Human Immunodeficiency Virus (HIV) infection initiates major metabolic and cell- survival complications. Anti-retroviral therapy (ART) is the current approach to suppress active HIV replication to a level of undetected viral load, but it is not a curative approach. Newer and sophisticated gene editing technologies could indeed be a potent antiviral therapy to achieve a clinical sterilization/cure of HIV infection. Chronic HIV patients, even under a successful ART regimen, exhibit a low-grade inflammation, immune senescence, premature aging, telomeric DNA attrition, T cell apoptosis, and cellular homeostasis. In this dissertation, we investigated CD4 T cell homeostasis, degree of T cell apoptosis, an associated telomeric DNA damage, DNA damage repair signaling, and the apoptotic pathways in CD4 T cells during HIV infection with or without ART treatment. Our data support a DNA damage accumulation, and impaired DNA damage repair in chromosome ends via recruitment of 53BP1 protein to the damaged foci. We found that a key player of DNA damage and repair enzyme, ATM, and its associated checkpoint proteins (CHK1, CKH2) are affected by HIV infection. HIV infection also altered another multifunctional master regulator protein AKT that is crucial in maintaining cellular homeostasis. Curing HIV is the ultimate redemption against HIV-associated complications. To explore the possibility of a functional cure, we investigated the use of a transient and a non-viral CRISPR/Cas9-based gene editing technology targeting the latently incorporated HIV provirus. After performing a nucleofection/electroporation using an in vitro formulated ribonucleoprotein (RNP) constituting a synthetic guide RNA (gRNA) and Cas9 nuclease protein, we demonstrated a significant (maximum 97%) reduction of HIV-mRNA and p24-capsid protein expression, upon stimulation (using PMA) and latency reactivation of latently HIV-infected CD4 T cells and latent-monocytes. Notably, the RNP treatment did not induce any cytotoxic effects, without affecting the abilility of cell proliferation. A sequence specific cleavage of HIV-provirus in two crucial gene locations (targeting vpr/tat genes) showed the most significant suppression of HIV reactivation or latency reversal. We have used DNA sequencing, and T7EI assay to confirm the target-site-specific cleavage of the HIV-proviral genome. Our data confirm the activation of non- homologous end joining (NHEJ) pathway to repair the double-stranded DNA break created by the CRISPR/Cas9 treatment. Taken together, this study provides a new gene therapeutic approach using synthetic gRNA/Cas9 targeting HIV genome, which warrant further in vivo animal and human studies.

Preterm birth is the number one cause of death in neonates, accounting for 35% of neonatal mortality. The preterm birth rate in the U.S. in 2021 was 10.5%, disproportionally affected by race and ethnicity. Obese women have an increased risk of preterm pregnancy, and if delivered before 37 weeks of gestation, the offspring have higher rates of complications that extend from the neonatal period into life-long metabolic and immune adverse outcomes. In the early months after delivery, preterm infants have higher rates of adverse health outcomes than term infants, including infections, extrauterine growth restrictions, respiratory, metabolic, and neurological complications, necrotizing enterocolitis (NEC), and bronchopulmonary dysplasia (BPD). Diet for the preterm infant is crucial for infection prevention, and maternal breast milk is most beneficial when given in the first few days after birth. Expectant and breastfeeding mothers should consume appropriate food and supplements to optimize their nutrition. In addition to nutrients, bioactive components, vitamins, and minerals found in breast milk (BM), there is evidence that microbes (microbiome) are a significant factor in infant development, contributing to protection against pathogens and playing a role in the development of the immune and nervous systems. Maternal BM composition and microbiome are affected by many factors, including maternal body mass index (BMI), maternal health, antibiotics use, mode of delivery, maternal parity, gestational age, and time and duration of lactation. Maternal body composition, however, and not maternal nutritional status, is associated with breast milk nutritional composition. Altogether, these maternal factors may modify the premature infant gut microbiome. We examined the role of maternal adiposity and how it impacts the composition of human breast milk, specifically hormones, nutrient composition, short and long chain fatty acids, and microbiome. We also examined the role of maternal adiposity and how it impacts the short-chain fatty acids and microbiome in infant fecal samples. We found that maternal adiposity affects breast milk hormones, potentially modulating infant metabolism. Additionally, we found that maternal adiposity does not alter the nutrient composition of breast milk; however, differences in both short and long chain fatty acids and maternal adiposity were detected. In our small cross-sectional cohort of preterm infants, we did not observe differences in short-chain fatty acids in the preterm infant stool samples compared to maternal adiposity. Concerning maternal adiposity and its impact on the microbiomes of breast milk and infants, we observed differences in phyla and genera between the maternal BMI groups on the outcome of breast milk and preterm infant microbiomes but no statistical significance in alpha and beta diversities between the groups. Thus, our results indicate that maternal adiposity impacts hormonal, microbial composition, and short-chain fatty acid profiles in breast milk, which tremendously influences infant growth and development.

