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Regulation of IgA Class Switch Recombination in the I.29μ B Cell Lymphoma by Cytokines and Inhibitors of Poly(ADP-ribose) Polymerase: A ThesisShockett, Penny E. 01 September 1993 (has links)
Heavy chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region which will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and the degree to which the regulation of germline transcripts correlates with the regulation of switching, we studied this process in the murine B-lymphoma cell line I.29μ, which in the presence of bacterial lipopolysaccharide (LPS) switches primarily to IgA and less frequently to IgE. Levels of α-germline transcripts initiating upstream of α switch (Sα) sequences are elevated in clones of this line which switch well as compared to clones which switch less frequently. TGFβ1 has been shown to increase α-germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B-cells. We now demonstrate in I.29μ cells that TGFβ also increases switching to IgA and increases the level of α-germline transcripts 5 to 9 fold. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGFβ appears to direct switching to IgA by inducing transcription from the unrearranged Sα- CαDNA segment. Germline α RNA is quite stable in I.29μ cells, having a half life of about 3 to 5 hours, and we find only slight stabilization in the presence of TGFβ. Levels of ε-germline transcripts are not increased by TGFβ . IL-4, which modestly increases switching to IgA in I.29μ cells, slightly increases trancription of α-germline RNA. However, we present evidence suggesting that endogenously produced IL-4 may also act at additional levels to increase switching to IgA. IFNγ, which reduces IgA expression in these cells, also reduces the level of α-germline transcripts. IFNγ also reduces the level of ε-germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines in turn regulates the specificity of recombination.
In studies aimed at identifying other signalling pathways that promote class switching, we discovered that inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) increase lipopolysaccharide (LPS)-induced switching to IgA in the B cell lymphoma I.29μ and to IgG1 in LPS + IL-4-treated splenic B cells. PARP, which binds to and is activated by DNA strand breaks, catalyzes the removal of ADP-ribose from NAD+ and poly(ADP-ribosylation) of chromatin-associated acceptor proteins. This enzyme is believed to function in cellular processes involving DNA strand breaks as well as in modulating chromatin structure. In I.29μ cells, PARP inhibitors increase IgA switching by day 2 and cause a 5-fold average increase in switching on day 3 as assayed by immunofluorescence microscopy. The PARP inhibitor, nicotinamide, also causes a reduced intensity of hybridization of Cμ and Cα specific probes to genomic DNA fragments containing the expressed VDJ-Cμ and the unrearranged Sα - Cα segments, respectively, indicating that PARP inhibition increases rearrangment of these fragments. Induction of switching by PARP inhibitors is not mimicked by treatment with cAMP analogs or reduced by inhibitors of protein kinase A (PKA). Induction of switching by PARP inhibitors does not appear to involve increased levels of transcription of the unrearranged Cα gene, although TGFβ is required for optimal induction by PARP inhibitors, consistent with a requirement for transcription of the unrearranged CH gene. PARP inhibitors do not overcome the requirement for endogenously produced IL-4.
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Cathosis: Cathepsins in Particle-induced Inflammatory Cell Death: A DissertationOrlowski, Gregory M. 01 May 2015 (has links)
Sterile particles underlie the pathogenesis of numerous inflammatory diseases. These diseases can often become chronic and debilitating. Moreover, they are common, and include silicosis (silica), asbestosis (asbestos), gout (monosodium urate), atherosclerosis (cholesterol crystals), and Alzeihmer’s disease (amyloid Aβ). Central to the pathology of these diseases is a repeating cycle of particle-induced cell death and inflammation. Macrophages are the key cellular mediators thought to drive this process, as they are especially sensitive to particle-induced cell death and they are also the dominant producers of the cytokine responsible for much of this inflammation, IL-1β. In response to cytokines or microbial cues, IL-1β is synthesized in an inactive form (pro-IL-1β) and requires an additional signal to be secreted as an active cytokine. Although a multimolecular complex, called the NLRP3 inflammasome, controls the activation/secretion of IL-1β (and has been thought to also control cell death) in response to particles in vitro, the in vivo inflammatory response to particles occurs independently of inflammasomes. Therefore, I sought to better understand the mechanisms governing IL-1β production and cell death in response to particles, focusing specifically on the role of lysosomal cathepsin proteases. Inhibitor studies have suggested that one of these proteases, cathepsin B, plays a role in promoting inflammasome activation subsequent to particle-induced lysosomal damage, however genetic models of cathepsin B deficiency have argued otherwise. Through the use of inhibitors, state-of-the-art biochemical tools, and multi-cathepsin-deficient genetic models, I found that multiple redundant cathepsins promote pro-IL-1β synthesis as well as particle-induced NLRP3 activation and cell death. Importantly, I also found that particle-induced cell death does not depend on inflammasomes, suggesting that this may be why inflammasomes do not contribute to particle-induced inflammation in vivo. Therefore, my observations suggest that cathepsins may be multifaceted therapeutic targets involved in the two key pathological aspects of particle-induced inflammatory disease, IL-1β production and cell death.
