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Title: [Delta]2079-tetrahydrocannabinol-mediated suppression of the Interferon-alpha (IFNalpha) response by plasmacytoid dendritic cells and IFNalpha-mediated activation of T cells in healthy and human immunodeficiency virus (HIV) infected human subjects
Creator: Henriquez, Joseph Edgar
Date: 2018
Collection: Electronic Theses & Dissertations
Description: Δ9-tetrahydrocannabinol (THC) is the primary psychoactive cannabinoid congener in Cannabis sativa and is a well characterized modulator of immune activation. In murine models, treatment with THC can exacerbate viral and bacterial infection, in part, by suppression of the inflammatory cytokine response. One of the key classes of cytokines suppressed by THC is type I interferons (IFN), a group of cytokines consisting of IFNα and IFNβ. The primary source of IFNα during acute antiviral immune...
Show moreΔ9-tetrahydrocannabinol (THC) is the primary psychoactive cannabinoid congener in Cannabis sativa and is a well characterized modulator of immune activation. In murine models, treatment with THC can exacerbate viral and bacterial infection, in part, by suppression of the inflammatory cytokine response. One of the key classes of cytokines suppressed by THC is type I interferons (IFN), a group of cytokines consisting of IFNα and IFNβ. The primary source of IFNα during acute antiviral immune responses is the Plasmacytoid Dendritic Cell (pDC), which can secrete 1000-fold more IFNα than other circulating peripheral blood mononuclear cells (PBMC). Paradoxically, patients infected with human immunodeficiency virus (HIV), a chronic viral infection that causes immunodeficiency via infection and depletion of CD4+ T cells, have fewer circulating pDC with a reduced capacity to secrete IFNα. Furthermore, circulating pDC number has been correlated with CD4+ T cell number and treatment with IFNα can reduce HIV-mediated CD4+ T cell depletion. Conversely, hyperactivation of pDC is associated with T cell exhaustion and is implicated in HIV-associated neurocognitive disorders (HAND). Interestingly, many HIV patients utilize medicinal cannabinoids to combat the effects of chronic HIV infection. The focus of this project was to determine if IFNα-mediated stimulation of T-cells can be suppressed by THC by testing the following hypothesis: THC will suppress TLR-9-dependent activation of pDC, subsequent efficacy of pDC-mediated T cell activation, and CD8+ T cell-mediated activation of astrocytes. These studies revealed that CpG-ODN-induced IFNα secretion and expression of CD83, a costimulatory molecule, by pDC is suppressed by THC in a concentration dependent manner. Furthermore, key intracellular signaling events required for inflammatory cytokine secretion by pDC were suppressed by treatment with THC and CBR2-specific agonists in pDC from healthy donors. Additionally, pDC from HIV+ donors were more sensitive to THC-mediated suppression than pDC from healthy donors. Treatment with THC also inhibited IFNα-mediated activation of CD4+ and CD8+ T cells from healthy and HIV+ donors. Specifically, treatment with THC diminished IFNα-induced IL-7R expression, cognate signaling, and subsequent proliferation. Interestingly, and in contrast to the results in pDC, T cells from HIV+ donors were less sensitive to the suppressive effects of THC. Finally, stimulation by CD3/CD28/IFNα induced the secretion of IFNγ and TNFα by CD8+ T cells from healthy donors. Further, IFNγ and TNFα induced secretion of inflammatory cytokines by U251 astrocytes. Coculture of CD8+ T cells with U251 astrocytes and direct stimulation of U251 astrocytes with recombinant TNFα and IFNγ revealed that treatment with THC reduced both the activation and secretion of cytokines from CD8+ T cells and the subsequent cytokine-mediated stimulation of the U251 astrocytes. Collectively, these studies have provided evidence for the use of cannabinoids in ablating the type of neuroimmune interactions which can lead to HAND by demonstrating that THC can suppress the activation of pDC, and subsequent activation of T cells and astrocytes.
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Title: Identifying the underlying mechanisms of Marek's disease vaccine synergy
Creator: Umthong, Supawadee
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Marek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more...
