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Endogenous, Controlled Expression Of Anti-Hiv-1 Broadly Neutralizing Antibody, Darshit Patel

Electronic Thesis and Dissertation Repository

Recently, researchers have identified a number of anti-HIV broadly neutralizing antibodies (bNAbs), such as VRC01 and N6, capable of targeting a broad range of HIV-1 strains. Passive immunization using these patient-derived bNAbs could provide temporary protection but are limited by the short antibody half-life. While current gene transfer technology allows sustained bNAb expression, it lacks the ability to control bNAb production in vivo resulting in possible autoimmunity. To address this issue of achieving controlled bNAb expression in vivo, we hypothesize that bNAb expression from transduced Flu-specific B cells can be activated and modulated by subsequent Flu immunizations in the …

Mucosa-Associated Invariant T Cell Roles And Local Responses In Cell-Mediated Immunity To Influenza A Virus, Christine Marie Wardell

Electronic Thesis and Dissertation Repository

Mucosa-associated invariant T (MAIT) cells are innate-like T cells that are activated by microbial vitamin B metabolites and/or cytokine stimulation to produce pro-inflammatory cytokines and cytotoxic granules. Influenza A viruses (IAVs) activate MAIT cells in a by-stander manner, and their abundance negatively correlates with infection-induced morbidity and mortality. However, how MAIT cells directly contribute to anti-viral immunity is ill-defined. I hypothesized that MAIT cells’ presence in mice enhances antigen-specific CD8⁺ T cell responses. I observed a trend that MAIT cell-deficient mice generally developed smaller populations of antigen-specific CD8⁺ T cells following IAV exposure than mice abundant in MAIT …

Immunization With Recombinant Myelin Oligodendrocyte Glycoprotein Identifies Autoreactive T Cells As A Limiting Factor In Autoreactive Germinal Center Maintenance, Rajiv William Jain

Electronic Thesis and Dissertation Repository

Germinal center (GC) responses are responsible for the protection provided by immunizations but can also drive autoimmunity. B and T cells collaborate in the GC to target the same antigen (Ag) to inform B cell differentiation; however, the properties of Ags differ substantially in autoimmunity and foreign-Ag driven immunity. Currently, it is not well understood how properties of the Ag itself influence the initiation or progression of GC responses, limiting our ability to develop effective vaccinations and predict the progression of autoimmune responses. The purpose of this thesis is to assess how GC responses initiate and progress when immunizing with …

The Role Of Pu.1 In Lipid Metabolism And Cell Cycle Regulation In Myeloid Progenitor Cells, Jess Rhee

Electronic Thesis and Dissertation Repository

PU.1 is a transcription factor essential for myeloid development. High PU.1 levels promote cell cycle arrest and differentiation. Low levels promote proliferation and have been associated with leukemia. BN mice are homozygous for a hypomorphic allele of Spi1 that results in expression of PU.1 at 20% of normal levels. Induction of PU.1 expression in BN myeloid progenitor cells causes cell cycle arrest, differentiation, and the upregulation of microRNAs targeting lipid metabolic genes. Acly encoding ATP citrate lyase (ACL) was one of these targets. ACL produces acetyl-CoA which is essential for fatty acid synthesis. We hypothesized that inhibiting ACL would cause …

Characterization Of Interleukin (Il)-1Β Regulatory Elements And Chromatin Conformation In Macrophage Activation, Woohyun Cho

Electronic Thesis and Dissertation Repository

IL-1β is a potent inflammatory cytokine promptly expressed in activated myeloid cells. PU.1 is a lineage-determining transcription factor that regulates myeloid-specific genes by activating distal enhancers. This study addresses the functional importance of a potential enhancer of IL-1β, and how PU.1 regulates its activity state. A putative enhancer RNA (eRNA) was transcribed from the enhancer, and eRNA knock-down with antisense oligonucleotides inhibited IL-1β production. Furthermore, enhancer-promoter interactions in stimulated macrophages were detected via chromatin conformation capture (3C) analysis. The role of PU.1 was examined in PU.1-overexpressed B16-BL6 cells, which responded to LPS and expressed IL-1β mRNA and eRNAs. Enhancer knock-out …

Counteracting Roles For The Related E26 Transformation Specific Transcription Factors Spi-B And Spi-C In Regulating Antibody-Forming Responses, Anne-Sophie Laramee

