2018 Summer Undergraduate Research Participants
Student at Bellarmine University
Mentor:Juhi Bagaitkar, PhD
Project Title: P. gingivalis infection debilitates anti-viral immune responses in gingival epithelial cells
The oral mucosa and epithelial tissues are frequent sites of viral infections. While most viral infections are benign and self-resolving, chronic viral infections can lead to considerable morbidity and may enhance risk for oral epithelial cancers in susceptible individuals. Epidemiological and clinical studies correlate prevalence of chronic oral viral infections with periodontitis, a chronic inflammatory disease associated with profound inflammation elicited by a dysbiotic oral microbiome. However mechanistic data underpinning an association between periodontitis and susceptibility to viral infections are lacking. We investigated whether infection of gingival epithelial cells (GECs) with the periodontal pathogen and oral colonizer Porphyromonas gingivalis hindered subsequent immune responses to viral agonists. Interferons are pleiotropic cytokines that play an integral role in anti-viral defenses at the gastro-intestinal and respiratory mucosal surfaces. We found that resting GEC actively produce low levels of IFN-l - a cytokine recently described to play key role in anti-viral responses specifically at the barrier surfaces. Additionally, challenge of GECs with the synthetic viral agonist ORN (single stranded viral nucleic acid analogue) strongly activated Type I (IFN-b) and further augmented Type III (IFN-l) production in a dose dependent manner. While P. gingivalis infection alone did not elicit any IFN responses in GECs, pre-infection with P. gingivalis significantly debilitated host responses to the viral agonists. Reduced IFN responses in P. gingivalis infected GECs correlated with the down-regulation of interferon-stimulated genes such as IRF-7. Our studies demonstrate that periodontal pathogens may impair host anti-viral responses at the oral mucosal barrier by inhibiting interferon production and the expression of interferon stimulated genes.
Student at Eastern Kentucky University
Mentor: Douglas Borchman, Ph.D.
Project Title: The Role of Lipids in Vitreous Syneresis
Vitreous liquefaction causes retinal detachment. The cause of vitreous liquefaction is unknown. The vitreous contains mainly 3 components, water, hyaluronan and collagen. Our lab has found lipids in human vitreous. The KBRIN student measured lipid hyaluronan binding in vitro to determine if lipids found in the vitreous could cause liquefaction. The student used NMR spectroscopy to measure the binding. Seven different lipid moieties were tested.
Student at Kentucky Wesleyan University
Mentor: Nicholas Mellen, Ph.D
Project Title: Optimization of µ-opioid Receptor Immunohistochemistry to Evaluate Efficacy of 6-β-naltrexol in the Treatment of Neonatal Abstinence Syndrome.
This project’s objective is to evaluate the efficacy of 6-β-naltrexol (6βN), a metabolite of naltrexone, in the treatment of neonatal abstinence syndrome (NAS) by selectively blocking the fetal µ-opioid receptors (MORs). NAS describes postnatal symptoms of infants born to mothers addicted to opioids, and closely parallels opioid withdrawal symptoms in adults. Here the rat is used as the animal model. NAS is induced by maintaining pregnant rats on drinking water containing methadone (MTD), administered at a dose sufficient to induce dependence (3 mg/kg/day), based on normal water intake levels in pregnant rats (40 ml/day). We hypothesize that exposure to chronically elevated opioid levels will lead to down-regulation of MOR expression in the fetal CNS, to return neuronal excitability to its homeostatic set-point. Thus, efficacy of 6βN is hypothesized to result in normal MOR expression levels. MOR expression was characterized via immunohistochemistry. Two antibodies were tested: Abcam Anti-Mu Opioid Receptor antibody (monoclonal, rabbit) and ImmunoStar MOR antibody (polyclonal, rabbit). To detect the bound antibody, a fluorescent secondary antibody was used (Thermo Fisher goat anti-rabbit). Brain tissue were obtained from P3, P6, and P10 rat pups, stored in 4% paraformaldehyde for 12 h, sucrose-protected and cryo-sectioned at 40 μm. Sections were exposed to goat serum phosphate buffered saline (PBS) to block non-specific binding sites for the secondary antibody; washed in PBS, and then incubated overnight in primary antibody. Sections were then washed in PBS and incubated with secondary antibody for 2 h, then washed again and cover-slipped and examined under an upright epifluorescence microsocope (MODEL). MOR labeling was optimized by varying primary and secondary antibody concentration, incubation time and volume. In addition, various permeabilization methods were tested, including acetone, 0.1% Triton 100X solution, and 0.5% Tween 20 solution. Images fore quantification were obtained using a confocal microscope (MODEL HERE), and image stacks were subsequently analyzed using in-house machine vision segmenting software. At the time of writing results are in the process of being collated and analyzed.
