Biomedical Sciences Theses

Inflammatory bowel disease (IBD) is a chronic inflammatory condition which can affect all parts of the gastrointestinal tract and increase the risk of developing colitis-associated cancers. Researchers have attempted to create therapies that can alleviate their symptoms and potentially reduce the severity of the disease. Currently, anti-TNF-alpha is a biologics therapy proven to be effective within IBD-afflicted patients, yet its drawbacks include its loss of efficacy and high likelihood for side effects. Some researchers have investigated anti-inflammatory natural products as alternative drug candidates to treat IBD. Ginger is a great resource as it exhibits strong anti-inflammatory effects, mainly due to its active components including 6-shogaol. In addition, the in vivo metabolite of 6-shogaol, M13, has demonstrated higher anti-inflammatory properties but is more hydrophilic than 6-shogaol. In this study, we investigated the potential efficacy of M13 analogs to see if they exhibit more favorable properties than M13. After testing the drugs’ in vitro efficiency, we found several more potent drug candidates (effective in reducing cancer cell growth). Further wound-healing tests of these analogs are still ongoing, but for now, we can conclude some of the M13 analogs show promising effects as treatment against IBD and colitis-associated cancer.

Vaccine development has been a cornerstone of modern medicine and the use of adjuvants in vaccine development has been a phenomenon present since the early 1900’s. Nanoparticles offer unique advantages as adjuvants due to their adjustable properties and their ability to serve as antigen carriers. In this study, we investigate the influence of polyethyleneimine-modified graphene oxide nanoparticles (GO-PEI) on the activation of the inflammasome, a crucial component of the innate immune system, in macrophage cells. Narrowing our focus on testing the successful activation of the inflammasome and its mechanism of activation, we found that GP nanoparticles could induce the activation of the inflammasome in vitro and its mechanism of action involved cathepsin B release and generation of reactive oxygen species as downstream secondary activating signals. This finding is significant and illustrates GP nanoparticles' usefulness in the development of more effective vaccines due to their unique properties.

Influenza remains a major global health issue, demanding the need for more effective vaccine strategies. This thesis searches into a novel approach, employing cell-derived extracellular vesicles (EVs) to boost influenza vaccine effectiveness. It dives into the impact of EVs on the innate immune system and their role on a cellular basis. Using cell culture and in vitro immune assays, the composition and immunomodulatory traits of EVs from antigen-presenting cells are researched. The study systematically examines the interactions between these EVs and key components of the innate immune system, unveiling the mechanisms driving their immunostimulatory effects. This research yields crucial insights into EVs' potential as immunomodulators in influenza vaccines. Additionally, determining insights into the identification and validation of adjuvants that enhance EV-mediated immune responses marks a pivotal stride toward the advancement of novel influenza vaccines, increasing their efficacy and protection against diverse viral strains.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak originating in Wuhan, China in late 2019, declared a pandemic in March 2020 by the World Health Organization, and led to widespread hospitalizations and deaths, prompting a global shutdown. As time progressed, variants emerged, and a solution was needed to prevent the spread. Vaccines and therapeutics were developed, but current vaccines are not able to neutralize all variants and do not have the capability to induce effective mucosal immunity. This study investigates two proteins targeting the receptor-binding domain of the wild-type strain and Omicron variant that are glycosylated and tagged with the Fc portion of human IgG. Three vaccine groups are studied and administered intranasally to induce local and systemic immunity. The findings show that both protein vaccines and their prime-boost vaccination induce high-level titers of specific antibodies with a balanced IgG1/IgG2 level, which could have the potential to be good vaccine candidates.

Background: Aortic aneurysms and dissections (AD) are common in aging populations, and their development is tightly associated with vascular inflammation. Aortic dissection and rupture are associated with high mortality. Currently, there is no effective drug treatment for AD progression and rupture except surgical treatment. Although ketone bodies have an anti-inflammation effect, it remains unclear whether ketone restrains AD and the risk of rupture.

Methods: C57BL/6J mouse underwent β-aminopropionitrile (BAPN) treatment with drinking water for 28 days, followed by angiotensin II (Ang II) mini-pump implantation for 3 days to induce AD. Ketone-ester (KE) was administered in drinking water starting at 17days after initiation of BAPN treatment. There were four groups: Sham, BAPN+Ang II, BAPN+Ang II+KE 20 g/L, and BAPN+Ang II+KE 50 g/L. The blood ketone level was monitored by a Precision Xtra meter. Ultrasound was employed to measure the aorta strain.