Polyamines, specifically putrescine, spermidine, and spermine, are small cationic molecules found in all organisms. Cells can biosynthetically make these molecules, or alternatively, they can be transported from the extracellular environment. Malignant cells have been shown to require relatively high amounts of polyamines. There is a chemotherapeutic agent, DFMO, used to block the biosynthesis of polyamines. Many malignant cells can circumvent DFMO therapy by activating their transport system. A potential solution is to simultaneously block biosynthesis and transport of polyamines. However, little is known about the polyamine transport system in higher eukaryotes. This thesis aims to add to the basic biological understanding of the polyamine transport system, as well as contribute to our understanding of the way in which malignant cells are able to sustain rapid growth. This was done by screening six candidate genes believed to be involved in the polyamine transport system. These six genes were identified using various bioinformatics databases. They were screened using RNAi to knock down each gene of interest and by using an assay developed in our lab. One of the genes, RabX6, may play a possible role in the transport of putrescine.

<p>In atherosclerotic plaques, macrophages ingest modified LDL and turn to foam cells. Others have shown that SR-BI expression levels inversely correlated with cellular cholesterol levels, and is independent of well characterized cholesterol sensing pathways; SREBP and LXR. Thus the mechanism of regulation of SR-BI is unclear. In this study, we showed that treating macrophage with agents known to increase cellular cholesterol levels, namely acLDL, LDL, MβCD:Cholesterol, resulted in reduction in HMGCoAR mRNA and SR-BI expression levels. In contrast, acLDL did not reduce SR-BI mRNA levels in macrophages from acLDL SR-A KO mice, demonstrating that acLDL mediate suppression of SR-BI was dependent on SR-A. Fucoidan, a known competitive inhibitor of acLDL binding to SR-A, and subsequent degradation, also suppressed SR-BI expression levels. Unlike acLDL, however, fucoidan induced mRNA levels corresponding to the pro-inflammatory genes iNOS and IL-6 mRNA levels, and its effects were not altered by the lack of SR-A. Instead, fucoidan mediated stimulation of iNOS and IL-6 and suppression of SR-BI mRNA levels was prevented by an anti-CD14 blocking antibody, demonstrating that the fucoidan mediated effects were dependent on CD14. Interleukin-15, a pro-inflammatory cytokine that binds to a distinct receptor, also induced iNOS and IL-6 mRNA levels and reduced SR-BI expression, suggesting that inflammatory signaling in general can reduce SR-BI expression levels. Treatment of macrophages with the lipoproteins acLDL, LDL or HDL suppressed the induction of iNOS and IL-6 mRNA by fucoidan or IL-15. Macrophages foam cells in an atherosclerotic plaque may have reduced SR-BI due to exposure with modified LDL or inflammatory cytokines or both in an atherosclerotic plaque. SR-BI expression in macrophages protects against atherosclerosis development. Our data suggests that modified lipoproteins as well as inflammatory stimuli suppress SR-BI expression in macrophages and this may contribute to their pro-apoptotic properties.</p> / Master of Science (MSc)

<p><strong>Introduction</strong></p> <p>Asthma is a chronic respiratory disease that is becoming more prevalent. Airway hyperresponsivness, a key feature of asthma, involves increased narrowing of the airways in response to bronchoconstricting agents. Airway smooth muscle (ASM) functioning is largely responsible for hyperresponsiveness yet the mechanisms behind excitation-contraction coupling are not fully understood. Src tyrosine kinase contributes to contraction in other smooth muscle types. Furthermore, STIM1, Orai1, IPLA₂b and RyRs play a role in ASM excitation-contraction coupling.</p> <p><strong>Aim</strong></p> <p>We sought to determine whether Src activity is involved in serotonin (5-HT)- and acetylcholine (ACh)-induced ASM contraction. We also examined whether the gene expression of molecules involved in sarcoplasmic reticulum emptying and refilling is altered during airway hyperresponsiveness.</p> <p><strong>Methods</strong></p> <p>Bovine tracheal ASM strips were pre-treated with the non-specific tyrosine kinase inhibitor genistein (10^-4M), src kinase family inhibitors PP1 (10^-5M) and PP2 (10^-5M) or vehicle and challenged with either 5-HT or ACh to determine the involvment of Src in contraction. Western blotting was used to examine Src activity following 5-HT or ACh treatment. Female BALB/c mice were exposed to an intranasal injection of [1.7mg/ml] HDM extract or saline. Real time, reverse-transcriptase polymerase chain reaction was used to examine gene expression.</p> <p><strong> </strong></p> <p><strong>Results</strong></p> <p>Genistein, PP1 and PP2 significantly reduced 5-HT-induced ASM contractions and Src activity was significantly increased in response to 5-HT. ACh-induced contractions were significantly reduced by genistein, but not PP1 and PP2. However, Src activity was significantly increased by ACh. RyR3 mRNA expression was significantly increased, Orai1 was significantly decreased, and STIM1, IPLA₂b, RyR1 and RyR2 were unchanged by the house dust mite treatment.</p> <p><strong>Conclusion</strong></p> <p>These data suggets 5-HT-induced ASM contraction involves Src activity. However, ACh-induced ASM contractions might not require Src. The changes in RyR3 and Orai1 expression might alter Ca²⁺-handling in such a way as to potentiate airway hyperresponsiveness but further investigation is required.</p> / Master of Science (MSc)