Student at Western Kentucky University College
Mentor: Rifaat El-Mallakh, Ph.D.
Project Title: Glutamate-Induced Apoptosis on Olfactory Neuroepithelial Progenitors Derived from Bipolar Subjects and Non-bipolar Subjects
Post-mortem studies have linked bipolar disorder (BD) with increased brain matter loss due to higher levels of cellular apoptosis. Apoptosis may be related to glutamatergic cytoxicity and studies suggest that BD subjects have higher levels of glutamate. Higher levels of glutamate increases cytoxicity and activates the cellular apoptotic process. Drugs that normalize glutamate levels may slow the apoptotic process and lead to improved outcomes for BD subjects. Previous work suggests that lithium may prevent glutamate induced excitotoxicity in olfactory neuroepithelial progenitor cells (ONPs) cultured from BD subjects.
The aim of the current study is to examine if ONPs from BD subjects are more sensitive to glutamate-induced apoptosis and to quantify the rate of ONPs undergoing glutamate-induced apoptosis within a cell population.
Student at Elizabethtown Community and Technical College
Mentor: Sham Kakar, Ph.D.
Project Title: Can securin regulate the epithelial mesenchymal transition (EMT) process?
Cancer stem cells are minor population in solid cancer. These cells are responsible for cancer initiation, drug resistance and recurrence of cancer. Currently used therapeutics target cancer cells but not the cancer stem cells that undergo amplification and result in recurrence of cancer. Epithelial mesenchymal transition (EMT) is an important intermediate step for the transformation of normal cells/stem cells to CSCs. There exist a relationship between EMT process and securin and oncogene which is highly expressed in most of tumors analyzed to date. In our present studies, we explored the importance of securin in regulation of EMT process in ovarian cancer cell line A2780 by knocking out securin using securin-specific siRNA. Gene expression of various genes involved in EMT process were anylyzed by real-time PCR. Our results showed that down regulation of securin resulted in down regulation of expression of TGFβ, Snail, Slug, and Zeb1, suggesting that securin may regulate the EMT process and hence CSCs population.
Student at Eastern Kentucky University
Mentor: Michal Hetman, PhD
Project Title: Effects of spinal cord injury on circadian rhythms.
Sleep, activity, food intake and metabolism of nutrients are example of processes that are regulated in a circadian fashion i.e. they are turned on in oscillations that have a period of about 24 h. Such oscillations are driven by time cues that come from the environment including light, food intake or activity. In addition, all cells are endowed with an intrinsic mechanism of counting time that relies on oscillatory activity of the heterodimeric transcription factor CLOCK/BMAL1. That intrinsic mechanism that regulates gene expression is synchronized by the external time cues to produce a large spectrum of co-ordinated rhythmic changes in body functions. The key cells for such co-ordination are neurons that are located in the suprachiasmatic nucleus (SCN) of the hypothalamus and receive direct innervation from the retina. Those neurons regulate rhythms in the brain and other organs by promoting oscillatory changes in brain activity (sleep/wake), hormone secretion as well as activity of the autonomous nervous system that innervates peripheral organs. Disruption of the circadian rhythms either by environmental changes such as jet lag or intrinsic genetic defects in the CLOCK/BMAL1 pathway has a wide spectrum of negative effects on health. Circadian rhythm perturbations increase risk of insomnia, metabolic syndrome, diabetes, obesity, cardiovascular disease, immune deficits and systemic inflammation. Such co-morbidities also occur more frequently in patients who suffered traumatic spinal cord injury (SCI). Therefore, one could wonder whether disruption of circadian rhythmicity occurs after SCI and contributes to its systemic complications. While anecdotal clinical reports suggested disruption of various biological rhythms in SCI, no systematic animal studies were reported that addressed effects of SCI on circadian rhythms. We have recently conducted such a pioneering study using mice as model animals to determine circadian rhythm outcomes of moderate thoracic level spinal cord contusion. Sleep/wake cycle of those animals were recorded 24/7 over several weeks initially under 12:12 h dark:light cycle and then in constant darkness. In this summer project, a participating student will analyze data from those recordings to determine whether SCI affects diurnal patterns of sleep/activity. The student will become familiar with video digitalization, mouse sleep behavior and statistical approaches to analyze circadian rhythms. In addition, a student will be offered an opportunity to observe surgical procedures to model SCI in rodents and analyze locomotor outcomes of those injuries. The planned analyses will be instrumental in interpreting other data that were collected from that study including effects of SCI on activity of the CLOCK/BMAL1 genes in SCN, liver and muscles.
Student at Murray State University
Mentor: Michael Merchant, PhD
Project Title: Experimental and informatics approaches to study the role of protein modifications by urea in kidney disease.
