Biology Theses

Zika virus (ZIKV) was declared as “Public Health Emergency of International Concern” in 2016. Differentiating ZIKV recent infections (< 12 weeks post symptom onset) from past infections (> 12 weeks post symptom onset) is a significant challenge of serological test, which is widely used to detect ZIKV infections. We developed ZIKV NS1 IgM and IgG capture ELISAs and applied four indicators: IgM values, capture IgM/IgG ratios, IgG avidity, combined IgG avidity and capture IgM/IgG ratios, to differentiate ZIKV recent and past infection for a cohort of serum collected in ZIKV epidemic regions of Bogota, Colombia. The sensitivity for differentiating ZIKV recent and past infections using each of the four indicators is: 54.78%, 54.88%, 40.68%, and 31.25%, respectively. The specificity for differentiating ZIKV recent and past infection using each of the four indicators is: 62.50%, 84.21%, 58.33%, and 47.37%, respectively.

Approximately 83% of the world’s population is seropositive for human cytomegalovirus (HCMV) and will endure lifelong latency. An immunocompromising disease such as HIV/AIDS increases susceptibility to reactivation and HCMV diseases. If an AIDS patient’s CD4+ T cell count falls below 50 cells/μL of peripheral blood, HCMV is reactivated which causes a sight-threatening retinitis. The disease affects up to 42% of AIDS patients and causes retinal necrosis. To further investigate the mechanisms of AIDS-related HCMV retinitis we studied RIPK3 stimulation in HCMV-infected ARPE-19 cells under oxidative stress, through pursuit of two experimental hypotheses: (1) HCMV-infected ARPE-19 cells will upregulate RIPK3 in the presence of oxidative stress induced by H2O2 and (2) HCMV-infected MRC-5 cells will not express RIPK3 stimulation in the presence of oxidative stress induced by H2O2. Our data suggests that RIPK3 stimulation presents a cell-specific dose-dependent response to H2O2 concentrations in ARPE-19 cells during HCMV infection.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels form an integral part of the plasma membrane, existing as tetramers and enclosing ion conducting pores. Ion channels are often regulated by auxiliary subunits and one such protein interacting with the HCN channel is TPR-containing Rab8b interacting protein (TRIP8b). TRIP8b is known to play a crucial role in controlling voltage dependence of HCN channel activation, channel trafficking and surface expression. Small Ubiquitin-like Modifier (SUMO) is a peptide that can be conjugated to a lysine residue on a target protein and modify protein- protein interactions. HCN2 channel can be SUMOylated and possible SUMOylation sites on TRIP8b exist. SUMOylation at these sites may strengthen or weaken the association between these proteins. In this study we use Pull-down assay technique to determine whether SUMO influences this interaction. By altering the baseline SUMOylation level, changes in the degree of HCN channel protein pulled down by TRIP8b is analyzed.

Heavy metal contamination is of growing concern as industrialization causes increasing levels to be released into the environment affecting all organisms. Cellular damage and coping mechanisms due to heavy metal toxicity vary depending upon the species of metal and organisms exposed. It is assumed that cellular damage can be attributed to the destructive effects of metal-induced ROS production, which is ameliorated, by the non-protein tripeptide glutathione, cycling between reduced (GSH) and oxidized (GSSG). We investigate the effects of acute cadmium and copper treatment on Saccharomyces cerevisiae and determine some of the immediate cellular responses to the exposure of these metals and demonstrated ameliorative effects of free form amino acid precursors (FAAPP). FFAAPs reduced cellular oxidative stress and served to offset the immediate oxidative stress that the presence of both metals caused within the cell within the first hour of exposure. FFAPs effected cellular responses differentially for each metal.

GABA is the prominent inhibitory neurotransmitter in the brain and defects in the GABA system are attributable to several genetic diseases including Rett Syndrome (RTT). People with RTT show characteristic breathing disorders, suggesting a link with defects in the GABA system. Noradrenergic neurons in the locus coeruleus (LC) are CO₂ chemosensitive and play a role in breathing regulation. We conducted experiments to find out if activation of a novel group of dorsomedial LC GABAergic neurons causes instantaneous firing rate changes in LC neurons. Spike train recordings with cross-correlation analysis suggests that the dmLC neurons made monosynaptic and oligosynaptic connections with the LC neurons. The GABAergic inhibition appeared to have major effects on medullary respiratory neurons, as our results showed that THIP, the extrasynaptic GABA receptor agonist, suppressed excitations in these cells. The results from these experiments provide new evidence for the GABAergic inhibition of brainstem neurons involved in breathing control.

