Masters of Science in Molecular Biosciences Theses
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Item Investigating minocycline’s anti-inflammatory role in alveolar macrophages(Albany College of Pharmacy and Health Sciences Theses, 2023-08) Scholl, Erica '23Minocycline has been FDA-approved for 50 years and is utilized to treat a wide range of infections. More recently, minocycline has been used to treat several sterile inflammatory conditions due to its non-antimicrobial anti-inflammatory properties. While minocycline is a unique and beneficial therapeutic, its immunomodulatory impact on inflammation during an active infection is not well understood. Elucidating the molecular mechanism of minocycline’s ability to modulate the inflammatory response can lead to the development of novel therapeutic strategies that take advantage of the antibiotic capabilities of minocycline while also protecting the host against post-infection inflammatory tissue damage. We hypothesized that minocycline exerts anti-inflammatory properties that suppress the cytokine production after macrophage activation, altering the host immune response to pathogens and limiting tissue damage. Our data indicates that minocycline is nontoxic to alveolar macrophages even at doses far beyond therapeutic potential. Additionally, pretreatment with minocycline significantly decreased the activation of nuclear factor kappa beta (NF-κB), as well as the secretion of several inflammatory cytokines following treatment with ligands to activate specific toll-like receptors. Total levels of key signaling molecules MyD88 and NF-κB were not altered following minocycline pretreatment, suggesting that minocycline may be altering the activation status within the cascade. Lastly, the pretreatment of macrophages with minocycline appears to have no significant effect on the production of oxidative stress following ligand treatment at 24 hours. Our long-term goal is to identify immunomodulatory mechanisms exerted by minocycline in the presence of an infection, as it could be the key to managing infections by not only eliminating the pathogen but also minimizing host inflammatory tissue damage, which is a driver of poor patient outcomes.Item HIV-1 infection by cell-to-cell transmission induces human innate immune response.(Albany College of Pharmacy and Health Sciences Theses, 2023-07) Sanchez, Anthony '23HIV-1 can spread from infected cells to intact cells frequently through close cellular contact in human lymphoid tissues/organs. Compared to cell free virus infection, HIV-1 cell-to-cell transmission yields a significantly higher multiplicity of infection and requires both cellular membrane and skeleton changes, however the impact of HIV-1 cell-to-cell transmission to host innate immune response, particularly the IFN initiation by cGAS-STING pathways, has not been reported previously. In this study, it was found that HIV-1 GFP+ (∆env) virus in infected MT2 cells could transmit to uninfected MT2 cells and other immune cells predominantly by cell-to-cell transmission. The infection by cell-to-cell transmission of HIV-1 started to increase 12 hours and peaked at 36 hours after coculture of infected donor MT2 cells and uninfected recipient cells. Syncytia was observed in HIV-1 cell-to-cell transmitted MT2 cells by using fluorescence microscopy. In addition, the flow cytometry result indicated cell morphology and structure changes in MT2 cells after HIV-1 cell-to-cell transmission. Importantly, in HIV-1 GFP+ infected MT2 cells, phosphorylation of both STING and IRF3 proteins were detected at 36 hours post infection, which was virus infection dosage dependent. The nuclear translocation of phosphorylated IRF3 was also identified. Furthermore, transcriptional upregulation of IFNB1 was confirmed by a real time PCR experiment. These data strongly demonstrated HIV-1 cell-to-cell transmission in MT2 cells triggered cGAS-STING pathway and activated IFN gene expression. Meanwhile, the influence of host cell autophagy process to HIV-1 cell-to-cell transmission was also studies. It was found that the induction of autophagy by rapamycin mildly inhibited HIV-1 cell-to-cell transmission; while autophagy inhibitors 3MA, Baf. A and chloroquine also blocked HIV-1 cell-to-cell transmission, except E64D/ pepstatin which inhibits final proteolytic stage of autophagy. Together, our results have provided compelling evidence that HIV-1 cell-to-cell transmission activates IFN and interacts with autophagy system, which might have significant impact on HIV-1 pathogenesis through the host innate immune response and inflammatory signaling.Item Understanding the Mechanism by which Sulforaphane Promotes SAMHD1 Activation to Protect Macrophages Against HIV Infection(Albany College of Pharmacy and Health Sciences Theses, 2022-08) Fazzari, Vincent A. '22The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). To date, HIV/AIDS has killed an estimated 40 million people. There is no cure, no vaccine, and current therapies are burdened with high cost and toxicity. Therefore, further study of HIV is necessary. Sulforaphane (SFN), a compound found in cruciferous vegetables, was shown to block HIV infection of macrophages through activation of the transcription factor Nrf2. Follow up, unpublished, work by H. John Sharifi, PhD and Dakota Paine, M.S. found that SFN promotes the absence of an inhibitory phosphate from the antiviral protein SAMHD1. The exact mechanism of unphosphorylated SAMHD1 by SFN is unclear. The goal of this work was to uncover potential ways that SFN can yield unphosphorylated SAMHD1. Our central hypothesis is that SFN, acting through Nrf2, upregulates p21 and drives SAMHD1 into the nucleus to promote SAMHD1 dephosphorylation and protection against HIV. This hypothesis is based on an observation by Dr. Sharifi and Mr. Paine that SFN upregulates p21, also known as cyclin-dependent kinase inhibitor 1, in primary macrophages. p21 is a cell cycle control protein that has been previously shown to inhibit