Show moreMarek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more virulent MDV strains have repeatedly emerged in vaccinated flocks. Thus, there is a need to understand how MD vaccines work in order to design future vaccines that are more protective, especially against more virulent MDVs. One promising insight for vaccine development is based upon protective synergism, a phenomenon where two vaccines when combined provide greater protection compared to either original vaccine when administered alone as a monovalent vaccine. The mechanism that underlines the synergistic effect between SB-1 (a Gallid herpesvirus 3, aka, serotype 2 strain) and HVT (herpesvirus of turkey, aka, Meleagrid herpesvirus 1 or serotype 3), two of the most widely used MD vaccines, has never been investigated, and thus, provides a highly relevant and useful model to explore. To investigate the mechanisms of protective synergy of SB-1 and HVT, we used three approaches. First, we investigated how monovalent SB-1 or HVT replicates when they were alone in the host or together as a bivalent vaccine. We observed that the replication patterns of SB-1 and HVT were different with respect to time after administration into the bird and the organs that they were found to replicate in regardless if the other vaccine were present. Based on the observation that HVT replicated primarily early in the bursa, we found that this organ was necessary for protection using both HVT and bivalent HVT + SB-1 vaccines. Second, we measured the effects of CD8 T cells in monovalent SB-1, HVT, and bivalent SB-1+HVT vaccine treatment. Specifically, we reduced CD8 T cells to see their effect of CD8 T cells on MD incidence and vaccinal protection by injecting the chickens with a monoclonal antibody directed against chicken CD8 T cells. In this study, we found that CD8 T cells were necessary for protection induced by vaccines. Third, we identified the cytokine profiles induced by SB-1, HVT, and the bivalent vaccine to see if cytokine synergy could be one of the mechanisms to explain protective synergy. We found that SB-1 induced an innate anti-viral response typified by IFN-α, IFN-β, IL-1β, T-cell proliferation cytokine IL-21, and Th2 cytokine IL-5, while HVT suppressed TGF-β3 and TGF-β4. The early stimulation of IL-1β and IL-21 (IFN-γ-promoting cytokines) at 4 days post vaccination (DPV) by SB-1 combined with the suppression of TGF-β (IFN-γ- suppressing cytokine) at 1 day post challenge (DPC) by HVT could result in the strong induction of IFN-γ found in the bivalent vaccine at 10 DPC. The induction of IFN-γ supports the synergistic effect of cytokines by a cooperative action mechanism where multiple cytokines work together to enhance the signal. Based on these findings, we propose a model to explain bivalent SB-1 and HVT vaccine synergy, which combines the replication of vaccines, T cell response to vaccinations, and cytokine synergy between SB-1 and HVT vaccine. Our proposed mechanism provides insights on how to generate rationally designed MD vaccines.
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Title: THE ROLE OF LCK AND PD-1 IN TCDD-MEDIATED SUPPRESSION OF THE IgM RESPONSE BY HUMAN CD5+ INNATE-LIKE B CELLS
Creator: Zhou, Jiajun
Date: 2019
Collection: Electronic Theses & Dissertations
Description: The aryl hydrocarbon receptor (AHR) is a cytosolic ligand-activated transcription factor involved in xenobiotic sensing and cell regulation. The activation of AHR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been shown to impair immunoglobulin M (IgM) responses in all laboratory animals. Previous studies in mouse splenocytes and purified mouse B cells revealed that AHR activation leads to a decrease of IgM production. It has been widely assumed that the molecular mechanisms responsible...
Show moreThe aryl hydrocarbon receptor (AHR) is a cytosolic ligand-activated transcription factor involved in xenobiotic sensing and cell regulation. The activation of AHR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been shown to impair immunoglobulin M (IgM) responses in all laboratory animals. Previous studies in mouse splenocytes and purified mouse B cells revealed that AHR activation leads to a decrease of IgM production. It has been widely assumed that the molecular mechanisms responsible for AHR-mediated suppression of the IgM response would be similar across animal species. However, no direct comparison has been conducted between mice and humans. Therefore, the first part of this dissertation is focused on comparing AHR-mediated suppression of IgM responses in mouse and human B cells. Contrary to the observations in mouse B cells, TCDD treatment results in a significant suppression of the number of IgM secreting cells, but it is not due to a decrease in IgM molecules in human B cells. These results suggested that AHR-mediated suppression of the IgM response involves different mechanism between mice and humans.The second part of this dissertation is focused on elucidating the role of lymphocyte-specific protein tyrosine kinase (LCK) in AHR-mediated suppression of the IgM response in human B cells. LCK is a well-characterized tyrosine kinase in T cell biology. In contrast, limited research has been done to understand the role of LCK in human B cells. An upregulation of LCK protein has been observed in AHR-activated human B cells. Treatment with an AHR antagonist reversed the AHR-mediated increase of LCK. Furthermore, LCK specific inhibitors also reversed the AHR-mediated suppression of the IgM response by human B cells. Collectively, the studies demonstrate a novel role of LCK in IgM secretion and provide new insights into the mechanism for AHR-mediated impairment of immunoglobulin secretion by human B cells.The third part of this dissertation is focused on understanding the role of LCK and program cell death protein-1 (PD-1), in CD5+ innate-like B cells (ILBs). Human CD5+ ILBs express high levels of LCK and PD-1 compared to CD5- B cells. Therefore, studies were conducted to determine the role of LCK and PD-1 in AHR-mediated suppression of the IgM response in CD5+ ILBs. In the current studies, AHR activation significantly upregulated total LCK and PD-1 proteins in CD5+ ILBs. LCK inhibitor treatment prevented the PD-1-mediated suppression of the IgM response in CD5+ ILBs. Furthermore, PD-1 blocking antibody prevented the suppression of the IgM response in CD5+ ILBs. Collectively, results from these studies support the critical role of LCK and PD-1 in AHR-mediated suppression of the IgM response by human CD5+ ILBs. Taken together, the results from these studies indicate that a) AHR-mediated suppression of the IgM responses is mechanistically different between mouse and human B cells; b) in humans, activation of AHR suppresses the IgM response through the modulation of LCK; c) LCK and PD-1 play a critical role in AHR-mediated suppression of the IgM response in CD5+ ILBs.