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Structural Studies of the Anti-HIV Human Protein APOBEC3G Catalytic Domain: A DissertationShandilya, Shivender 12 August 2011 (has links)
HIV/AIDS is a disease of grave global importance with over 33 million people infected world-wide and nearly 2 million deaths each year. The rapid emergence of drug resistance, due to viral mutation, renders anti-retroviral drug candidates ineffective with alarming speed and regularity. Instead of targeting mutation prone viral proteins, an alternative approach is to target host proteins that interact with viral proteins and are critical for the HIV life-cycle. APOBEC3G is a host anti-HIV restriction factor that can exert tremendous negative pressure by hypermutating the viral genome and has the potential to be a promising candidate for anti-retroviral therapeutic research.
The work presented in this thesis is focused on investigating the A3G catalytic domain structure and implications of various observed structural features for biological function. High-resolution crystal structures of the A3G catalytic domain were solved using data from macromolecular X-ray crystallographic experiments, revealing a novel intermolecular zinc coordinating motif unique to A3G. Major intermolecular interfaces observed in the crystal structure were investigated for relevance to biochemical activity and biological function.
Co-crystallization with a small-molecule A3G inhibitor, discovered using high-throughput screening assays, revealed a cysteine residue near the active site that is critical for inhibition of catalytic activity by catechol moieties. The serendipitous discovery of covalent interactions between this inhibitor and a surface cysteine residue led to further biochemical experiments that revealed the other cysteine, near the active site, to be critical for inhibition.
Computational modeling was used to propose a steric-hinderance based mechanism of action that was supported by mutational experiments. Structures of other human APOBEC3 homologs were modeled using in-silico methods examined for similarities and differences with A3G catalytic domain crystal structures. Comparisons based on these homology models suggest putative structural features that may endow substrate specificity and other characteristics to the APOBEC3 family members.
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Exploring Molecular Mechanisms of Drug Resistance in HIV-1 Protease through Biochemical and Biophysical Studies: A DissertationBandaranayake, Rajintha M. 20 May 2010 (has links)
The human immunodeficiency virus type-1 (HIV-1) is the leading cause of acquired immunodeficiency syndrome (AIDS) in the world. As there is no cure currently available to treat HIV-1 infections or AIDS, the major focus of drug development efforts has been to target viral replication in an effort to slow down the progression of the infection to AIDS. The aspartyl protease of HIV-1 is an important component in the viral replication cycle and thus, has been an important anti-HIV-1 drug target. Currently there are nine protease inhibitors (PIs) that are being used successfully as a part of highly active antiretroviral therapy (HAART). However, as is with all HIV-1 drug targets, the emergence of drug resistance substitutions within protease is a major obstacle in the use of PIs. Understanding how amino acid substitutions within protease confer drug resistance is key to develop new PIs that are not influenced by resistance mutations. Thus, the primary focus of my dissertation research was to understand the molecular basis for drug resistance caused by some of these resistance substitutions.
Until recently, the genetic diversity of the HIV-1 genome was not considered to be important in formulating treatment strategies. However, as the prevalence of HIV-1 continues, the variability of the HIV-1 genome has now been identified as an important factor in how the virus spreads as well as how fast the infection progresses to AIDS. Clinical studies have also revealed that the pathway to protease inhibitor resistance can vary between HIV-1 clades. Therefore, in studying the molecular basis of drug resistance in HIV-1 protease, I have also attempted to understand how genetic variability in HIV-1 protease contributes to PI resistance.