Urea is a metabolic by-product of living cells and is eliminated through renal excretion. Urea is also a chemical denaturant that is routinely used in proteomic sample handling to improve sample proteolysis and to eliminate the presence of detergents through the filter-assisted sample preparation (FASP) protocol. Increases in blood urea have been correlated with loss of kidney function and with chemical derivatization of blood proteins. Sequence specific urea-based modifications (carbamylation) of human serum albumin have been shown to be predictive of response to growth factors and to be associated with increased risk of mortality. Differentiation of biologically-based and methodologically-based carbamylation would be important for kidney disease biomarker discovery studies.
We hypothesized the use of the urea analogue, thiourea, would enable use of the FASP protocol and enable identification of endogenous sequence specific carbamylation of proteins and peptides. Our immediategoals were to conduct in silico and in vitro experiments to address the effects of urea in the FASP protocol on proteomic studies. The objectives were to (A) use published lung and breast cancer studies incorporating the modified FASP protocols and use informatics to determine if urea had a meaningful and significant detrimental effect on the proteomic study results and (B) to conduct original studies on fetal bovine serum (FBS) using urea and thiourea to establish a method for mitigating the effects of experimentally introduced carbamylation.
In silico analysis of a published datasets for breast cancer and for lung cancer determined addition of the carbamylation modification increased numbers of identified proteins significantly (breast cancer study – 73 unique proteins; lung cancer study- normal tissue 148 and lung cancer tissue 1,269 new proteins). The results of the experimental in vitro data from FBS studies demonstrated sample handling dependent carbamylation modifications can be eliminated by substitution with thiourea.
Sample dependent introduction of amino group carbamylation by use of urea was significant, led to large numbers missed identifications, but was averted through use of alternative chemical denaturants. Our experimental protocol presents a viable, alternative work-around for the traditional FASP method of sample preparation. These results successfully addressed the study objectives. The long-term goals for these improvements would be application to human serum proteomic studies of patients with end-stage kidney disease and determining if carbamylation of specific components of the serum peptidome may function as surrogate markers of mortality.
Student at Carlton College
Mentor: Ann Morris, PhD
Topic: Effect of Her9 mRNA on the craniofacial and retinal defects in mutant embryos and larvae
Student at Murray State University
Student at Centre College
Mentor: Doug Harrison, PhD
Title: Genetic analysis of soma-germline communication during spermatid differentiation
Sperm are perhaps the most highly specialized and modified cells in the animal world. They are specifically designed to be efficient couriers of the male’s genetic information. Animal sperm vary in shapes and sizes to carry out this task. Nonetheless, the general process of sperm production is largely similar from insects to mammals. Construction of a sperm is accomplished with the assistance of other cells in the testis. While many studies have uncovered genes that are active in sperm during their development, little is known about the genes and functions of the support cells in the testis. In the fruit fly, Drosophila melanogaster, each 64-spermatid cluster develops as an interconnected cyst and is encapsulated by a pair of somatic support cells, called cyst cells. Recent work in the lab has uncovered that communication between these somatic cyst cells and the spermatids is necessary to direct the separation of spermatids within a cyst during the late stages of spermatogenesis. To better understand the roles of the somatic support cells, this project will identify factors required in cyst cells to complete sperm development. For this project, we will specifically impair the functions of particular candidate genes only in the support cells of the testis to examine the impact on sperm formation. Because the process of spermatogenesis is similar across species, this work will not only aid in understanding cellular collaboration in Drosophila spermatogenesis, but it will contribute to our understanding of spermatogenesis in general.
Student at Bellarmine University
Student at Centre College
Mentor: Vincent Cassone, PhD
Topic: Investigation of the gut microbiome in the European House Sparrow, Passer domesticus
The European House Sparrow, Passer domesticus, are commonly found in urban and suburban areas, and have adapted well to the presence of humans. Indeed, they are often found scavenging food from dumpsters at Taco Bell and McDonald's restaurants! As such, we believe that the gut microbiome of these birds may be more similar to that of humans than the current model, the domesticated lab mouse, Mus musculus. In both humans and mice, we know that the gut microbiome exhibits circadian fluctuations in both diversity and abundance of bacteria. We also know that disruptions to the circadian mechanism in both humans and mice (i.e., jet-lag or light-dark schedules simulating shift-work) also disrupt gastrointestinal function related to the microbiome. Using previously collected gut tissue, we will now attempt to characterize the gut microbiome in house sparrows across time of day, which has not been done before. In addition to measuring abundance and diversity indices, we will also investigate whether a specific member of the human gut microbiome, Enterobacter aerogenes, displays rhythmicity in the house sparrow gut. Our lab has previously shown that this bacterium can sustain its own circadian rhythm of motility and gene expression independent of its host. This bacterium is not found in the lab mouse, and so this investigation would provide a direct validation for studying the sparrow microbiome as a translational model for human gut microbe interactions.