Results: KE treatment (both 20 g/L and 50 g/L) reduced the incidence of both thoracic aortic dissection and abdominal aortic dissection. KE at 50 g/L increased the survival rate of BAPN plus AngII-treated mice. KE treatment did not reverse the Ang II-induced aortic stiffness demonstrated by reduced aorta strain. BAPN and KE did not affect mice weights, but Ang II slightly reduced the mouse body weight. BAPN and KE treatment had no effect on water consumption. These results suggest that ketone appears to decrease aortic dissection and risk of rupture and provides a potential therapeutic strategy for aortic dissection.

Background: Atherosclerosis is a chronic inflammatory condition that affects the arteries and is distinguished by the formation of plaques. This can lead to constricted blood flow and conceivably lethal cardiovascular events. The disease progressed over the years and is impacted by various risk factors, including high blood pressure, diabetes, smoking, obesity, and high cholesterol. Atherosclerosis contributes to a quarter of the world's deaths globally. Therefore, in this article, we put together an impartial, systematic, and organized bioinformatics system to understand the biomarkers and possible regulatory targets involved in treating Atherosclerosis and plaque development to understand better the disease's pathogenesis, which can help reduce the global risk of cardiovascular disease.

Material/Methods: Microarray datasets GSE43292 and GSE28829 were obtained from the Gene Expression Omnibus database (GEO). Limma analysis was done in R version 4.2.2 using the Bioconductor package version 3.16 by dividing GSE28829 into two groups, advanced and early plaque, to find the DEGS, whereas GSE43292 was divided into two groups, namely advanced plaque, and microscopically intact tissue to find the DEGS. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of these modules were performed by Enrichr. Differentially expressed genes (DEGs) were mapped into calcification-related genes using the STRING database; two PPI networks were generated. These PPI genes were used to extract. Transcription factors (Tf) from CHea3 and MiRNA from MIRwalk were sorted about Atherosclerosis to do the FFL analysis. Further experimental studies were done to verify the key miRNAs, i.e., that showed a strong association with the gene and transcription factor (TF).

Results: The genes were found in the DEGS list in GSE28829 and GSE43292. The PPI genes were identified as. GO and KEGG enrichment analysis revealed that genes in GSE28829 were enriched. At the same time, the genes in GSE43292 were enriched.

Conclusions: Overall, the DEG analysis of GSE28829 and GSE43292 provides insights into the molecular mechanisms underlying the progression of Atherosclerosis from early to advanced stages. The upregulation of genes involved in an extracellular matrix organization and adhesion and the downregulation of genes involved in immune and inflammatory responses suggest that changes in the plaque microenvironment may contribute to disease progression. These findings have important implications for the development of new therapeutic strategies for the treatment of Atherosclerosis

Diets rich in fiber are known to have significant health benefits, but patients with inflammatory bowel disease (IBD) have reported an increase in disease symptoms while on such diets. Thus, IBD patients are recommended to eat low fiber foods and are unable to gain health benefits from fiber. While it has been shown that genetic factors can play a role in the occurrence of IBD, environmental factors are thought to further induce IBD, specifically diet and its effect on the gut microbiota. The self-reporting from IBD patients has been supported with studies in which the fiber inulin added to a low fiber diet has caused increased severity of colitis; conversely, a diet of a standard mouse chow containing naturally occurring dietary fiber has shown resistance to the same colitis murine models. Similarly, metabolic syndrome is a cluster of health conditions related to inflammation and diet, with studies finding dietary fiber capable of reducing metabolic syndrome symptoms. From these findings, wheat and oat fibers have been analyzed to determine if they provide protection against colitis and diet-induced obesity in murine models. After performing the in vivo models, wheat fiber provided protective benefits against IBD and metabolic syndrome. With these results, patients with IBD or metabolic syndrome will be able to consume a dietary fiber that will significantly reduce their disease severity or potentially eliminate manifestations of IBD and reverse metabolic syndrome conditions.