In Chapters II, III and Appendix 1, I have examined how clade specific amino acid variations within HIV-1 CRF01_AE and clade C protease affect enzyme structure and activity. Furthermore, I have examined how these sequence variations, which are predominantly outside the active site, contribute to inhibitor resistance in comparison to clade B protease. With the results presented in Chapter II, I was able to show that sequence variations within CRF01_AE protease resulted in structural changes within the protease that might influence enzyme activity. In Chapter III, I focused on how sequence variations in CRF01_AE influence protease activity and inhibitor binding in comparison to clade B protease. Enzyme kinetics data showed that the CRF01-AE had reduced catalytic turnover rates when compared to clade B protease. Binding data also indicated that CRF01_AE protease had an inherent weaker affinity for the PIs nelfinavir (NFV) and darunavir (DRV). In work described in Chapter III, I have also examined the different pathways to NFV resistance seen in CRF01_AE and clade B protease. Using x-ray crystallographic studies I have shown the molecular mechanism by which the two different pathways confer NFV resistance. Furthermore, I provide a rational for why different resistance pathways might emerge in the two clades. In Appendix I, I present results from a parallel study carried out on clade C protease.
In Chapter IV, I have examined the role of residue 50 in HIV-1 protease in modulating inhibitor binding. Patients failing amprevavir (APV) and DRV therapy often develop the I50V substitution while the I50L substitution is often observed in patients failing atazanavir (ATV) therapy. This indicates that by making subtle changes at residue 50 the protease is able to confer differential PI resistance. With binding data presented in this chapter I have shown that substitutions at residue 50 change the susceptibility profiles of APV, DRV and ATV. Furthermore, from analyses of protease-inhibitor complexes, I have described structural insights into how substitutions at residue 50 can modulate inhibitor binding.
This thesis presents results that reveal mechanistic insights into how a number of resistance substitutions within protease confer drug resistance. The results on non-B clade proteases demonstrate that clade specific sequence variations play a role in modulating enzyme activity and influence the pathway taken to confer PI resistance. Furthermore, the results provide structural insights into how amino acid substitutions outside the active site effectively alter inhibitor binding.
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Nucleic Acid Sensing by the Immune System: Roles For the Receptor For Advanced Glycation End Products (RAGE) and Intracellular Receptor Proteins: A DissertationSirois, Cherilyn M. 14 July 2011 (has links)
As humans, we inhabit an environment shared with many microorganisms, some of which are harmless or beneficial, and others which represent a threat to our health. A complex network of organs, cells and their protein products form our bodies’ immune system, tasked with detecting these potentially harmful agents and eliminating them. This same system also serves to detect changes in the healthy balance of normal functions in the body, and for repairing tissue damage caused by injury. Immune recognition of nucleic acids, DNA and RNA, is one way that the body detects invading pathogens and initiates tissue repair. A number of specialized receptor proteins have evolved to distinguish nucleic acids that represent “threats” from those involved in normal physiology. These proteins include members of the Toll-like receptor family and diverse types of cytosolic proteins, all of which reside within the confines of the cell. Few proteins on the cell surface have been clearly characterized to interact with nucleic acids in the extracellular environment. In this dissertation, I present collaborative work that identifies the receptor for advanced glycation end products (RAGE) as a cell surface receptor for nucleic acids and positions it as an important modulator of immune responses. Molecular dimers of RAGE interact with the sugar-phosphate backbones of nucleic acid ligands, allowing this receptor to recognize a variety of DNA and RNA molecules regardless of their nucleotide sequence. Expression of RAGE on cells promotes uptake of DNA and enhances subsequent responses that are dependent on the nucleic acid sensor Toll-like receptor 9. When mice deficient in RAGE are exposed to DNA in the lung, the predominant site of RAGE expression, they do not mount a typical early inflammatory response, suggesting that RAGE is important in generating immune responses to DNA in mammalian organisms. Further evidence suggests that RAGE interacts preferentially with multimolecular complexes that contain nucleic acids, and that these complexes may induce clustering of receptor dimers into larger multimeric structures. Taken together, the data reported here identify RAGE as an important cell surface receptor protein for nucleic acids, which is capable of modulating the intensity of immune responses to DNA and RNA. Understanding of and intervention in this recognition pathway hold therapeutic promise for diseases characterized by excessive responses to self nucleic acids, such as systemic lupus erythematosus, and for the pathology caused by chronic inflammatory responses to self and foreign nucleic acids.
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Investigating the Peptide-MHC Specificity of Alloreactive T Cells and Natural T Regulatory Cells Using a Self-peptide Display LibraryDuke, Brian R. 27 November 2017 (has links)
T cells use their highly variable T cell receptor (TCR) to engage major histocompatibility molecules (MHC) presenting peptides on the surface of antigen presenting cells during an immune response. The TCR repertoire of developing T cells is shaped by thymic selection, resulting in a self-tolerant and foreign peptide specific naïve T cell population. However, naive T cells are alloreactive and generate immune responses towards foreign MHC alleles in clinical settings involving transplantation. While T cell immune responses towards foreign pathogens are peptide specific, the overall specificity of allo-responses is still debated.