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Title: [Delta]2079-tetrahydrocannabinol suppresses human monocyte activation and monocyte-mediated astrocyte inflammation : implications for HIV-associated neuroinflammation
Creator: Rizzo, Michael Denton
Date: 2019
Collection: Electronic Theses & Dissertations
Description: A hallmark of human immunodeficiency virus (HIV) infection is chronic immune activation and is believed to be one of the major contributors to neuroinflammation and HIV-associated neurocognitive disorder (HAND). Circulating activated monocytes, including those that are CD16⁺, have been implicated in HIV-associated neuroinflammation. These activated monocytes become infected with HIV in the periphery, cross the blood-brain barrier (BBB) and release inflammatory factors, HIV virions and viral...
Show moreA hallmark of human immunodeficiency virus (HIV) infection is chronic immune activation and is believed to be one of the major contributors to neuroinflammation and HIV-associated neurocognitive disorder (HAND). Circulating activated monocytes, including those that are CD16⁺, have been implicated in HIV-associated neuroinflammation. These activated monocytes become infected with HIV in the periphery, cross the blood-brain barrier (BBB) and release inflammatory factors, HIV virions and viral proteins. These factors lead to HIV infection and activation of brain-resident cells, including microglia and astrocytes, driving a pro-inflammatory environment in the brain. Ultimately, these processes contribute to neuronal dysfunction and death, ultimately resulting in cognitive decline in up to 50% of the HIV-infected population. Cannabis is widely used by the HIV-infected population at an estimated prevalence of 23-56% in the United States. [Delta]2079-Tetrahydrocannabinol (THC) and cannabidiol (CBD), two major constituents of cannabis, are known to have immune suppressive and anti-inflammatory properties. The overall objective of this project was to determine whether the cannabinoids, THC and CBD, could suppress monocyte activation and monocyte-mediated astrocyte inflammation, which are key processes implicated in chronic neuroinflammation and HAND. Herein, it is shown that HIV-infected donors using cannabis displayed a lower level of circulating activated (CD16⁺) monocytes and plasma IP-10 compared to non-using HIV-infected donors. Furthermore, in vitro studies revealed that THC but not CBD suppressed monocyte expression of CD16 and secretion of IP-10, suggesting that THC is the major cannabinoid in cannabis promoting the anti-inflammatory effects. To determine whether activated monocytes could promote inflammatory functions of brain-resident glial cells, we developed a human co-culture system utilizing primary monocytes and cell-line/primary fetal astrocytes with viral-related stimulators (IFNa a0333nd a TLR7 agonist - R837). Monocytes, together with IFNa a0333nd/or R837, promoted astrocyte secretion of MCP-1, IL-6 and IP-10. Furthermore, monocyte-derived IL-1v0333 was critical for astrocyte secretion of pro-inflammatory factors, as neutralization of IL-1v0333 strongly hampered the astrocyte response, while direct addition of recombinant IL-1v0333 to astrocyte monocultures mimicked the actions of monocytes. In vitro THC treatment of the R837-stimulated co-culture resulted in decreased astrocyte production of MCP-1 and IL-6, while CBD increased IL-6 production and had no effect on MCP-1 production. With the use of separate monocyte and astrocyte monocultures, THC and CBD were shown to directly target both cell types. Interestingly, THC and CBD were both shown to decrease the percent of astrocytes producing IL-6 and MCP-1, which for THC, is concordant with the co-culture observation. However, the CBD-mediated decrease in IL-6 and MCP-1 production in the astrocyte monoculture differed from the observations in the CBD-treated co-culture. Our findings were explained when THC and CBD were shown to suppress and augment monocyte production of IL-1v0333, respectively. Furthermore, the CBD-mediated augmentation of monocyte-derived IL-1v0333 was able to override the direct CBD suppression on the astrocytes. Collectively, THC but not CBD, impairs monocyte activation and monocyte-driven astrocyte inflammatory responses. In the context of HAND, cannabis use, in particular THC, may decelerate monocyte processes that are implicated in neuroinflammation and cognitive dysfunction.
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Title: The CA19-9 antigen and sTRA glycans define independent pancreatic ductal adenocarcinoma subpopulations improving diagnostic accuracy and approach to prognostic classification
Creator: Barnett, Daniel Mark
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Pancreatic cancer is the third deadliest cancer annually in the United States. Although the vast majority of pancreatic cancer belongs to a single type called pancreatic ductal adenocarcinoma (PDAC), tremendous heterogeneity exists within and between PDACs in their biology and clinical behavior, making it difficult to optimize treatment strategies and therapeutics research. The possibility exists that the heterogeneity results from the fact that PDACs actually encompass several distinct...