With the rise in diagnoses of social deficit disorders comes an increased demand in elucidating the neural mechanisms that underlie social behavior. In the central nervous system arginine-vasopressin (AVP) has been shown to effect social communication, such as aggression, pair bonding, and maternal behavior, and many AVP cell bodies and fibers are distributed in a sexually dimorphic fashion. One such area is the bed nucleus of the stria terminalis (BNST), with males having more AVP cells than females, thus making it a likely candidate in the control of male specific social behavior. We found that activation of this specific cell population using optogenetics in socially-naïve males does not induce a place preference, affect male territorial aggression, or investigation towards females.

L- Asparaginase is capable of hydrolyzing L-asparagine into L-aspartic acid and ammonia. This catalytic function is used for the treatment of children with acute lymphoblastic leukemia resulting in the depletion of L- asparagine in the leukemia cells. Bacterial asparaginase used in current leukemia therapies has side effects and short serum half-life. This study presents the purification and characterization of nitrile hydratase (NHase) with asparaginase (ASNase) and glutaminase (GLNase) from Rhodococcus rhodochrous DAP 96253and 96622.The influence of pH, lysis buffer composition, dialysis process, anion exchange chromatography’s flow rates and pH were incorporated into the approach to purify NHase exhibiting ASNase activity.A NHase purified at pH 7.6, using 50 mM phosphate buffer, 5mM 2-mercaptoethanol as a lysis buffer, then dialyzed, followed by anion exchange chromatography and subsequent size exclusion chromatography showed a higher ASNase and GLNase activity and lower NHase activity. This enzyme shows potential as leukemia treatment.

Bi-parental care and pair bonding often coincide in nature. The reproductive success of the organisms that apply this strategy is dependent upon defensive behaviors and territorial aggression. Some of these organisms also display affiliative behavior within the pair pond during the time of parental care. The behavioral dynamics that occur over the course of the pair bond and their relationship to the reproductive success of the organism is not well understood. Convict cichlids (Amatitlania siquia) form pair bonds during the breeding season and provide bi-parental care; their behavioral repertoire is ideal for studying pair bonding. The androgen profile of organisms that provide parental care through aggressive means is also not fully understood. We provide a context based comparative view of the role of behavior and androgens in the pair bonds of these organisms. Observations were conducted in both the lab and the field; samples of 11-ketotestosterone (KT) were collected from the males. We found that there is an effect of fry stage on the behavior of the parent. Data suggests that there is some effect of fry stage on androgen levels in males.

Asparaginase derived from Eschericia coli or Erwinia crysanthum is used in the treatment of juvenile Acute Lymphoblastic Leukemia (ALL). ALL cells cannot make asparagine due to the lack of L-asparagine synthetase gene. Asparaginase is therefore used as a chemotherapeutic treatment for ALL through hydrolysis of the L-asparagine to aspartic acid, which leads to starvation of the ALL tumor cells, then apoptosis. In addition, the asparaginase enzyme is used in the food industry. Asparaginase can be used to reduce acrylamide in baked food formed during the Milliard reaction. In this study, in this study, Rhodococcus rhodochrous DAP 96253 asparaginase enzyme activity is measured at varying temperatures (27 °C and 37 °C) and substrate concentrations, using colorimetric reagents, to determine the enzyme reaction stability with respect to these variables. Asparaginase enzyme activity increases by increasing the temperature to a specific optimal point, after that it declines. This accelerates the hydrolysis of L-asparagine to L-aspartic acid. The goal is to determine the optimum temperature for the enzyme function.

Naturally occurring perinatal cell death is a major developmental process in the formation of the brain. Microglia, the primary immunocompetent cells of the brain, have recently been implicated in actively contributing to perinatal cell death. However, the current research is contradictory. Some studies suggest that microglia induce neuronal death, while others suggest that microglia protect neurons from intrinsic cell death programs. These studies utilize many methods of manipulating microglia, they examine single regions of interest, and analyze at different times in development. These differences in analysis may be responsible for the different conclusions. In this study, we selectively reduced microglia brain wide via clodronate liposomes and measured the amount of microglial reduction and the subsequent effects on cell death. We also analyzed regional differences in cytokine expression as a possible underlying mechanism for effects of microglia on cell death.

