Browsing by Subject "RNA, Messenger"
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Item Assessment of Gamma/Delta T Cell Functionality Following Pathogenic HIV/SIV and Non-Pathogenic SIV Infections(2007-05-22) Kosub, David Alan; Sodora, DonaldPathogenic HIV/SIV infection induces high viral loads, aberrant immune activation, and dysfunction in numerous immunologic cells (including gamma/delta (gamma delta ) T cells) leading to opportunistic infections. gamma delta T cells bridge the innate and adaptive immune responses primarily via cytokines produced in response to microbial phosphoantigens. gamma delta T cells have also been implicated in the control of an SIV challenge infection as evidenced by increased numbers and beta -chemokine expression at mucosal sites in vaccinated macaques. The goal of Aim 1 of this thesis was to assess the impact of an acute SIV infection on the levels of gamma delta T cells at mucosal and lymphoid sites in macaques utilizing quantitative PCR. At two days post-infection, a decrease in gamma delta T cell levels was observed at mucosa sites whereas increased levels were present at regional lymph nodes. Also, an increase in lymphoid homing molecules was observed at these lymph nodes, indicating a mechanism whereby gamma delta T cells migrate away from mucosal sites towards secondary lymphoid tissues following an acute SIV infection. The redistribution of gamma delta T cells may be important for the initiation of an anti-viral immune response and control of rapid viral spread. The goal of Aims 2 and 3 was to assess the ability of gamma delta T cells in HIV-infected patients to express cytokines and compare these results to analysis of the non-pathogenic SIV infection of sooty mangabeys. Following stimulation with the non-specific activators PMA/Ionomycin or the gamma delta TCR specific ligand isopentenyl pyrophosphate, a decrease in the percentages of gamma delta T cells expressing Th1 pro-inflammatory cytokines including TNF-alpha and IFN-gamma was observed in the HIV+ patients (regardless of CD4+ T cell levels). Highly active anti-retroviral therapy (HAART) partially restored the ability of gamma delta T cells from HIV+ patients to express Th1 cytokines. SIV infection of mangabeys results in high viral replication, low levels of immune activation, and generally no signs of progression to AIDS. Evidence for preserved or increased functionality of gamma delta T cells from SIV+ mangabeys (regardless of CD4+ T cell levels) was demonstrated by maintained percentages of gamma delta T cells that expressed Th1 cytokines following ex vivo stimulation. These data suggest that in the absence of aberrant immune activation, controlled Th1 responses by gamma delta T cells from mangabeys may assist in suppressing damage due to the SIV infection as well as inhibiting the onset of opportunistic infections. These data provide rationale for therapies aimed at increasing gamma delta T cell functionality in humans, particularly with regard to Th1 cytokine responses to augment protection against opportunistic infections and HIV disease progression.Item Regulatory RNAs at the Heart of Sugar Metabolism: New Mechanisms and Novel Discoveries(2011-02-01T19:35:28Z) Irnov; Winkler, WadeBacteria are adept at using a variety of posttranscriptional strategies to regulate gene expression. Specifically, various RNA-mediated genetic control elements have been discovered in the past decade through a combination of genetics, bioinformatics, and transcriptomic approaches. Together, these RNA elements control the expression of many genes involved in diverse cellular processes such as energy metabolism, stress response, biofilm formation, and pathogenesis. In the Gram-positive bacterium Bacillus subtilis, several RNA elements have been shown to be required for the precise coordination of genes involved in various sugar utilization pathways. These genetic switches typically regulate gene expression by modulating the formation of a transcription termination element in a ligand-dependent manner. Interestingly, two unique elements, the glmS ribozyme and the eps-associated RNA (EAR), are missing the signature elements required for control of transcription termination or translation initiation. The latter mechanism is more commonly found in Gram-negative bacteria. Our objective is to study the mechanisms by which these two RNAs control gene expression. Additionally, we would like to identify other regulatory RNAs that are important for sugar metabolism in Bacillus subtilis. Both the glmS ribozyme and EAR are positioned at the center of the sugar metabolism pathways in B. subtilis. The glmS RNA is a glucosamine-6-phosphate responsive element that regulates the expression of the GlmS enzyme, which directs sugar precursors from glycolysis into the cell wall biosynthesis pathway. The EAR element resides within the 16-kb eps operon that is required for biofilm exopolysaccharide production. Our data demonstrates that both RNAs employ novel mechanisms: the glmS ribozyme utilizes a ligand-specific RNase-mediated degradation event, while EAR uses a processive antitermination mechanism for complete synthesis of the long operon. Furthermore, by using high-throughput sequencing approach we have successfully identified many new regulatory RNA candidates, including various long 5`-UTR, toxin-antitoxin systems, prophage-encoded RNAs, and several developmentally regulated small RNAs. Their functions are still under investigation. Collectively, our studies provide important insights into the different aspects of bacterial physiology, including RNA decay pathways, transcription of long operons and cellular differentiation. We argue that posttranscriptional regulation is of greater importance to Bacillus subtilis (and probably all bacteria) than previously realized.Item The Role of NS1-BP in Influenza Virus Replication(2012-08-13) Tsai, Pei-Ling; Fontoura, Beatriz, M.A.Influenza A viruses are negative-sense, segmented RNA viruses which cause about 500,000 deaths worldwide per year. Genomic studies have shown that the non-structural protein (NS1) of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 is a multifunctional protein localized in the nucleus and in the cytoplasm. In the cytoplasm, NS1 inhibits host signaling pathways that result in down-regulation of interferon expression and innate immune response. In the nucleus, NS1 represses host gene expression. I have shown that NS1 binds an mRNA complex containing NXF1/TAP, NXT/p15, Rae1, and E1B-AP5, which are key components of the mRNA export machinery. By targeting this complex, NS1 blocks host mRNA export, and cells become highly permissive to viral replication. Another intranuclear pool of NS1 was found to interact with a host protein termed NS1-BP, which has been suggested to play a role in pre-mRNA splicing. However, the functions and mechanisms of NS1-BP involved in influenza life cycle remain to be elucidated. To investigate the function of NS1-BP, I first identified its binding partners by immunoprecipitation followed by mass spectrometry. I found interactions of NS1-BP with viral polymerase complex and host RNA polymerase II indicating that NS1-BP has a role in regulating viral RNA transcription and replication. I further showed that low levels of NS1-BP led to a decrease in viral polymerase activity resulting in inhibition of virus replication. Thus, I identified NS1-BP as a novel pro-viral factor required for proper replication of influenza virus. Since NS1 is a key contributor to the virulence of influenza viruses, discovering the function of NS1 interacting partners has major implications for antiviral therapy. [Keywords: NSI-BP, influenza, virus, polymerase, RNA]