Recent studies have demonstrated that the Valosin-containing protein (VCP), an ATPase – associated protein, plays a protective role in the heart against cardiomyopathies and injuries caused by various cardiac stresses, including ischemia and pressure overload. However, the underlying molecular mechanisms remain largely unknown. It has been shown that VCP presents in stressed hearts. Our hypothesis is that VCP plays an essential role in cardiomyocyte growth and survival through a dual regulatory effect on the signaling of the mechanistic target of rapamycin (mTOR). Thus, we used a functional gain and loss strategy to determine the critical role of VCP in regulating mTOR signaling in cardiomyocytes in vitro. By using recombined adeno-virus system (Ad-CMV-GFP-VCP for overexpression and Ad-U6- siRNA-VCP for knockdown), we successfully overexpressed VCP and knocked down VCP in H9C2 cells respectively. This study aimed to better understand how VCP overexpression and knockdown affected certain proteins involved in the mTOR pathway – in particular phosphorylated Akt (pAkt T308 and pAkt S473), Raptor, Rictor and mTOR. Western blots were also done to confirm VCP knockdown and overexpression with GaPDH as the loading control. This preliminary study, although did not yield substantial results, can be designed as an in vitro system to better understand VCP and mTOR interactions.

Introduction: Pathogenic transformation of vascular smooth muscle cells (VSMCs) into osteochondrogenic phenotypes is a key feature of advanced atherosclerotic cardiovascular disease and is driven by transcription factors, like SOX9 and RUNX2. Here, we explore the previously uncharacterized role of SMYD2, a histone lysine methyltransferase, in VSMC phenotypic switching during atherosclerotic plaque formation.

Methods: C3H/10T1/2 (10T1/2) cells, wildtype (WT) and Smyd2-depleted (Smyd2KO), were cultured and treated with 5 mmol/L inorganic phosphate (Pi) for 24 or 72 hours, followed by Western blot (WB) analysis. For RUNX2 inhibition, 10T1/2 cells were treated with CADD522 and 5 mmol/L Pi for 8, 24, 48, or 72 hours, with protein expression analyzed by WB. Co-immunoprecipitation (Co-IP) was performed on WT or Smyd2 KO 10T1/2 cells. Immunoprecipitation was conducted using an antibody against RUNX2, followed by immunoblotting with a SUMO-2/3 antibody to assess RUNX2 SUMOylation. For SUMO inhibition, 10T1/2 cells were treated with ginkgolic acid and 5 mmol/L Pi for 24 hours, with protein expression analyzed by WB.

Results: Western blotting revealed lower RUNX2 protein levels in Smyd2KO 10T1/2 cells. Co-IP demonstrated increased SUMOylation of RUNX2 in Smyd2KO cells compared to wild-type cells. RUNX2 inhibition with CADD522 reduced SMYD2, while SUMOylation inhibition with ginkgolic acid had no effect on SMYD2 and RUNX2 expression.

Conclusion: Smyd2 promotes atherosclerotic plaque formation by regulating SUMOylation of essential transcription factors involved in VSMC osteochondrogenic phenotypic switching. Targeting SMC-specific Smyd2 and SUMOylation pathways may offer a novel therapeutic strategy in preventing vascular calcification in atherosclerosis.

Autoimmune diseases are diverse disorders arising from abnormal cellular and humoral responses to self-antigens in the immune system. However, the underlying cause of autoimmunity remains unclear. In this study, ready-made autoreactive CD4⁺ T cell hybridomas (containing autoreactive CD4⁺ lymphocytes from scurfy TCR^mini mice with BWα⁻β⁻ thymoma line) were cloned and their antigen receptors (TCRs) were characterized to decipher the specificity of autoreactive TCRs. This approach was used to understand the mechanisms of autoreactivity in the immune system and assist in redirecting abnormal CD4⁺ T cells present in the peripheral repertoire of healthy individuals for therapeutic advantage. Some hybridoma clones expressed CD4⁺ and Vα2⁺ TCRs with unique amino acid sequences and they upregulated Nur77^GFP expression upon autologous DCs stimulation and a stronger expression of Nur77^GFP in the presence of aCD3 stimulation. Our data suggests that these activated hybridoma clones TCRs have an elevated binding affinity for self-peptides/MHCII complex and are autoreactive.

Background: Over the last decade, the prevalence, morbidity, and mortality of cardiovascular diseases (CVDs) have increased worldwide. Ferroptosis being an iron-dependent cell death has been recently recognized to be triggered by lipid peroxides buildup and is increasingly linked to the onset and progression of CVDs. Vascular smooth muscle cells (VSMCs), which are critical for the proper functioning of the vasculature are particularly susceptible to ferroptosis damage. Recent studies suggest that tryptophan metabolites may modulate ferroptosis in cancer cells, but their effects on VSMCs remain completely unexplored. Therefore, this study seeks to determine the effect of 3-hydroxyanthranilic acid (3-HAA), a key metabolite of the kynurenine pathway, on ferroptosis in VSMCs.