Show morePancreatic cancer is the third deadliest cancer annually in the United States. Although the vast majority of pancreatic cancer belongs to a single type called pancreatic ductal adenocarcinoma (PDAC), tremendous heterogeneity exists within and between PDACs in their biology and clinical behavior, making it difficult to optimize treatment strategies and therapeutics research. The possibility exists that the heterogeneity results from the fact that PDACs actually encompass several distinct subtypes. Recent research has uncovered much evidence for such subtypes, but so far, the research has not produced clear definitions of the subtypes or associated biomarkers that define them. PDACs express a unique set of glycans derived largely from their origins as duct cells with a protective glycocalyx, including the CA19-9 antigen sialyl-Lewis A (sLeA), which serves as the only approved biomarker of pancreatic cancer, and its near relative sTRA. I hypothesized that the neoplastic cells of pancreatic ductal adenocarcinoma can be separated into subpopulations by their specific glycan expression of sTRA and CA19-9 and that these subpopulations have different functional characteristics and risk for disease dissemination. To test this hypothesis, I used several methods involving both primary specimens and model systems. First, I used multimarker immunofluorescence to detect sTRA and CA19-9 and compare their cellular locations, morphologies, and protein co-expression in tumor and matched adjacent uninvolved tissue, lymph nodes, and metastases. Immunofluorescence was detected by automated microscopy and quantified by novel automated software developed specifically for this project. Clear differences were observed between cancer cells that expressed only CA19-9 and those that expressed only sTRA, as well as a third cell subpopulation represented by dual expression. Dual expression represented a well differentiated epithelial population of cells in well-formed glandular tissue; CA19-9-only expression represented poor to moderately differentiated cell subpopulations of epithelial and flat (mesenchymal) characteristics; and sTRA-only expression represented poor to moderately differentiated cell subpopulations present in "foamy cytoplasm" and flat (mesenchymal) cell features. The co-expression of MUC5AC and beta-catenin was different between the subsets, indicating differences in differentiation. The differences were preserved in cell-line and patient-derived mouse xenografts. I next tested for differences in metastatic propensity. Xenograft tumors expressing sTRA were more strongly correlated with metastasis than those expressing CA19-9, and primary tumors showed differential correlations with lymph-node or liver metastasis depending on glycan expression. Finally, we tested whether blood plasma levels of these glycans correlate with tissue expression and whether elevations occur in distinct subpopulations of patients. The secretion of glycans into cell-culture media, mouse sera, or plasma from human patients generally correlated with glycan expression in the cancer cells, indicating the value of the glycans as serological biomarkers to indicate the tumor type. Certain tissues expressing only CA19-9 did not secrete to blood plasma, particularly in hyperglandular and very high stromal tissue, suggesting a new cause of false negative CA19-9 patients in PDAC detection. CA19-9 and sTRA were elevated in separate subgroups of patients, each with low false-positive rates. As a result, CA19-9 and sTRA together gave better accuracy of PDAC diagnosis than CA19-9 alone (97% specificity, 65% sensitivity vs. 96% and 46%). In summary, these studies support the concept that distinct subtypes of PDAC can be identified by the expression of sTRA or CA19-9. Additionally, sTRA co-expression with CA19-9 also identified a third subpopulation of PDAC with different morphology, likely aggressiveness, and secretion characteristics. Clinical translation is potentially enabled by the detection of these biomarkers in blood plasma, which provides a new approach to improve diagnosis, prognosis and treatment development.
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Title: The microbiome of acute bacterial gastroenteritis and the functional role of intestinal bacteriophages
Creator: Nohomovich, Brian
Date: 2019
Collection: Electronic Theses & Dissertations
Description: "Acute gastroenteritis has a major disease burden worldwide. There are 2.3 billion cases of acute gastroenteritis worldwide each year that accounts for 8% of all deaths in children under the age of 5. In the United States, there are an estimated 179 to 375 million cases annually. Gastroenteritis can have acute and chronic effects on human health. Pathogens often are not identified in cases of acute gastroenteritis due in part to the wide range of causative agents and the difficulties with...
Show more"Acute gastroenteritis has a major disease burden worldwide. There are 2.3 billion cases of acute gastroenteritis worldwide each year that accounts for 8% of all deaths in children under the age of 5. In the United States, there are an estimated 179 to 375 million cases annually. Gastroenteritis can have acute and chronic effects on human health. Pathogens often are not identified in cases of acute gastroenteritis due in part to the wide range of causative agents and the difficulties with standard culturing practices. The advent of next-generation sequencing has allowed the study of the intestinal microbiome to detect alterations in the composition as specific disease signatures. There have been few studies on the microbiome of gastroenteritis, but none have to date have studied both the virome and bacteriome together. Through this combined analysis, a deeper understanding of gastroenteritis can be generated.In this dissertation, the Microbiome (Virome and Bacteriome) of 79 cases and 125 member controls were examined. It was found that cases had lower diversity and richness in and increased abundances in Enterobacteriaceae. Additionally, associations with severe illness were made to a specific cluster of samples. Differential abundance analysis identified the involvement of both viruses and bacteria. Analysis of the same 79 cases in a recovery state (n = 63), identified the changes that occur during and after infection. These changes agree with the case and control analysis. The functional aspects were analyzed of the viral communities. Three novel bacteriophages were isolated from stool samples and characterized. Two of the bacteriophages were determined to be lysogenic and were found in 23 additional E. coli O157:H7 strains based on BLAST alignments. One of the lysogenic bacteriophages (PHG003), harbors an SbcC gene which is a predicted exonuclease but it's important to the host bacterium remains unknown. Additionally, a lytic bacteriophage (PHG001) was also isolated and exhibited a relatively broad host range and was incredibly virulent to E. coli O157:H7. Additionally, PHG001 exhibits a phage-antibiotic synergism with the use of ampicillin and mitomycin c. Either antibiotic with the bacteriophage exhibited a drastic reduction in bacteria growth. PHG001 also reduced shiga toxin expression compared to control levels."--Pages ii-iii.
