M.S. - Biomedical Sciences (Tropical Medicine)
Permanent URI for this collection
Browse
Recent Submissions
Item ASSESSING THE IMPEDIMENTS ASSOCIATED WITH SARS-COV-2 WHOLE GENOME SEQUENCING(2024) Salomon, Renn Silve Chua; Nerurkar, Vivek R.; Biomed Science (Tropical Medicine)Item Dysregulation of Complement Components Associated with Inflammation and Coagulation in Virally Suppressed People Living with HIV (PLWH)(2024) Subia, Natalie Tejero; Park, Juwon; Biomed Science (Tropical Medicine)Item USING AND OPTIMIZING DROSOPHILA S2 EXPRESSION SYSTEMS AND AFFINITY CHROMATOGRAPHY FOR RECOMBINANT ANTIGEN PRODUCTION(2024) Eiser, Isabelle Elaine Yazel; Lehrer, Axel T.; Biomed Science (Tropical Medicine)Item Investigating Antibody Avidity In SARS-CoV-2 Naïve And Convalescent Vaccinees(University of Hawaii at Manoa, 2022) Odo, Troy Kazuma; Lehrer, Axel T.; Biomed Science (Tropical Medicine)Over the past two years, SARS-CoV-2 has had a devastating impact across the world. Despite the historic success of the multiple SARS-CoV-2 vaccines, additional research is needed to further understand the underlying mechanisms of protection. While many studies focus primarily on neutralizing antibody titers, another aspect of humoral immunity that is often overlooked is antibody avidity. Avidity, the total binding strength of all interactions between an antibody and antigen, is important to consider in the context of vaccination because it provides an important measure of antibody maturation and development. To date, there is no “gold standard” approach to measure antibody avidity. Solid-state immunoassays such as ELISAs are commonly modified with a chaotropic agent capable of disrupting antigen-antibody interactions. This overall focus of this study is to measure and compare antibody avidity in SARS-CoV-2 naïve and convalescent vaccinees. To do so we optimized an ammonium thiocyanate-modified multiplex immunoassay that can be used to measure SARS-CoV-2 antibody avidity. We found that standardizing antibody concentrations is an important step when measuring antibody avidity. This helps to prevent oversaturation of the assay and ensures that avidity can be measured and compared between sera with different antibody populations. Additionally, we used this assay to measure longitudinal sera collected from SARS-CoV-2 naïve and convalescent vaccinees to investigate the relationship between natural infection and vaccination. Sera were collected prior to vaccination, >14 days post-dose 1, and >14 days post-dose 2 and individuals were grouped based on whether they had SARS-CoV-2 infection prior to vaccination. Additionally, in both the naïve and convalescent groups, antibody avidity increased following each vaccine dose. A single antigen exposure in the convalescent group (natural infection), generated higher antibody avidity than a single vaccine dose in the naïve group. Avidity was also higher following two antigen exposures in the convalescent group (natural infection + a single vaccine dose) compared to two vaccine doses in the naïve group. Our assay demonstrates differences in the antibody response following natural infection and vaccination which opens the door for further investigation into these differences.Item Development of Microfluidics for Use in a Novel Way to Characterize Latently HIV-Infected Cells(University of Hawaii at Manoa, 2022) Mak, Bradley; MacPherson, Iain; Biomed Science (Tropical Medicine)Antiretroviral therapy (ART) has been transformative in helping people living with HIV (PLWH) in the last 30 years. Despite the many advances in ART, it cannot eradicate HIV due to its inability to target the HIV latent reservoir. Therapeutic approaches have been considered for eradicating latent HIV such as “shock and kill” and “block and lock”but are still in development and have shown poor results in clinical trials thus far. Laboratory techniques such as STIP-Seq and HIV-Flow have been developed to quantify and characterize the HIV reservoir but do not aim to specifically identify and characterize genuine latently infected cells. Such techniques fall short due to the requirement to activate latently infected cells for identification and analysis, since activation changes cellular gene expression globally. Therefore, we introduce HIV-Seek, a novel approach to characterizing latently HIV infected cells in their