Student at Berea College
Mentor: Jessica Santollo, PhD
Topic: Role of gonadal hormones in cognitive impairment due to hypovolemic dehydration.
Estrogen improves performance on cognitive tests and has protective effects on fluid balance. We previously demonstrated that object recognition memory following hypovolemic dehydration was impaired in diestrous females (a time of low ovarian hormone release), but not estrous female (a time of high ovarian hormone release) or male rats. This suggests that ovarian hormones in females protect against cognitive impairment in the face of dehydration. To directly test this hypothesis we will manipulate gonadal hormones in both male and female rats to determine if object recognition memory after hypovolemic dehydration is impaired in gonadectomized rats. Thirty two male and 32 female Sprague Dawley rats will undergo castration (n=16), sham castration (n=16), ovariectomy (n=16), or sham ovariectomy (n=16) surgery. After a two week recover period rats will undergo training and testing in the Novel Object Recognition paradigm. This will involve one 10 min session of habituation to the open field (Day 1), three 5 min training sessions with two identical objects (Days 2-4), and a test day (Day 5) consisting of treatment with 20 µg/kg furosemide (to induce hypovolemia) or 0.9% saline control and 3 h later a 5 min test session with one copy of the original object and a novel object. Activity will be recorded during the test day and time spent interacting with the novel and original object will be quantified along with general activity. We predict, based on our previous experiments, that the control and intact animals will spend more time investigating the novel object, however, the dehydrated gonadectomized rats will not be able to discriminate between the novel and original objects.
Student at Centre College
Mentor: Jeramiah Smith, PhD
Topic: Examining the Role of ANAPC11 and HAT1 in DNA elimination..
The sea lamprey (Petromyzon marinus) is a living representative of the agnathans, a jawless lineage of basal vertebrates that diverged from the jawed gnathostomes about 550 million years ago. Sea lamprey are one of the few organisms to undergo a physical reengineering of its genome during early embryonic development, a process termed programmed genome rearrangement (or PGR). PGR results in the elimination of 20% from the somatic cell genome, with this same 20% being retained only within germ cells. During anaphase of early embryonic mitoses, DNA targeted for elimination forms bridges of lagging chromatin between the spindle poles of the retained chromosomes and is subsequently packaged into discrete subcellular structures, or micronuclei, prior to being degraded and eliminated from the cell entirely. To further investigate the functions of histone acetylation and cell cycle-dependent regulatory elements in PGR, I will specifically test the hypothesis that two protein coding genes: ANAPC11 (Anaphase Promoting Complex Subunit 11) and HAT1 (Histone Acetyltransferase 1) participate in DNA elimination. To test this hypothesis I will disrupt the function of these genes via Cas9-mediated site-specific mutagenesis and observe the formation of features associated with PGR (micronuclei and lagging chromatin) relative to wildtype embryos.
Student at Murray State University
Mentor: Jeramiah Smith, PhD
Topic: Investigating the Possible Role of DMNT3b and MBD2 in DNA elimination in Sea Lamprey
The sea lamprey (Petromyzon marinus) is an anadromous jawless fish, native to the Atlantic ocean and an invasive species in the Great Lakes. Unlike most vertebrates, the sea lamprey undergoes physical reengineering of its genome during early embryonic development via a process termed programmed genome rearrangement (PGR). PGR results in the physical elimination of 20% of the genome from somatic progenitor cells, with this same 20% being solely retained within the genome of germ cells. During anaphase of early embryonic mitoses, DNA slated for elimination forms bridges of lagging chromatin between the spindle poles of the retained chromosomes and is subsequently packaged into discreet subcellular structures (micronuclei) prior to being degraded and eliminated from the cell entirely. Though PGR only occurs in a limited number of species, elucidating its mechanism may reveal clues to the mechanisms of gene silencing in more complex vertebrates, such as humans, which has major implications for cancer and other forms of aberrant gene expression. It is known that common silencing mechanisms, including DNA methylation, participate in PGR. To further resolve the functions of DNA methylation in PGR, I will specifically test the hypothesis that two critical methylation genes: DMNT3b [DNA (Cytosine-5-)-Methyltransferase 3] and MBD2 (Methyl-CpG Binding Domain Protein 2) participate in DNA elimination. To test this hypothesis I will disrupt the function of these genes via Cas9-mediated site-specific mutagenesis and observe the formation of features associated with PGR (lagging chromosomes and micronuclei) relative to wildtype embryos. If positive results are obtained, this study is expected to provide not only general evidence for the participation of these genes in PGR, but also specific evidence for the stage(s) of PGR in which they participate.