequence different message is produced. This phenomenon is known as alternative splicing. It is now realized that nearly every Human gene undergoes alternative splicing, producing large variability in types and number of transcripts produced. In this thesis, we examine the functional and structural consequences of alternative splicing on proteins, we look into the mechanism of formation of complex splicing patterns, and examine the role of noise in the process.

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