pre-activated state. HIV-Seek aims to combine microfluidics, single cell RNA-sequencing, and molecular biology to identify biomarkers for immunological or pharmacological control of the viral reservoir. The focus of this thesis is to establish the development of microfluidics for both its fabrication and function in HIV-Seek alongside the integration of microfluidics and molecular biology. Designs and fabrication conditions were developed for the custom microfluidics for use in HIV-Seek. Encapsulation of beads and cells was performed using microfluidics and a coencapsulation rate of 4.38 ± 0.31% was achieved. Further experimentation with the integration of the molecular biology with the microfluidics is required but shows promise as shown with the dPCR.Item Elucidating The Antimicrobial & Antiviral Activity Of An Underexplored Class Of Organobismuth Compounds(University of Hawaii at Manoa, 2022) Faundo, Michael; Lehrer, Axel T.; Biomed Science (Tropical Medicine)Infections of antibiotic-resistant pathogens pose an ever-increasing threat to mankind. The investigation of novel approaches for tackling the issue of antimicrobial resistance (AMR) must be part of any global response to this problem if an unwanted reversion to the pre-penicillin era of medicine is to be avoided 1. One such promising avenue of research is looking into the potential of metallodrugs as a solution. Given the historical usage of bismuth compounds in the treatment of certain types of bacterial infections before the treatment of modern antibiotics and its interesting safety profile despite its heavy-metal grouping has made it a driving interest in its basic and applied research 2. Specific research efforts are focused on the development of novel Bi-based compounds, nanoparticles and composites with applications in treating cancer and microbial infections, imaging, theranostics and biosensing 3. Moreover, while metal agents have demonstrated their value as potential antimicrobial and anticancer agents, their antiviral activities have been rarely explored. Despite its growing interests in the medicinal chemistry landscape, organobismuth chemistry is far less developed in comparison to other main-group organometallic chemistry due to two obstacles. First, the C–Bi bond is weak (bond disassociation energy = 46 kcal/mol), making it prone to dismutation, a substituent scrambling process, which complicates the synthesis of unsymmetrical triarylbismuthanes of the general formula Ar21Ar2Bi (Ar1 ≠= Ar2) 4. However, the Hyvl lab at the University of Hawaii at Manoa has designed an efficient two-step synthetic protocol that affords the synthesis of a electronically diverse set of heteroleptic triarylbismuthanes without the formation of dismutated contaminants. The long-term goal of this study is to use the lead structures identified in this work to facilitate the development of novel antimicrobials and antivirals. The immediate objective of the study is to elucidate the antimicrobial and antiviral activities of this group of organobismuth compounds. Given bismuth’s historical use in treating microbial infections before the advent of modern antibiotics, such as syphilis and Helicobacter pylori, we hypothesize that these organobismuth compounds may exhibit a comparable level of antimicrobial activity. Moreover, while metal compounds have demonstrated their applications as antimicrobial and anticancer agents, their antiviral activities have rarely been explored. Due to the novelty of the structure of this underexplored class of compounds, we also hypothesize that these compounds may exert their activity through other modes of action rather than the better understood mechanisms of Bi(III). In the first aim, we screened thirty-one unique organobismuth compounds for their various levels of antimicrobial activities in the context of multiple Streptococcus spp., Staphylococcus aureus strains, and several species of Gram-negative organisms. Additionally, we determined the minimum inhibitory concentration (MIC) for each individual compound that passed initial antimicrobial activity screenings. Additionally, we also determined a range of concentrations at which each compound displays to be cytotoxic to Vero and human embryonic kidney (HEK293T) cells. We found that of the thirty-one unique compounds, twenty-eight exhibited some level of antimicrobial