Electronic Thesis and Dissertation Repository

The activation of B cells culminates in a fate decision between plasma cell or memory B cell differentiation pathways. Mice lacking the Ets transcription factor Spi-B exhibit impaired secondary antibody (Ab)-forming responses. The role of the related factor Spi-C in Ab-forming responses remains unknown. Using Spib^-/-Spic^+/- mice, we showed that heterozygosity of Spi-C rescued reductions in secondary IgG₁-secreting cell frequencies. Spib^-/- and Spib^-/-Spic^+/- B cells generated increased frequencies of CD138⁺ cells, relative to WT B cells. Spib^-/- B cells underwent accelerated differentiation compared to WT B cells, while Spib …

Characterizing Ferroportin Trafficking In Macrophages During Phagocytosis, Tayler J. Farrell

Electronic Thesis and Dissertation Repository

Macrophages are important mediators of innate immunity and nutritional immunity via modulation of essential nutrients like iron during bacterial infection. Ferroportin (Fpn), an iron-exporting protein, is found on the plasma membrane of macrophages and, if not modulated during phagocytosis, would transport iron into phagosomes and supply phagocytosed bacteria with iron. Interestingly, the fate of Fpn during phagocytosis and bacterial infection remains unknown. We generated a Fpn-GFP fusion protein and, using fluorescence microscopy, demonstrated that, during phagocytosis in RAW264.7 macrophages, Fpn is removed from phagosomes containing IgG-coated beads or Staphylococcus aureus. Further, Fpn is present on Rab5-containing phagosomes but absent …

Evaluation Of Mertk Evolution And Efferocytosis Signalling, Amanda L. Evans

Electronic Thesis and Dissertation Repository

The TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases allow phagocytes to engage in the phagocytic removal of apoptotic cells. Although all three members of the TAM family are structurally homologous and function in a similar fashion, both human genome-wide association studies and knockout mice models have demonstrated that MERTK is the critical member of the TAM family for maintaining homeostasis. In this thesis, an evolutionary analysis was used to provide insight into the function of MERTK. Selection analysis in primates unexpectedly revealed a high degree of recent positive selection in MERTK’s signal peptide and transmembrane domain, …

The E26 Transformation-Specific Transcription Factors Pu.1, Spi-B, And Spi-C Regulate Transcriptional Activation And Repression Of Nfkb1 To Control B Cell Development And Function, Stephen Ka Ho Li

Electronic Thesis and Dissertation Repository

PU.1, Spi-B, and Spi-C are highly related E26 transformation-specific family transcription factors that can bind nearly identical DNA sequences. PU.1 and Spi-B (encoded by Spi1 and Spib respectively) are important for B cell development and function, but the function of Spi-C (encoded by Spic) in B cells is not clear. The objective of this study was to determine PU.1, Spi-B, and Spi-C’s function during B cell development, and during TLR-mediated responses. It was hypothesized that PU.1 and Spi-B were required for positively regulating components of TLR responses, and Spi-C inhibited PU.1 and Spi-B targets. Spi1^+/-Spib^-/- ( …

Elucidating The Signalling Pathway Of Mer Tyrosine Kinase Receptor In Efferocytosis, Ekenedelichukwu Azu

Electronic Thesis and Dissertation Repository

Efferocytosis is the clearance of apoptotic cells and is necessary for homeostasis. Mer Tyrosine Kinase (MerTK) is a crucial efferocytic receptor whose loss is associated with chronic inflammatory diseases and autoimmunity. While previous studies have shown that MerTK mediates efferocytosis through a unique mechanism that requires integrins, MerTK signalling pathway remains unknown. Given this unusual internalization mechanism, I hypothesized that MerTK signals and engages integrins through a novel signalling pathway different from that used by other phagocytic receptors. Therefore, this study aimed to identify the signalling pathways activated by MerTK, utilizing conventional cell biology and pharmacological approaches.

I found that …

Characterization Of Efferosome Maturation And The Processing Of Apoptotic Bodies, Yohan Kim

Electronic Thesis and Dissertation Repository

Every day billions of cells in our bodies undergo apoptosis and are cleared through efferocytosis – a phagocytosis-like process in which phagocytes engulf and degrade apoptotic cells. Proper processing of efferosomes prevents inflammation and immunogenic presentation of antigens. In this thesis I determined that the early stages of efferosome maturation parallel that of the pro-immunogenic phagocytosis of pathogens. Mass spectrometry analysis of later maturation stages identified unique regulatory proteins on efferosomes and phagosomes. Keys among these were Rab17 and Rab45 on efferosomes versus Rab6b and PI-4-Kinase on phagosomes. The later would allow for antigen presentation from phagosomes, while the former …

Transcriptional Regulation Of Peptidylarginine Deiminase Type Iv: Implications For Rheumatoid Arthritis, Ali Abbas