Methods: Ferroptosis was induced in primary human VSMCs using ferroptosis inducers, erastin and Ras- selective lethal 3 (RSL3). 3HAA, or other tryptophan metabolites, 3-hydroxykynurenine (3-HK), and Kynurenine (L-KYN) was administered to assess its effect. The C11-BODIPY581/591 dye was used to assess lipid peroxidation in the cultured VSMCs. Western blotting and reverse transcription polymerase chain reaction (RT-qPCR) were performed to evaluate the protein and gene expression levels of ferroptosis markers, including transferrin receptor 1 (TFR1), SLC7A11, glutathione peroxidase 4 (GPX4). The involvement of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway in mediating protection was also assessed.

Results: RSL3 treatment in VSMCs resulted in significant lipid peroxidation and morphological changes, such as cellular deformation and cell shrinkage. Fluorescence analysis using the BODIPY581/591 C11 dye revealed a marked increase in fluorescence intensity, indicating elevated lipid peroxidation in ferroptosis-induced cells. Treatment with 3-HAA rescued the cells from ferroptosis by upregulating GPX4 and SLC7A11 expression levels at both transcriptional and protein levels, confirming its protective role. In contrast, neither 3-HK nor L-KYN mitigated RSL3-induced ferroptosis. The protective effect of 3-HAA may be linked to the activation of the NRF2 antioxidant pathway, while 3-HK and L-KYN had no significant effect on this pathway.

Conclusion: This study demonstrates that 3-HAA protects VSMCs from ferroptosis by reducing lipid peroxidation, which may be associated with NRF2 pathway activation. The findings suggest that 3-HAA shows promise as a potential therapeutic agent for mitigating ferroptosis-related vascular damage in CVDs, such as atherosclerosis and aortic aneurysm.

Neisseria gonorrhoeae, the etiological agent of gonorrhea, remains a significant global health threat, exacerbated by rising antibiotic resistance. For its survival, N. gonorrhoeae utilizes the TonB-dependent transporters to acquire metals from the host. Zinc acquisition is essential for the bacterium's survival, with the TonB-dependent transporter TdfH playing a pivotal role by binding to human calprotectin to extract and acquire zinc thereby circumventing host-imposed nutritional immunity. This study aimed to map the epitopes of TdfH recognized by monoclonal antibodies (mAbs) using an overlapping peptide-ELISA approach. Four mAbs—5H-1C2, 4H-3B11, 5H-1E10, and 5H-6B12—were used to probe synthesized overlapping TdfH peptides, identifying distinct immunodominant regions. mAbs 5H-1C2 and 4H-3B11, belonging to the IgG1 subclass, recognized overlapping epitopes on peptides 61, 62, and 63 with common amino acid sequence NLAPQYDITP. While mAbs 5H-1E10 and 5H-6B12 (IgG2a) both recognized peptide 178 with the amino acid sequence CNDDNTLCNGKYGGT. Notably, peptides 176 and 177 were missing due to peptide synthesis challenges. The conserved nature of these epitopes among disseminated and urogenital strains of N. gonorrhoea underscores these region as a viable target for immune-based interventions, with implications for both therapeutic monoclonal antibody development and vaccine design. This work advances the understanding of N. gonorrhoeae immune evasion strategies and provides a framework for developing novel therapeutic interventions, particularly in the context of rising antibiotic resistance.

Lipid nanoparticles (LNPs) represent a significant advancement in mRNA delivery, especially regarding the recent Coronavirus Disease 2019 (COVID-19) pandemic. The use of LNPs in COVID-19 mRNA vaccines allows for the particles to have a larger carrying capacity, meaning an easier and safer delivery of nucleic acids with little to no cytotoxicity. While most LNPs target hepatocytes, their specific formulation allows for a more tunable delivery system, as well as increased stability, distribution, and bioavailability. This study investigates various lipid combinations for the purpose of mRNA-LNPs that show high protein expression in vitro and are cost-effective. These formulations were tested in two different human cell lines to assess the expression of mRNA-encoded luciferase protein. The findings indicate that an optimized lipid formulation utilizing a cationic lipid does potentiate strong protein expression in both cell lines tested, meaning that this formulation may have potential to be optimized even further for vaccination use