activity in a dose-dependent manner. While the observed activity showed variability between different species and strains of Gram-positive organisms, there did appear to be stronger levels of inhibition skewed towards strains of S. aureus, more so towards strains of methicillin-resistant S. aureus (MRSA) in certain compounds; however, we do not see any inhibitory activity against any Gram-negative bacteria. These data suggest that the targets of for some of these compounds may be specific to S. aureus and even more specifically to MRSA strains in certain contexts. Moreover, we found that twenty-one compounds demonstrate very low levels of cytotoxicity, being well tolerated even at the highest concentration tested (30 µM) displaying no noticeable toxicity when compared to healthy untreated controls measured by MTT assay. In the second aim, we investigated the potential antiviral activity for each of the thirty-one unique compounds. Using recombinant vesicular stomatitis viruses (rVSV), we identified a total of twenty positive hits. Additionally, we found seventeen compounds that demonstrate significant levels of antiviral activity while simultaneously showing no noticeable levels of cytotoxicity even at the highest assayed concentration. Of these positive hits, we did see some overlap with lead compounds described in the previous aim assessing antimicrobial activity. However, we were also able to identify nine hit compounds that solely demonstrated antiviral activity to rVSV pseudotyped viruses. In these structures we are able to observe preliminary structure activity relationships (SARs) and how substitution at certain positions affects compound activity. Preliminary work has qualitatively shown that some compounds are capable of also inhibiting Dengue virus serotype 2 (DENV-2). These results suggest that the activity conferred by these compounds may have the potential to affect additional virus families beyond what has been investigated here. Moreover, when examining compounds under pre-treatment conditions versus virucidal conditions, we found that the compounds are more likely to act directly on the cell to mediate inhibition of viral replication and not directly on the virus itself; however, the specific mechanisms of how they do so remain unclear. If this is indeed the case, these findings may implicate their capacity to act as broad-spectrum antiviral agents.Item Role of WNV NS4A and NS4B Proteins in Reducing Type I IFN Response via Interactions with Host IKKe and TBK1 Proteins(University of Hawaii at Manoa, 2022) Awamura, Thomas Ken; Kaufusi, Pakieli H.; Biomed Science (Tropical Medicine)West Nile virus (WNV) is a zoonotic flavivirus, with the potential to cause severe virally induced meningitis and encephalitis in humans. Currently, there are no specific antivirals or vaccine options for treatment or prevention of West Nile virus infection. Research on the closely related Dengue virus (DENV) has shown that both nonstructural proteins 4A and 4B (NS4A and NS4B) play key roles in the inhibition of type I interferon (IFN) by infected cells, aiding in the virus’ ability to evade the innate immune system. Despite this, the mechanism by which NS4A and NS4B inhibits the production of IFN-β is inadequately understood, especially for WNV. Recent findings for DENV suggest that the virus may target TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa B kinase subunit epsilon (IKKe), both potent activators of the type I IFN pathway. The expression of IKKe or TBK1 is required for full expression of IFN-β in cell models, and are a potential target for many viruses. To elucidate if WNV NS4A and/or NS4B proteins interacted with TBK1 and/or IKKe, we examined potential interactions between viral and host cell proteins and determined the degree of IFN inhibition in TBK1 and IKKe overexpressing cells transfected with WNV protein. Human endothelial kidney 293T (HEK293T) cells were transfected with WNV NS4A or NS4B, and IKKe or TBK1 plasmids (n=10-15). Pearson correlation coefficient (PCC) values were used to determine degree of colocalization of WNV proteins to host cell proteins shown in immunofluorescence assay (IFA) images. Calculations of PCC were done using FIJI (version 2.1.0/1.53c), an application within ImageJ, utilizing the coloc2 plugin. To corroborate the findings from the IFA imaging, Western