<p>Streptomycetes biology and genetics encompasses a variety of interesting features including multicellular growth, rich secondary metabolite production, and extensive environmental sensory and response systems. The characteristically large genomes of streptomycetes makes studying the diverse external stimuli intricate and internal regulation of these gene systems a challenge. Currently, there does not exist an efficient, cost-effective method of high throughput gene expression analysis in streptomycetes. Luciferase reporters have been used successfully in <em>Streptomyces coelicolor</em> to measure select promoter activity, however, they have demonstrated limited success in other strains and are not favourable to gene expression studies on a larger scale. Here, I present pLHR, a novel luciferase-based reporter tool designed specifically for high throughput gene expression studies in streptomycetes as well as the preliminary results which support the further development of this tool for gene expression profiling in <em>S. coelicolor</em>. Once developed, pLHR may be used to generate libraries of <em>Streptomyces</em> reporter stains to measure promoter activity repeatedly under variable conditions for the duration of the organism’s complex lifecycle.</p> / Master of Science (MSc)

<p>Parkinson’s disease (PD) is a complex neurodegenerative movement disorder involving protein misfolding, mitochondrial dysfunction, and oxidative stress. The current dissertation, motivated by a lack of valid biomarkers and sustainable therapies, examined the potential application of a novel target for therapeutics and diagnostics of PD — the multifunctional, heat-shock like protein Catecholamine-Regulated Protein 40 (CRP40). The goal of this program of research was to elucidate further the implications of CRP40 in PD using a variety of molecular biology, bioinformatics, and clinical approaches through integrative collaborations with academia, government, and industry partners to translate scientific findings into real world solutions. Chapters 2 and 3 explored the potential therapeutic use and structure-function relationships of CRP40 through elucidating the smallest functional piece of this protein that was six times smaller, and validating a negative control for these experiments (Heat-Shock Protein 47). These initiatives could eventually lead to a small drug that could cross the blood-brain barrier and be targeted to the specific brain regions affected in PD. Chapter 4 examined the potential mechanisms of CRP40, and suggested that this protein may protect neurons from oxidative stress, maintain energy levels, and mitochondrial homeostasis, with important future implications for a variety of disorders. Finally, Chapter 5 presented compelling evidence for the potential use of CRP40 as a valid biomarker for early detection of PD and monitoring of disease progression. Overall, findings suggest that CRP40 may be a critical target for future breakthroughs in the diagnosis and treatment of PD.</p> / Doctor of Science (PhD)

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading frame which encodes a putative serine hydroxymethyltransferase (SHMT). The revelation of this shared open reading frame resulted in the proposal that this putative SHMT catalyzes an aldol-type condensation reaction utilizing glycine and uridine-5’-aldehyde, resulting in the GlyU core. The enzyme LipK involved in A-90289 biosynthesis was used as a model to functionally assign this putative SHMT to reveal its functions as an l-threonine: uridine-5’-aldehyde transaldolases. Biochemical analysis indicates enzymatic activity is dependent upon pyridoxal-5’-phosphate, is non-reactive with alternative amino acids, and produces acetaldehyde as a co-product. Structural characterization of the enzymatic product is consistent with (5’S,6’S)-GlyU indicating that this enzyme orchestrates a C-C bond breaking and formation resulting in two new stereocenters to make a new l-a-amino acid. The same activity was demonstrated for the LipK homologues involved in the biosynthesis of muraminomicin, A-503083, and A-102395. This l-threonine: uridine-5’-aldehyde transaldolase was used with alternative aldehyde substrates to prepare unusual l-a-amino acids, suggesting the potential for exploiting this enzyme to make new compounds.

First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway. First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway.

Huntington’s disease is an incurable, progressive neurological disorder characterized by loss of motor control, psychiatric dysfunction, and eventual dystonia leading to death. Despite the fact that this disorder is caused by a mutation in one single gene, there is no cure. The mutant Huntingtin (mHtt) protein is expressed ubiquitously throughout the brain but frank cell death is limited to the striatum. Recent work has suggested that Rhes, Ras homolog enriched in striatum, which is selectively expressed in the striatum, may play a role in Huntington’s disease neuropathology. In vitro studies have shown Rhes to be an E3 ligase for the post-translational modification protein SUMO. Rhes increases binding of SUMO to mHtt which competes for the same binding site as Ubiquitin. SUMOylation of mHtt leads to disaggregation and cellular death, whereas ubiquitination leads to aggregation and cellular protection. In a previous study we showed that deletion of Rhes caused a decrease in the Huntington’s disease phenotype in mice. We hypothesized that mice lacking Rhes would also show increased aggregation in the striatum and this increased aggregation would correlate in a rescue of behavioral symptoms. Despite the prior in vitro and in vivo evidence, deletion of Rhes in vivo did not alter the aggregation of mHtt in the striatum of mice however deletion of Rhes still showed a rescue from the diseased phenotype. This result would indicate that deletion of Rhes alters the neurobehavioral phenotype of Huntington’s disease through a different pathway than promoting aggregation in striatal cells.