ABSTRACT Evidence from knockout studies in male mice and from experiments in male rats,in which expression of the estrogen receptor alpha (ERα) gene was inhibited in the medial preoptic area (MPO), suggests that ERα is important in the control of male rat mating behavior. Therefore, in this experiment, we tested the hypothesis that activation of ERα in the MPO is sufficient to maintain mating behavior in castrated male rats receiving subcutaneously (s.c.) dihydrotestosterone (DHT), a non-aromatizable androgen. Accordingly, castrated rats treated with DHT s.c. received MPO implants of either: (i) propyl-pyrazole-triol (PPT) (Stauffer, et al 2000; Katzenellenbogen, et al 2000), a selective ERα agonist, (ii) E2 (positive controls) or (iii) cholesterol (negative controls)and sexual behavior was monitored. PPT was as effective as E2 at maintaining mating behavior suggesting that, in the MPO, ERα is sufficient to mediate responses to E2 that underlie male rat mating behavior.

Naturally occurring perinatal cell death is a major developmental process in the formation of the brain. Microglia, the primary immunocompetent cells of the brain, have recently been implicated in actively contributing to perinatal cell death. However, the current research is contradictory. Some studies suggest that microglia induce neuronal death, while others suggest that microglia protect neurons from intrinsic cell death programs. These studies utilize many methods of manipulating microglia, they examine single regions of interest, and analyze at different times in development. These differences in analysis may be responsible for the different conclusions. In this study, we selectively reduced microglia brain wide via clodronate liposomes and measured the amount of microglial reduction and the subsequent effects on cell death. We also analyzed regional differences in cytokine expression as a possible underlying mechanism for effects of microglia on cell death.

Recurrence of distinct strains of seasonal influenza viruses requires the development of universal vaccines. Previous studies have demonstrated the cross-protection effects of influenza virus A-derived extracellular domain of ion channel matrix protein 2 (M2e) vaccines in an adult mouse model. However, M2e vaccine efficacy of wide-range protection against influenza remains largely unknown in aged mice. I hypothesized that co-immunization with M2e virus-like particle (M2e-VLP) vaccine and hemagglutinin-based VLP vaccine (HA-VLP) can provide superior protection against homologous virus and antigenically different strains of influenza viruses in aged (15-18 months old) BALB/c mice compared to the current HA-based vaccine. In this study, I examined a strategy of co-immunization of HA-VLP and M2e-VLP as a potential approach for effective vaccination for the immunosuppressed and elderly against a wide-range of influenza strains.

Sight threatening human cytomegalovirus (HCMV) retinitis still remains a cause for concern among AIDS patients who do not respond to or do not have access to current antiretroviral (ART) therapy [1]. However, little is currently known about the degenerative mechanisms behind HHCMV retinal destruction during retroviral immunosuppression. The well-established murine AIDS (MAIDS) related murine cytomegalovirus (MCMV) retinitis model closely mimics disease progression seen in AIDS patients [4]. Previous work using this model has shown that while the cell death pathway apoptosis is involved in disease progression, it is not fully responsible [1]. It has been found that the mRNA of molecules associated with two other cell death pathways, necroptosis and pyroptosis, are also correlated with the pathophysiology of MAIDS-related MCMV retinitis [1]. We propose that retinal degeneration seen during CMV retinitis involves necroptosis/pyroptosis programmed cell death correlating with the increased mRNA/protein expression of associated molecules observed in this study.

Cancer is the modern, most persistent serial killer. Cancer cells intelligently recruit and utilize all the biological functions to support their survival, proliferation, and invasion, which require altering and reprogramming a lot of the normal signals and pathways. Calcium (Ca²⁺) is a fundamental secondary messenger involved in most of the regular cellular functions, not only the survival processes but also, the apoptotic death. Cancer cells exhibit unique expression levels of T-type Ca²⁺ channels (TTCC) that vary according to the tumor type, stage, and the channel isoform. The aberrant expression of the TTCC family (Cav3.1, Cav3.2, and Cav3.3) genes in cancer cells significantly contribute in the remodeled Ca²⁺ signaling that mediates many cancer cell functions including mitochondrial energy production, proliferation, angiogenesis, metastasis, and sensitivity to cell death. Accordingly, the TTCC blockers were proposed for their anti-cancer effect. Mibefradil (MBF) (Posicor^®) is a TTCC blocker initially introduced into the pharmaceutical market in 1997 as an FDA-approved antihypertensive drug but voluntarily withdrawn due to drug-drug interactions. 10 years later, MBF was qualified for drug orphan designation by the FDA for the treatment of pancreatic, ovary, and brain cancers. Herein, we investigate the anti-cancer effect of MBF on different cell lines by determining its IC₅₀ and indicate MBF-induced endoplasmic reticulum Ca²⁺ release as a key for the anticancer effect of MBF. Moreover, we identify the effect of MBF on important cytoplasmic organelles such as mitochondria and lipid droplets (LDs). Further, we demonstrate that MBF reduces the number of the lipid droplets and affects the cellular lipid storage by inducing lipolysis in cancer cells.