blots of sucrose gradients from transfected cell lysate were performed to visualize recruitment of IKKe and TBK1 away from its intended location in the cytoplasm towards fractions containing WNV protein. To determine whether NS4A and NS4B were able to inhibit type I IFN production in cells overexpressing IKKe and/or TBK1, a dual-reporter luciferase assay was conducted to determine the degree to which these proteins could inhibit IKKe and TBK1 activation of IFN-β production. IFA imaging data indicated that cells expressing WNV proteins saw a significant degree of colocalization with IKKe and TBK1 (PCC ≥0.5) with NS4B seeing the highest degree of colocalization. Additional wWestern blot imaging taken from sucrose gradients conducted on transfected cells lysates showed a clear recruitment of IKKe and TBK1 proteins from the cytoplasm in control cells, towards the rough endoplasmic reticulum (RER) in cells expressing WNV protein. Luciferase assay data showed that in the presence of NS4A or NS4B, in comparison to other WNV NS proteins, IFN-β production was remarkably reduced (p<0.0001). Our study has identified interactions between NS4A and NS4B with both IKKe and TBK1 proteins and potential sequestration of these cytoplasmic host cell proteins towards the RER where the WNV proteins are located. Additionally, we have demonstrated that NS4A and NS4B significantly inhibit IFN-β production in cells expressing high levels of TBK1 and/or IKKe, likely via the targeting of said proteins. These findings provide further evidence that NS4A and NS4B may be optimal candidates for further antiviral development to combat WNV infection.Item Characterization Of Circulating Fibrocytes In People Living With HIV(University of Hawaii at Manoa, 2022) Dean, Logan Skyler; Shikuma, Cecilia; Biomed Science (Tropical Medicine)Fibrosis is estimated to account for 45% of all-cause mortality in the developed world. Immune dysregulation and chronic inflammation are potent contributors to fibrotic disease, increasingly so in people living with HIV (PLWH), independent of viral suppression. Fibrocytes are bone marrow-derived fibroblast-like cells whose increased numbers have been correlated with incidence and severity of fibrotic diseases. Despite their relevance to fibrosis, studies characterizing the fibrocyte in PLWH are scarce. Using 34 HIV+ individuals on suppressive antiretroviral therapy and 34 healthy controls (HC) we identified and quantified circulating fibrocytes in peripheral blood mononuclear cell (PBMC) samples. Participants were similar in age with a median age of 59.96 (56-63) years in HIV+ and 59.23 (56-64) years in HC and both groups were 88.2% male. No significant difference in non-infectious comorbidity prevalence was noted between the groups. HIV+ participantsʻ median CD4 count was within normal limits at 747.5 (601-898) cells/μL. Fibrocyte and activated fibrocyte numbers were not different between HIV+ and HC (p=0.257 and p=0.866, respectively). Similarly, the percentage of fibrocytes and activated fibrocytes from total CD45+ cells did not significantly differ (p=0.235 and p=0.710, respectively). However, fibrocyte numbers and the percentage of fibrocytes from CD45+ cells were significantly associated with increasing age in both HIV+ and HC groups (r=0.558, p=<0.0001 and r=0.575, p=<0.0001, respectively). Cultured fibrocytes isolated from HIV+ PBMC exhibited an increasing trend of mRNA expression of collagen-1 (COL-1), α-smooth muscle actin (α-SMA), vimentin, CCR5, and CCR7 with a trend towards decreased mRNA expression of CXCR4 and CCR3 compared to HC fibrocytes. Cultured HIV+ and HC fibrocytes treated with TGF-β1 at 5ng/mL and 20ng/mL enhanced fold change expression in COL-1, α-SMA, and chemokine receptor genes. Interestingly, fibrocytes from HIV+ PBMC exhibited decreased susceptibility to TGF- β1-induced COL-1 and CCR7 expression compared to HC. Our study demonstrates that circulating fibrocyte and activated fibrocyte populations are comparable between and strongly associated with age in virally suppressed PLWH and HC. In addition, cultured fibrocytes from PLWH demonstrated different responses to TGF- β1 stimulation compared to HC.Item Development of the Molecular Biology of HIV-Seek: A Novel Approach to Characterizing Latently Infected CD4+ T Cells(University of Hawaii at Manoa, 2022) Gamez, Eduardo