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Title: The tissue angiotensin system in the lung : roles in human pulmonary fibrosis
Creator: Dang, My-Trang Thi
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Idiopathic Pulmonary Fibrosis (IPF) is the most common form of interstitial lung disease with a 3-year median survival upon diagnosis. The lack of effective therapies in treating this disease highlights our incomplete understanding in the pathogenesis of IPF. The prevailing hypothesis is that IPF is a result of abnormal wound healing which consists of persistent injury and apoptosis to alveolar epithelial cells (AECs), aberrant fibroblast proliferation, and the accumulation of extracellular...
Show moreIdiopathic Pulmonary Fibrosis (IPF) is the most common form of interstitial lung disease with a 3-year median survival upon diagnosis. The lack of effective therapies in treating this disease highlights our incomplete understanding in the pathogenesis of IPF. The prevailing hypothesis is that IPF is a result of abnormal wound healing which consists of persistent injury and apoptosis to alveolar epithelial cells (AECs), aberrant fibroblast proliferation, and the accumulation of extracellular matrix proteins. Our laboratory has implicated a role of the angiotensin (ANG) system in these events. In IPF, both angiotensinogen (AGT), the only known precursor to angiotensin II (ANGII), and Transforming Growth Factor-Beta (TGF-β₁) mRNA and protein are up-regulated, as well as the profibrotic peptide, ANGII. In human pulmonary fibroblasts, TGF-β₁-inducible AGT transcription is mediated by the core promoter spanning from -46 to +22. At the -20, -18, and -6 positions lies single nucleotide polymorphisms (SNPs) that have been shown to influence its transcription rate in hepatocytes. Our results in human pulmonary fibroblasts parallel those observed in hepatocytes where the CA haplotype at -20 and -6 respectively, had about a 1.5-fold increase in AGT transcription compared to the AG haplotype (p = 0.011). The increase in AGT transcription would result in an increase in ANGII, which we predict to be associated with greater severity of IPF as measured by pulmonary function tests. Studies in IPF cohorts from the United States and Spain demonstrated that the CC genotype at -20 (p = 0.0028 for U.S. and p = 0.017 for Spain), the AA genotype at -6 (p = 0.021 for U.S.), and the CA haplotype (p = 0.0048 for U.S. and p = 0.014 for Spain) predicted lower diffusing capacity. Additionally, the Proline/Proline variant at codon 10 in TGF-β₁ was also associated with lower diffusing capacity (p = 0.0014). Surprisingly, the results of both studies were only significant in males, reflecting the male bias of this disease. Preliminary data indicates that in addition to inducing AGT transcription, TGF-β₁ also up-regulates cathepsin D and down-regulates ACE-2. Cathepsin D and AGT are both part of the rate-limiting step in the generation of ANGII whereas ACE-2 functions in its removal. This suggests that TGF-β₁ may cause an imbalance in the ANG system by favoring the ANGII producing axis. The mechanism by which ACE-2 is down-regulated has not been well studied. However, results from our lab suggests that this down-regulation may be related to ACE-2 ectodomain shedding or through a JNK-mediated mechanism as seen with inducers of ER-stress and cell-cycling in AECs.
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Title: The identification of ATPAF1 as a novel asthma susceptibility gene and the characterization of functional regulatory variants
Creator: Schauberger, Eric Michael
Date: 2011
Collection: Electronic Theses & Dissertations
Description: Asthma, the most common chronic disease of childhood, is driven by genetic and environmental determinants. To identify genes that increase the risk of asthma in children, a multiple stage genome-wide association study was conducted in a nested case-control study of a whole-population birth cohort from the Isle of Wight, UK. This study resulted in the identification of a cluster of associated SNPs and SNP haplotypes in the ATPAF1 gene (ATP synthase mitochondrial F1 complex...
Show moreAsthma, the most common chronic disease of childhood, is driven by genetic and environmental determinants. To identify genes that increase the risk of asthma in children, a multiple stage genome-wide association study was conducted in a nested case-control study of a whole-population birth cohort from the Isle of Wight, UK. This study resulted in the identification of a cluster of associated SNPs and SNP haplotypes in the ATPAF1 gene (ATP synthase mitochondrial F1 complex assembly factor 1) on human chromosome 1p33, with two SNPs achieving significance at a genome-wide level (P=2.26E-5 to 2.2E-8). SNP, haplotype, and gene-level associations were confirmed in three of four replication populations. The ATPAF1 gene contains 303 reported variants, which were assessed using in silico techniques and prioritized through annotated function in public databases, and/or inferred function based on their location in experimentally reported or predicted functional DNA sequences. The in silico screen prioritized 27 variants, of which several had predicted function as coding, splicing, and/or gene expression regulation. These prioritized variants were targeted in addition to exons, conserved, and regulatory regions for selective resequencing in 40 cohort individuals using Sanger sequencing.Selective resequencing of 14.6 kb resulted in the identification of 35 total variants. This included validation of 9 (of the 27) prioritized variants from the in silico screen and 9 new rare variants, including 1 nonsynonymous mutation. Three variants with gene expression regulatory potential were found to be clustered within 600 bp of each other in the promoter/exon 1 of ATPAF1 in four haplotypes. This region was targeted for analysis using luciferase reporter gene assays in BEAS-2B and COS-7 cell lines. These cell culture assays confirmed promoter functionality and indicated a statistically significant difference in luciferase expression (means ranging between 2-3 fold differences) among the promoter haplotypes.In conclusion, ATPAF1 was identified as a childhood asthma susceptibility gene. In silico studies coupled with selective resequencing of the ATPAF1 region provided an efficient method to identify functional variants. DNA variant haplotypes within the ATPAF1 promoter demonstrated the ability to differentially regulate gene expression. However, the roles of these and other functional variants in ATPAF1 and their ability to modulate asthma susceptibility need further study.