Under normal circumstances, immune system homeostasis and self-tolerance is maintained by specialized natural T regulatory cells (nTregs) that develop in the thymus. nTregs respond to self-peptide MHC they encountered in peripheral tissues with immune-suppressive activities. However, the identify of self-peptides that stimulate nTregs, specificity towards these self-peptides, and the method nTreg TCRs engage self-peptide MHC molecules is not clear.
Here, we built a library of defined MHC-linked self-peptides eluted from the I-Ab MHC molecule to screen alloreactive T cells and self-reactive nTregs for activating self-peptides. We used this library to show that negative selection shapes the TCR repertoire’s specificity to self-peptides. We also provide evidence that alloreactive T cells have degenerate self and foreign peptide recognition if the foreign MHC allele is largely different from the host’s MHC allele. Finally, we identified a self-peptide that activates an nTreg, and present protein crystal structures that reveal its TCR engages self and foreign peptide MHC complexes via fairly conventional mechanisms.
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Latino Men Managing HIV: An Appraisal Analysis of Intersubjective Relations in the Discourse of Five Research InterviewsCaston, Will 06 November 2014 (has links)
Latino men, particularly those who have sex with other men, have been disproportionately affected by HIV/AIDS. Scholars have sought for nearly two decades to understand how various social and cultural factors in the Latino community exacerbate HIV risk among these men. Although following the advent of life-sustaining medications in 1996, HIV is often regarded as a manageable chronic illness, as opposed to a death sentence, scant attention has been devoted to how HIV-positive Latino men experience managing the illness. Among studies that have focused on HIV-positive persons' illness management, few Latino men have participated.
Using the Appraisal framework from Hallidayan Systemic Functional Linguistics, with Bucholtz and Hall's theory of social identity (2004, 2005), this discourse analysis sought to explore intersubjective relations as reported by five HIV-positive Latino men, three native-born and two immigrants, in semi-structured interviews that attempted to avoid preconceived expectations about salient structures. While structures such as homophobia, machismo, and stigma emerged in each interview, the native-born men's discourse differed from that of the immigrants in that the former did not address financial concerns with regard to HIV medications, whereas the latter represented their agency as having been constrained by low income requirements for obtaining assistance in accessing expensive HIV medications. This finding tentatively suggests that the issue could be more salient for immigrants than native-born Latinos and warrants additional, more focused research on the effects of the structures of benefit programs on HIV-positive Latino immigrants.
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HIV Tat and Morphine-induced Neurodegeneration in a Beclin 1 Hemizygous Mouse ModelLapierre, Jessica A 08 November 2018 (has links)
Early in infection, HIV crosses the blood-brain barrier and induces neuropathology. Viral presence in the CNS coupled with secretion of neurotoxic proteins causes neuroinflammation, glial dysfunction, excitotoxicity, and neuronal death. Despite advances in combined antiretroviral therapy, HIV-infected patients present with a spectrum of cognitive and psychomotor deficits collectively referred to as HIV-associated neurological disorders (HAND). A subset of HAND patients abuses drugs such as opiates like heroin and morphine show an exacerbation and rapid progression of HIV neuropathology; however, the mechanisms of this synergy are not well understood. Autophagy is a lysosomal degradative process which eliminates and recycles cytosolic components and is implicated in facilitating HIV-1 replication in the CNS and periphery, and in Tat-induced neurodegeneration. When a key initiator of autophagy Beclin 1 was silenced using siRNAs, there was a marked reduction of HIV-1 replication in human microglia and astrocytes and the corresponding inflammatory response. As such, the goal of the current study is to determine if diminished Beclin 1 is neuroprotective against Tat and morphine-induced neurodegeneration using heterozygous Beclin 1 (Becn1+/-) mice. Examination of Tat and morphine-induced inflammatory molecule secretion revealed that Becn1+/- mixed astrocyte and microglia (glia) exhibited attenuated secretion of cytokine IL-6 and chemokines RANTES and MCP-1 compared to control (C57BL/6J) glia, an effect mediated through the μ-opioid receptor. Dysregulation of autophagy-related gene expression and excessive intracellular calcium accumulation were limited in Becn1+/- glia. When determining the effects of Tat-and morphine co-exposure on neuronal survival in vitro, we found Becn1+/- neurons were particularly sensitive to injury, excitotoxicity, and toxic exposures; however, when C57BL/6J neurons were exposed to conditioned media of C57BL/6J and Becn1+/- glia treated with Tat and morphine, neurons treated with Becn1+/- supernatant had better outcomes than those treated with C57BL/6J conditioned media. Furthermore, despite minimal difference between strains in locomotor assessment, we observed significantly greater striatal neuron losses in adult C57BL/6J mice exposed to intrastriatal Tat-and systemic morphine compared to Becn1+/- mice. Our studies demonstrate the potential of targeting Beclin 1 in glia for the prevention of Tat and opiate-induced CNS dysfunction.