The ability of cancer cells to synthesize and store lipids in the form of lipid droplets (LDs) gives them a proliferative advantage over normal cells. Here, we elucidate the effect of monoethanolamine (Etn) on the lipid metabolism of LD-rich prostate carcinoma (PC3) cells. Etn is a ubiquitous molecule that serves as a phospholipid precursor and has been shown to have anticancer activity, although its mechanism of action is not fully understood. PC3 cells were treated with Etn and evaluated with a combination of biochemical and cell biology techniques. Treatment with Etn caused a decrease in cytosolic LD abundance induced by Ca²⁺-stimulated lipolysis. Our study effectively implicates the disruption of lipid metabolism as a contributor to Etn-induced cell death. This finding opens new avenues for the development of LD-targeted anticancer therapy.

C. elegans provides a number of tools for understanding cellular networks and neural connections. We identified jd1500 in previous reports as a mutation that affects forward locomotion, which is unusual. Our aims were to: 1) identify the gene responsible for the phenotype that jd1500 exhibits and 2) distinguish the basis for the locomotive asymmetries. Using next-gen whole genome sequencing, we were able to identify specific genes that are likely responsible for the phenotype it shows. Our results suggest that gap junction mutations mask jd1500 activity, but also suggest that jd1500 masks acr-2 activity.

The purpose of this study is to explore the use of stress physiology biomarkers in wildlife rehabilitation. We examined collection, extraction and enzyme immunoassay protocols in order to identify the optimal methods for testing corticosterone levels in injured raptors at a wildlife rehabilitation center. Corticosterone levels were measured periodically during the raptors' rehabilitation using noninvasive techniques. This study focuses on three species: Red-Tailed Hawks (Buteo jamaicensis), Red-Shouldered Hawks (Buteo lineatus), and Barred Owls (Strix varia). Fecal samples were collected from patients from all three species from May 2017 to March 2018. This information will contribute to our understanding of the stress profile of raptors in rehabilitation, and will allow for future studies to employ the methods validated by this project.

In recent years, cancer treatments have progressed substantially. However, the tumor microenvironment is dynamic and continuously evolving thereby resulting in decreased efficacy to therapies, and resistant cancer cells. The development of drug resistance in cancer patients may potentially trigger or instigate disease progression in the presence of therapeutic interventions. In this study, we investigated effects of chronic Gefitinib treatments in a lung cancer cell line model. Gefitinib is a tyrosine kinase inhibitor targeting specific mutations within the epidermal growth factor receptor (EGFR). Lung cancer cells treated for extended periods of time with Gefitinib displayed resistant phenotypes, typically characterized by DNA damage, epithelial- mesenchymal transition (EMT) as well as heightened and sustained levels of reactive oxygen species (ROS). Furthermore, we uncovered novel cancer resistant phenotypes, namely dysregulated mitochondrial biogenesis, endoplasmic reticulum (ER) alterations, cellular calcium levels and propensity towards bypassing senescence.

Human papillomavirus (HPV)-negative (-ve) oropharyngeal squamous cell carcinomas (OPSCCs) are associated with poorer overall survival (OS) compared to HPV-positive (+ve) OPSCCs. The major obstacle to improving outcomes of HPV -ve patients is the absence of good biomarkers. Herein, we investigated the role of centrosome amplification (CA) as a prognostic marker in HPV –ve OPSCCs. We first quantitatively assessed CA in OPSCC tumor samples and found that HPV -ve OPSCCs exhibit higher CA compared to HPV +ve OPSCCs, and was associated with poor OS even after adjusting for potentially confounding variables. Further, the expression of genes associated with hypoxia and CA was significantly higher in HPV -ve OPSCCs than in HPV +ve OPSCCs. We further uncovered a mechanism by which hypoxia-induced HIF-1α downregulates miR-34a resulting in cyclin D1 overexpression and rampant CA in HPV -ve OPSCCs. Our findings demonstrate that assessment of CA may aid in therapeutic decision-making for these patients.