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Title: Tfap2a, Irf6 & Grhl3 : a novel network that regulates both neurulation and craniofacial development
Creator: Kousa, Youssef Ayoub Adly
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Interferon Regulatory Factors transcriptionally regulate development and differentiation of the innate and adaptive immune systems. Within this family, IRF6 is unique because it regulates cutaneous and orofacial development in humans and mice. Common variants in IRF6 are associated with 12% of all orofacial clefting risk. Critically, a DNA variant in the IRF6 enhancer MCS9.7, rs642961, is found in 30% of the world's population. Biochemically, we know that rs642961 abrogates one of four TFAP2a...
Show moreInterferon Regulatory Factors transcriptionally regulate development and differentiation of the innate and adaptive immune systems. Within this family, IRF6 is unique because it regulates cutaneous and orofacial development in humans and mice. Common variants in IRF6 are associated with 12% of all orofacial clefting risk. Critically, a DNA variant in the IRF6 enhancer MCS9.7, rs642961, is found in 30% of the world's population. Biochemically, we know that rs642961 abrogates one of four TFAP2a binding sites, suggesting regulatory function. Mutations in TFAP2a can lead to Branio-oculo-facial Syndrome, a dominantly inherited orofacial clefting syndrome that includes upper lip pits. However, functional studies have not shown if Tfap2a regulates MCS9.7 activity or endogenous Irf6 expression in the mouse. In addition, rare mutations in IRF6, located within 1q32-q41, lead to Van der Woude and Popliteal Pterygium Syndromes, dominantly inherited orofacial clefting disorders. Currently, 70% of VWS families have mutations in IRF6. While the remaining 30% have unknown etiology, prior linkage analysis suggest locus heterogeneity.We use a mouse models to determine how common variants in IRF6 may be associated with orofacial clefting and to investigate locus heterogeneity in VWS. We find that knocking out Tfap2a leads to loss of MCS9.7 enhancer activity and Irf6 expression in vivo. On the other hand, Irf6 also appears to stabilize Tfap2a protein in epidermis. The necessity of Tfap2a for Irf6 expression contributes to our understanding of the association between rs642961 and isolated orofacial clefting. Significantly, we also find that Irf6 transcriptionally activities Grhl3 in epithelium. Consistent with prior work showing locus heterogeneity, we find that mutations in GRHL3 can also led to Van der Woude Syndrome. These results suggest that TFAP2a, IRF6, and GRHL3 share a conserved genetic pathway that is required for proper development of the lip and palate in humans and mice. In the mouse, loss of Grhl3 and Tfap2a leads to skin, limb, craniofacial and neural tube defects. Because Irf6 is an intermediate node between Tfap2a and Grhl3 in oral epithelium, we predict and find that changes in Irf6 expression can lead to neural tube defects. Over-expressing Irf6 leads to rostral neural tube defects, including loss of the cranial vault, i.e. acrania, and a split face. In addition, both reducing and over-expressing Irf6 leads to caudal neural tube defects, a curled and kinked tail, respectively. Consistent with orofacial genetic regulation, we find that Irf6 represses Tfap2a in rostral neural tube development. In the caudal neural tube, we find that Irf6 activates both Tfap2a and Grhl3 expression and that Tfap2a and Grhl3 interact in caudal neurulation. Consistently, human sequencing reveals a rare IRF6 mutation in an individual with spina bifida. Finally, we show that Irf6 expression in skin development rescues perinatal lethality but not limb, tail and palatal development. These results suggest that Tfap2a-Irf6-Grhl3 regulate the development of multiple ectodermal lineages. We conclude that cross-fertilization in orofacial and neural tube development provides candidate genes and potential therapeutic strategies for two congenital diseases with significant morbidity and mortality.
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Title: The role of parkin in the recovery of central dopamine neurons from acute neurotoxicant exposure
Creator: Benskey, Matthew John
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Parkinson Disease (PD) pathology is associated with the selective degeneration of nigrostriatal dopamine (NSDA) neurons, while the tuberoinfundibular DA (TIDA) neurons of the hypothalamus remain intact. The same pattern of selective degeneration has been observed following exposure to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyradine (MPTP), a mitochondrial complex I inhibitor which recapitulates many of the molecular pathologies associated with PD. The purpose of this dissertation is to...