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Functional Genomics of Mammalian Innate ImmunityKiritsy, Michael C. 31 August 2020 (has links)
The breadth of genetic diversity in the mammalian immune response stands out amongst the ubiquity of variation seen in the genome, evidence that microbial infections have been a major driver of evolution. As technology has facilitated an understanding of the etiology of immunological diversity, so too has it enabled the assessment of its varied functions. Functional genomics, with its ability to assess both cause and effect, has revolutionized our understanding of fundamental biological phenomena and recalibrated our hypotheses. We build upon the model of host immunity established by rare genetic variants that are causative of immunodeficiencies, but that incompletely consider the complexities of the genome. To expand our understanding, we performed a series of forward genetic screens to identify regulators of distinct functions of the innate immune system. Our studies discovered genes with novel functions in antigen presentation and immunoregulation, including several involved in central metabolism. Studies in macrophages and dendritic cells identified mitochondrial respiration as a positive regulator of the interferon-gamma response, and cells incapable of respiration failed to activate T cells. Notably, human mutations in several of these genes are responsible for immune dysfunction. In summary, this work uses new methods in genetic engineering to systematically assess the regulation of innate immunity. Our results suggest that variation in these regulatory pathways is likely to alter immunity in states of health and disease. Thus, our work validates a new approach to identify candidate genes relevant to immune dysfunction.
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The Mechanisms of Mitochondrial Dysfunction in T Cell Aging during Chronic Viral InfectionSchank, Madison B. 01 December 2022 (has links)
Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections induce a myriad of disturbances to CD4 T cell functions, including mitochondrial compromise, excessive inflammation, increased telomeric DNA damage and attrition, cellular exhaustion and senescence, and accelerated aging. In this dissertation, the mechanisms underlying metabolic failure, accelerated aging, and cellular dysfunctions were evaluated in CD4 T cells from healthy subjects (HS) treated with a telomere-targeting drug (KML001) or HCV-infected individuals or people living with HIV (PLHIV) compared to HS. We observed that KML001-induced telomere injury resulted in mitochondrial swelling and decreased mitochondrial membrane potential, cellular respiration, mitochondrial DNA (mtDNA) copy number, and ATP production mediated by p53-mediated repression of the master mitochondrial regulators peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear respiratory factor 1 (NRF-1). We then investigated the mechanisms responsible for T cell dysfunction and metabolic failure during chronic viral infections (HCV, HIV). We observed that chronic HCV infection leads to elevated production of cellular and mitochondrial reactive oxygen species (ROS), impaired mtDNA, and altered levels of proteins responsible for mediating oxidative stress, apoptosis, and mtDNA maintenance, as well as mitochondrial regulators PGC-1α and mitochondrial transcription factor A (mtTFA), contributing to impaired cellular respiration and mtDNA content. Similarly, we demonstrated that latent HIV infection induced disruptions to CD4 T cell homeostasis and increased cellular exhaustion, senescence, and apoptosis and reduced proliferation. We also observed significant repression of mitochondrial respiration, mtDNA content, and mtTFA levels in CD4 T cells from PLHIV, which was reversed via ectopic expression of mtTFA. Finally, we observed elevated cellular and mitochondria ROS production in CD4 T cells from PLHIV, along with significant deregulation of levels of antioxidant defense (superoxide dismutase 1, SOD1) and oxidative stress-induced DNA damage repair (apurinic/apyrimidinic endonuclease 1, APE1) proteins, which were shown to be essential for cellular respiration independently of mtDNA content. Taken together, this research highlights novel multi-leveled mechanisms by which chronic viral infection induces accelerated T cell aging and mitochondrial compromise via deregulating master mitochondrial regulators and provides a diverse collection of novel therapeutic targets that may be applied to various infectious diseases.
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