Rafael; MacPherson, Iain; Biomed Science (Tropical Medicine)Although antiretroviral therapy (ART) is effective at managing HIV infection and extending the lives of people living with HIV (PLWH), it is limited by the inability to target the HIV viral reservoir, specifically in latently infected CD4+ T cells. Strategies for targeting the viral reservoir, including “shock and kill” and “block and lock,” while still in development, require more supportive clinical data to legitimize this approach. These strategies may benefit from an accurate characterization of latently infected cells. However, current techniques for characterization are limited due to the scarcity of latency and the stipulation to activate latently infected cells. Cell reprogramming in the activated state of CD4+ T cells obscures the biology of the cell in its latent state. Therefore, we introduce HIV-Seek, a novel approach to characterize the pre-activated transcriptome of latently infected resting memory CD4+ T cells. This characterization will reveal more information on an infected cell in its latent state and may then enable the identification of a biomarker unique to latently infected cells. In turn, a successful biomarker candidate could lead to immunological or pharmacological control of the viral reservoir. HIV-Seek utilizes a variety of technologies including microfluidics and single-cell transcriptomics to isolate and analyze the pre-activated transcriptome of latently infected cells. In this thesis, we establish bead-based molecular techniques that demonstrate the use of oligo-dT-conjugated beads to facilitate reactions involved in HIV-Seek.Item Scyllo-inositol And Other Brain Metabolites In Relation To Regional Brain Volumes, Cognition, And Inflammatory Markers In HIV(University of Hawaii at Manoa, 2021) Lee, Awapuhi; Kallianpur Tata, Kalpana J.; Biomed Science (Tropical Medicine)HIV-associated neurocognitive disorders (HAND) are a manifestation of neuropsychological (NP) impairment. Brain atrophy that exceeds normal age-related volumetric loss has been observed in HIV patients with NP impairment, and cerebral metabolites have been associated with reduced regional brain volumes and cognitive impairment. Low levels of scyllo-inositol (sI) were recently found to be related to NP impairment in a cohort of HIV patients with mild-to-moderate cognitive impairment. sI has not been extensively studied in the HIV population, so its role in HIV infection and cognitive impairment is unknown. To elucidate the potential roles of sI and other cerebral metabolites in cognitive impairment, we examined associations between metabolites, regional brain volumes, NP performance, and inflammatory markers in HIV+ individuals on combination antiretroviral therapy (cART) who had mild-to-moderate cognitive impairment. We also examined changes in metabolites among HIV patients before and after initiation of Cenicriviroc (CVC) or Maraviroc (MVC).Magnetic resonance imaging (MRI) was used to obtain T1-weighted scans, which were processed using FreeSurfer (version 6.0) to obtain regional brain volumes and total intracranial volume (ICV) of HIV+ participants (N=30) from the CVC and Maraviroc MVC studies from the Hawaiʻi Center for AIDS. Single-voxel magnetic resonance spectroscopy (MRS) and magnetic resonance spectroscopy imaging (MRSI) at 3T was used to measure brain metabolites. Participants underwent NP tests in multiple domains. Immune and inflammatory markers were measured by multi-parametric flow cytometry. Multivariable regression models, Pearson and Spearman correlations, Wilcoxon signed-ranked tests, and Mann-Whitney tests were performed using SPSS (versions 26 and 27). There were many significant associations and correlations between cerebral metabolites, regional brain volumes, NP domains, and inflammatory markers. Choline concentrations increased among patients treated with CVC. HIV patients in the MVC treatment group vs. the placebo group had higher mean differences in GABA and choline. Our study identified multiple associations between cerebral metabolites and regional brain volumes, including sI being positively associated with the putamen, cerebellar cortex and white matter, and the brain stem. This metabolite should be studied further as it could play a role in improving brain health and neurocognitive performance among HIV+ individuals.