Achimota Virus 2 (AchPV2) is a newly identified bat-derived pathogen belonging to the Pararubulavirus genus of the Paramyxoviridae family. The Pararubulavirus genus includes many emerging zoonotic pathogens such as Sosuga virus, Tioman virus, and Menangle virus. Achimota virus 2 was first isolated from urban bats, Eidolon helvum, in Ghana, West Africa. AchPV2 is capable of infecting a wide range of cell lines and is considered to be a potential zoonotic threat. Supporting this, previous serological evidence has suggested AchPV2 spillover events have already occurred. In this study, I have developed a novel reverse genetics system to generate recombinant AchPV2. The full-length viral genome was reconstituted in a T7 driven rescue system, along with all of the viral proteins necessary for viral replication. I have performed initial characterization of viral tropism, replication kinetics in immortalized cell lines, and sensitivity to known antivirals. The utilization of this system will provide us with an innovative platform to study these emerging zoonotic viruses and assist in the discovery of therapeutic measures to limit and control any future outbreaks.

The main driver of cardiovascular disease risk is atherogenic low density lipoprotein (LDL) particles in addition to other risk factors. Patients with elevated risk are prescribed lipid modifying drugs like statins and fibrates for preventing potential atherosclerotic plaque accumulation. In conjunction with a CDC study investigating association between atherosclerosis risk and lipoprotein levels, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and optimized to screen for the presence or absence of statins or fibrates in the plasma samples collected from study participants. The comprehensive panel of statins, fibrates, and their metabolites were identified with a high sensitivity, 4-minute-long method, using a low plasma volume of 10 µL. The presence and identification of the medications were compared to medical records. The comparison revealed many discrepancies that had significant impact on scientific conclusions concerning correlations between LDL levels and coronary artery disease (CAD) risk score determined by cardiac catheterization.

Introduction: Oxidative stress induced vascular remodeling promotes development and progression of atherosclerosis. By scavenging excessive Lipid ROS, GPX4 reduces atherosclerotic plaque severity and VSMC sensitivity, whereas BRD4 promotes plaque progression by facilitating lipid uptake associated with atherosclerosis. This study aimed to explore smooth muscle cell (SMC)-specific role of SMYD2 in modulating oxidative stress in vitro through regulation of GPX4/BRD4

Methods: To evaluate the effect of ferroptosis on SMYD2 and BRD4, C3H/10T1/2 (10T1/2 cells) mouse embryonic fibroblasts were treated with Ferrostatin-1 (Potent and selective inhibitor of ferroptosis), and 1S,3R-RSL3 (Ferroptosis inducer), PAz-PC (oxLDL), in dose and time dependent manner. Next, to evaluate if SMYD2’s mediated ferroptosis is dependent on BRD4, control and Smyd2-depelted 10T1/2 cells were treated with Ferrostatin-1/1S,3R-RSL3 and (+)-JQ1. Lipid hydroperoxides, and Glutathione assay along with Immunofluorescence C11-BODIPY581/591 staining was used to measure oxidative stress in the cellular in cultured cells. Primary VSMCs isolated from control and SMYD2 depleted mice and cultured C3H/10T1/2 cells were used for in vitro studies.

Results: Smyd2 depletion in C3H/10T1/2 and primary cells protected against oxidative stress by regulating GPX4 and BRD4 axis. Pharmacological inhibition (Ferrostatin-1, 2µM, 24hr) or activation (3R-RSL3, 0.8nM, 8hr) of GPX4 activity either by resulted in a significant upregulation and suppression of SMYD2 respectively. OxLDL (PAz-PC, 15ug, 48r) treatment resulted in elevated GPX4 expression in SMYD2-depleted cells compared to control, suggesting that SMYD2 deficiency mitigating ferroptosis by upregulating GPX4. Moreover, inhibiting BRD4 with (+)-JQ1 treatment in C3H/10T1/2 cells, regardless of SMYD2 presence, led to an increase in ferroptosis. Immunofluorescence and C11-BODIPY581/591 staining of these cells revealed higher GPX4 expression and lower BRD4 expression in SMYD2-depleted cells compared to controls. MDA assay results from both cells and culture media corroborated these findings. qPCR analysis and the re-examination of our RNA-seq data demonstrated that either depletion of SMYD2, or (+)-JQ1 treatment significantly downregulated the expression of ferroptosis-associated genes, such as Hmox1, Acsl3, Nox2, GPX4, and Slc40a1. This suggests that SMYD2 may govern lipid peroxidation by modulating the expression of ferroptosis-associated genes regulated by BRD4.

Conclusion: SMYD2 depletion protects against the severity of oxidative stress by regulating the GPX4/BRD4 axis, suggesting its therapeutic potential against lipid peroxidation and cardiometabolic disease.