Show moreParkinson Disease (PD) pathology is associated with the selective degeneration of nigrostriatal dopamine (NSDA) neurons, while the tuberoinfundibular DA (TIDA) neurons of the hypothalamus remain intact. The same pattern of selective degeneration has been observed following exposure to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyradine (MPTP), a mitochondrial complex I inhibitor which recapitulates many of the molecular pathologies associated with PD. The purpose of this dissertation is to identify early molecular events that underlie TIDA neuron recovery from toxicant exposure and adapt these mechanisms in an attempt to rescue NSDA neurons from toxicity. NSDA neurons show loss of axon terminal DA concentrations following acute (20mg/kg; s.c.) and chronic (10 x 20mg/kg; s.c. over 35 days) MPTP administration and exhibit cell death following chronic MPTP administration. In contrast, TIDA neurons show no loss of axon terminal DA concentrations or cell death following acute or chronic MPTP exposure. The recovery of TIDA neurons is independent of extrinsic factors such as decreased toxicant exposure or hormonal activation. TIDA neuron recovery is associated with an increase in the PD-associated proteins, parkin and ubiquitin carboxy-terminal hydrolase L-1 (UCHL-1) within the arcuate nucleus (ARC) 24 h following MPTP. Additionally, parkin protein concentrations remain elevated in the ARC for up to 22 days following chronic MPTP administration. In contrast, the susceptibility of NSDA neurons is associated with decreased expression of both parkin and UCH-L1. The high correlation between the presence of the parkin protein and the recovery of DA neurons from MPTP toxicity is consistent with a role of parkin in DA neuron survival. In order to determine if parkin is necessary and sufficient in the recovery of TIDA neurons following MPTP, recombinant adeno-associated viral (rAAV) vectors containing parkin shRNA or a scrambled shRNA were created. Mice received stereotaxic ARC injections of rAAV containing either parkin shRNA or scrambled shRNA (250nl/side; 3.5x1013vg/ml), or remained naïve to surgery, and were administered a single injection of MPTP (20mg/kg; s.c.) 30 days following rAAV surgery. Twenty-four h post-MPTP, TIDA neurons were able to recover axon terminal DA concentrations following MPTP in control and scrambled shRNA treated animals. However, axon terminal DA was significantly reduced 24 hr following MPTP exposure following knockdown of parkin in TIDA neurons. To determine if parkin overexpression would protect NSDA neurons from MPTP toxicity, mice received unilateral stereotaxic injection of rAAV containing parkin into the substantia nigra (SN) (500nl; 3.4x1013vg/ml) and were administered a single injection of MPTP (20mg/kg; s.c.) 30 days following rAAV surgery. Twenty-four hours post-MPTP, parkin overexpression was unable to rescue MPTP-induced loss of DA in the striatum (ST), but did rescue MPTP-induced loss of tyrosine hydroxylase (TH) in the SN and ST. These findings are consistent with the following conclusions: 1) TIDA neuronal recovery from acute MPTP exposure is independent of extrinsic factors and is mediated by an intrinsic ability to increase expression of neuroprotective proteins, 2) The ability of TIDA neurons to up-regulate parkin is at least partially responsible for recovery of axon terminal DA following MPTP, 3) toxicant-induced loss of parkin contributes to MPTP toxicity within NSDA neurons.
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Title: Modulation of HIVGP120 antigen-specific immune responses, in vitro and in vivo, by Delta9-tetrahydrocannabinol and cannabinoid receptors 1 and 2
Creator: Chen, Weimin
Date: 2014
Collection: Electronic Theses & Dissertations
Description: "Approximately 25% of HIV patients use marijuana for its putative therapeutic benefit; however, it is unknown how cannabinoids affect the immune function of immunocompromised HIV patients at the early stage. The goal of these studies was to investigate the immunomodulatory effects of cannabinoids on the early stage of the anti-HIV immune responses... " -- Abstract.

Title: Immunomodulatory properties of feline mesenchymal stem cells and their clinical application in treatment of feline chronic idiopathic cystitis
Creator: Parys, Maciej
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Mesenchymal stem cells (MSC) offer great promise for treatment of inflammatory disorders. Cats spontaneously develop a variety of disorders that can serve as translational model for human diseases. One such disorder is chronic feline idiopathic cystitis, which closely resembles human Interstitial Cystitis/ Painful Bladder Syndrome. These cystopathies in both cats and humans are characterized by inflammatory cell infiltrate within bladder wall. Since MSC have been reported to migrate to sites...