SAMHD1 phosphorylation. This hypothesis is additionally based on work showing that SAMHD1 accumulates in the cytosol under oxidizing conditions and that SFN promotes reducing conditions. This hypothesis was tested to determine the influence on SAMHD1 phosphorylation. Here, we first confirmed that SAMHD1 is required for the antiviral effect of SFN. We then established that p21 levels do not necessarily connect SFN to unphosphorylated SAMHD1. We further show that SFN does not change the intracellular location of SAMHD1.Item Mitochondrial Trafficking During the Hyperglycemic Shift From TNF-\316\261-Induced Apoptosis to Necroptosis(Albany College of Pharmacy and Health Sciences Theses, 2022-08) Truong, Phillip V. '22Necroptosis is a pro-inflammatory programmed cell death (PCD) pathway. Unlike apoptosis, necroptosis is caspase-independent and is instead mediated by the necrosome complex consisting of receptor-interacting protein 1 and 3 (RIP1, RIP3), and mixed lineage kinase domain-like pseudokinase (MLKL). We have previously discovered the hyperglycemic shift from TNF-\316\261-induced apoptosis to necroptosis and wish to further delineate its mechanism. As we have also noted a central role for mitochondrial reactive oxygen species (mROS) in this shift to necroptosis, this work serves to analyze the cellular trafficking of critical cell death factors as part of this mechanism. We show that during the hyperglycemic shift to necroptosis, executioner caspases (-3, -6, -7) do not traffic to the mitochondria. Instead, RIP1 and MLKL in their phosphorylated forms, Bak, Bax, and dephosphorylated Drp1 traffic to the mitochondria. Furthermore, we show that RIP1, MLKL, Bak, and Bax are oxidized and oligomerized in the mitochondria. We show that this mitochondrial trafficking is primarily driven by ROS in experiments utilizing superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), and antioxidant, N-acetylcysteine (NAC). DDC-induced ROS led to increased trafficking of RIP1 and MLKL in their phosphorylated forms to the mitochondria. DDC-induced ROS also led to increased trafficking of Bak, Bax, and dephosphorylated Drp1 to the mitochondria. Moreover, DDC-induced ROS promoted increased oxidization/oligomerization of RIP1, MLKL, Bax, and Bak in the mitochondria. Contrastingly, in hyperglycemic conditions, NAC-inhibition of ROS prevented the trafficking of RIP1 and MLKL in their phosphorylated forms to the mitochondria. NAC-inhibition of ROS also prevented the mitochondrial trafficking of Bak, Bax, and dephosphorylated Drp1. NAC-inhibition of ROS prevented the oxidization/oligomerization of RIP1, MLKL, Bax, and Bak in the mitochondria. Proteinase K assay analysis revealed that RIP1, MLKL, and Bax and Bak are localized to the mitochondrial matrix, both the inner and outer mitochondrial membrane, and solely to the outer mitochondrial membrane, respectively. Flow cytometric and fluorescent microscopy analysis revealed significant \316\224\316\250m loss driven by Bax during the hyperglycemic shift to necroptosis. This work serves to provide further insights into the specific molecular mechanisms involved during the hyperglycemic shift to and exacerbation of necroptosis.Item Alternative Treatment Options against Carbapenem-Resistant Acinetobacter baumannii Infections(Albany College of Pharmacy and Health Sciences Theses, 2022-08) Putra, Vibert '22Carbapenem-resistant Acinetobacter baumanii (CRAB) is a multidrug-resistant nosocomial gram-negative pathogen that has become increasingly prevalent in the United States. CRAB has been labelled by the U.S. Centre for Disease Control and World Health Organisation as an “urgent threat” and “critical priority”, respectively. Due to its plethora of resistance arsenals, current treatment options against CRAB are extremely limited and combination treatments have become the norm. The purpose of this study to explore alternative combination treatments by evaluating clinically used antibiotic combinations and identifying a novel non-antibiotic combination strategy. Polymyxin B plus meropenem and polymyxin B plus minocycline were evaluated by in vitro hollow fibre infection models against minocycline-susceptible clinical CRAB strains. Our results showed polymyxin B combination with meropenem have greater bacterial killing despite minocycline monotherapy susceptibility in vitro. Previous studies showed that a USFDA-approved rheumatoid arthritis drug, auranofin, inhibits bacterial thioredoxin reductase within the antioxidant system that restores protein structures damaged by oxidative stress and regulates downstream effector proteins. Thioredoxin reductase activates thioredoxin A (TrxA), which is the effector protein in the thioredoxin system. Our 24-hour static concentration time kill assays showed that auranofin and meropenem combinations were beneficial against clinical CRAB isolates but not meropenem-susceptible clinical isolate. Furthermore, disc diffusion assay showed that auranofin has additive effects with minocycline, colistin, and chloramphenicol. Our total antioxidant assay showed that the TrxA-deleted mutant showed has significantly increased total antioxidant activity under meropenem stress, suggesting that the pathogen can compensate for the lack of antioxidant activity from the thioredoxin system. Hence, we propose that the increased antibiotic susceptibility observed under auranofin treatment was due to thioredoxin modulation of resistance mechanisms. Our study showed that TrxA modulates tetracycline efflux pump activities, while TrxA only modulates baseline total β-lactamase activities and not its induction overtime under meropenem stress. Therefore, our study showed the two potential avenues of therapies against CRAB infections that can either be applied immediately in the clinics or further characterised in vitro. Further evaluation of polymyxin B plus meropenem combination dose ranges and determination of thioredoxin role in modulation of resistance are needed to develop optimal treatments.