Electronic Thesis and Dissertation Repository

High titers of anti-citrullinated protein antibodies have been detected in sera of rheumatoid arthritis (RA) patients, implicating citrullinating enzymes in the pathogenesis of RA. Peptidylarginine deiminase type IV (PAD4) is a member of the PAD family of enzymes that catalyze the post- translational modification of arginine to citrulline and has been linked with RA. However, little is known about its transcriptional regulation. Therefore, our aim was to determine how transcription of PAD4 is activated in the myeloid lineage. Using bioinformatics, a potential nuclear factor kappa B (NF-kB) binding site was identified on the PAD4 promoter. Luciferase assays were used to …

Role Of Branched-Chain Amino Acid Transporters In Staphylococcus Aureus Virulence, Sameha Omer

Electronic Thesis and Dissertation Repository

Branched-chain amino acids (BCAAs) act as effector molecules that signal a global transcriptional regulator, CodY, to regulate virulence factors in nutrient depleted environments. Staphylococcus aureus contains three putative BCAA transporters (BrnQ1, BrnQ2, BrnQ3) whose role in BCAA uptake is unknown. We hypothesize that BrnQ transporters are involved in BCAA uptake and contribute to virulence in S. aureus by modulating CodY activity. Results from radioactive uptake assays indicate that BrnQ1 is the predominant BrnQ transporter of isoleucine, valine and leucine. Meanwhile, BrnQ2 is more specific for isoleucine. Furthermore, only the lack of BrnQ1 hinders growth of S. aureus in chemically-defined media …

Human Adenovirus E1a Binds And Retasks Cellular Hbre1, Blocking Interferon Signalling And Activating Virus Early Gene Transcription, Gregory J. Fonseca

Electronic Thesis and Dissertation Repository

Upon infection, human adenovirus (HAdV) must block interferon signaling and activate the expression of its early genes to reprogram the cellular environment to support virus replication. During the initial phase of infection, these processes are orchestrated by the first HAdV gene expressed during infection, early region 1A (E1A). E1A binds and appropriates components of the cellular transcriptional machinery to modulate cellular gene transcription and activate viral early genes transcription. We have identified hBre1/RNF20 as a novel target of E1A. hBre1 is an E3 ubiquitin ligase which acts with the Ube2b E2 conjugase and accessory factors RNF40 and WAC1 to monoubiquitinate …

Cellular Adaptation Of Macrophages To Anthrax Lethal Toxin-Induced Pyroptosis Via Epigenetic Mechanisms, Chae Young Han

Electronic Thesis and Dissertation Repository

Cellular adaptation to microbial stresses has been demonstrated in several cell types. Macrophages (MФ) are sentinel immune cells fending off invading microbes. Anthrax lethal toxin (LeTx) is a key virulence factor released by Bacillus anthracis that causes rapid cell death, pyroptosis. A small number of RAW246.7 macrophages (~4%) exposed to a non-lethal dose of LeTx become resistant to LeTx-induced pyroptosis for ~ 4 weeks, termed “toxin-induced resistance (TIR)”. Here, I showed that high levels of DNA methyl transferase1 (DNMT1) expression were maintained although global genomic methylation levels were not high in TIR. TIR cells treated with the DNMT inhibitor 5-azacitidine …

The Effect Of Dendritic Cell Mobilization On Cd8+ T Cell Responses To Influenza A Virus, Adil N. Shivji

Electronic Thesis and Dissertation Repository

Influenza A viruses (IAV) cause respiratory infections with potentially catastrophic consequences. Neutralizing antibodies towards surface proteins of IAV prevent reinfection. However, mutations in these proteins allow the virus to evade these antibodies. Enhancing cytotoxic T cell-mediated immunity has been proposed as an attractive strategy to combat flu. Recombinant human FMS-like tyrosine kinase 3 ligand (FL) is known to mobilize dendritic cells (DCs) in mice. Given that DCs are the most potent antigen-presenting cells, I hypothesized that their mobilization by FL will improve the CD8+ T cell response to IAV. Quantification of CD8+ T cell responses to IAV epitopes by intracellular …

Restriction Of Hiv-1 Replication By Unique Trim22 Isoforms., Clayton Hattlmann

Electronic Thesis and Dissertation Repository

Understanding how the immune system reacts to HIV infection and why normal antiviral defenses are insufficient to fight infection is a key step towards creating better therapies. Several interferon-induced proteins, such as the tripartite motif protein TRIM22, are capable of restricting HIV-1 replication; however single nucleotide polymorphisms (SNPs) can dramatically impact the actions of these proteins. While the trim22 gene contains numerous SNPs, no study has addressed how these may affect TRIM22 functions. Here we provide the first direct comparison of two TRIM22 unique isoforms. Through confocal microscopy we observed these isoforms exhibit different patterns of localization. In vitro studies …