Show moreMesenchymal stem cells (MSC) offer great promise for treatment of inflammatory disorders. Cats spontaneously develop a variety of disorders that can serve as translational model for human diseases. One such disorder is chronic feline idiopathic cystitis, which closely resembles human Interstitial Cystitis/ Painful Bladder Syndrome. These cystopathies in both cats and humans are characterized by inflammatory cell infiltrate within bladder wall. Since MSC have been reported to migrate to sites of inflammation, bladder wall inflammation can potentially be targeted by MSC, whose immunomodulatory properties could beneficially affect disease expression. One of the initial steps needed to evaluate use of feline MSC, as a potential therapy is to investigate similarities between feline and human MSC in their immunomodulatory properties. We investigated expression of genes involved in MSC immunomodulation and how they react to stimulation with two cytokines INFγ and TNFα. Unstimulated MSC express similar immunomodulatory genes as human MSC except for FASL and IL10. The reaction of feline MSC to cytokine stimulation is similar to that of human MSC, including the upregulation of IDO in reaction to INFγ stimulation. Other genes upregulated by stimulation included IL-6, PD-L1 and HGF. IL-6 was also significantly upregulated at protein level after stimulation. However levels of HGF were lowered in cell culture supernatants under some conditions. Increased IDO expression was further confirmed through increase in kynurenine, a tryptophan degradation product. TNFα stimulation resulted in strong upregulation of IL-6 gene and protein expression. Interestingly PGE2 levels remained unchanged after stimulation, although basal expression of this factor was high relative to published human data. Feline MSC were also capable of blocking proliferation of activated peripheral blood mononuclear cells. Route of injection of MSC can potentially affect the efficacy of MSC therapy. To avoid pulmonary vascular trapping after IV injection we have investigated the intraperitoneal route of MSC injection, as an alternative route of MSC administration. Our studies show intraperitoneal administration of MSC is safe and associated with only mild short-term adverse effects. Reliable biomarkers are not currently available for FIC. This makes diagnosis dependent on exclusion of other lower urinary tract disorders and evaluation of patient responses difficult. We investigated urine and serum cytokine concentration as biomarkers. We have identified that FIC affected cats have increased urine concentrations of IL-6 and sFAS and increased serum IL-12, IL-18, SDF1 and FLT3. Lastly, we performed a pilot study to evaluate MSC based therapy for chronic FIC. Unfortunately, only one patient met inclusion criteria and received autologous MSC by IP injection. While reportedly showing an initial favorable response for the first 10 days, clinical signs returned and were progressive. This initial experience was very informative with regard to the many practical difficulties of assessing the effects of such a treatment in this complicated disease.
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Title: In vivo analysis of the molecular mechanisms of long- and short-range transcriptional repression
Creator: Li, Li
Date: 2010
Collection: Electronic Theses & Dissertations
Description: Transcription repression is essential for establishing precise patterns of gene expression during development. Studies of Drosophila transcriptional regulation reveal that transcriptional repressors fall into two classes: those acting locally as "short-range" repressors, and those acting dominantly as "long-range" repressors. The types of transcription factors and cofactors involved are highly conserved in all metazoans. One area in which our knowledge has lagged is in understanding how these...
Show moreTranscription repression is essential for establishing precise patterns of gene expression during development. Studies of Drosophila transcriptional regulation reveal that transcriptional repressors fall into two classes: those acting locally as "short-range" repressors, and those acting dominantly as "long-range" repressors. The types of transcription factors and cofactors involved are highly conserved in all metazoans. One area in which our knowledge has lagged is in understanding how these different classes of repressors function at a mechanistic level. Understanding of repression mechanisms in the developmental circuits will shed light on key biological processes including stem cell reprogramming, cancer, and development.In this work, I describe studies that reveal molecular mechanisms of long- and short-range repression during early Drosophila development. My chromatin studies of the prototypic long-range repressor Hairy and the short-range repressor Knirps suggest that these two modes of repression induce distinct chromatin states on the repressed genes. These results indicate that transcriptoinal corepressors can be deployed in a context-dependent manner to effect transcription. To discern the importance of individual corepressors, my studies have also examined the role of the conserved Groucho corepressor in Hairy-mediated long-range repression.
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Title: Metabolic remodeling and growth regulation of Mycobacterium tuberculosis at acidic pH
Creator: Baker, Jacob J.
Date: 2017
Collection: Electronic Theses & Dissertations
Description: Mycobacterium tuberculosis (Mtb), the leading cause of infectious disease death worldwide, remains a global health threat. The success of Mtb as a pathogen can be attributed to its ability to sense and adapt to the host environment. Understanding the mechanisms of Mtb adaptation to the host environment has the potential to inform the development of novel and effective tuberculosis treatments. An important aspect of Mtb adaptation is its ability to regulate growth rate, as slow growing and...
Show moreMycobacterium tuberculosis (Mtb), the leading cause of infectious disease death worldwide, remains a global health threat. The success of Mtb as a pathogen can be attributed to its ability to sense and adapt to the host environment. Understanding the mechanisms of Mtb adaptation to the host environment has the potential to inform the development of novel and effective tuberculosis treatments. An important aspect of Mtb adaptation is its ability to regulate growth rate, as slow growing and growth arrested Mtb has been shown to exhibit increased phenotypic tolerance to killing by the immune system or antibiotics. In response to the important host cue of acidic pH, I have observed that Mtb exhibits carbon source specific growth arrest. While the majority of carbon sources were unable to promote growth at acidic pH in minimal medium, carbon sources associated with the anaplerotic node and with the host relevant nutrient cholesterol were permissive for Mtb growth. Transcriptional profiling of Mtb at acidic pH demonstrated that Mtb induces genes involved in anaplerotic metabolism, lipid synthesis, and redox homeostasis. Furthermore, deletion of the two-component regulatory system phoPR that is induced at acidic pH led to enhanced growth at acidic pH, suggesting that slow growth is an aspect of Mtb adaptation to acidic pH. By performing growth curves and metabolic profiling of wild type Mtb as well as mutants lacking specific enzymes of the anaplerotic node, I have also sought to characterize the mechanisms of metabolic remodeling that occur at acidic pH and their role in growth regulation. Finally, through the isolation of mutants with enhanced growth at acidic pH, I have demonstrated that growth arrest at acidic pH is a regulated process in Mtb necessary for phenotypic tolerance.
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