• SYNTHESIS OF PEPTIDES WITH FLUORESCENT TAGS

      Weather, Angel; Sweatman, Zachary; Bae, Junsoo; Lebedyeva, Iryna; Department of Chemistry and Physics; Augusta University (2018-02-21)
      Covalently linked acceptor-spacer-donor (A-S-D) multichromophoric systems have previously been discussed largely in mechanistic terms, with emphasis on the roles of electronic and nuclear functions in determining the factors of internal electron transfer. Electron energy transfer between donor (D) and acceptor (A) molecules has long been applied to measure or estimate distance in macromolecular systems, molecular stacking, intramolecular changes caused by ionic strength or target molecule binding, to define protein folding and aggregation and finally to give information about conformational changes in the molecule. Coumarins represent a family of dyes that often play the role of electron donor in electron-charged systems. During tailoring of new chromophore dyads, coumarin derivatives have been linked to fullerene, peptides, and dyes (benzimidazol benzopyrans). In particular, 7-methoxy-2-oxo-2H-chromene-3-carboxylic acid is a commonly used tag that provides strong fluorescence and acts as an electron-donor. In current work we are developing chiral systems of 10 Å and more in order to establish a structure map whenever the donor-acceptor fluorophore is conjugating with another system (e. g. peptides). Our team enhances the fluorescence of the two coumarin-based fluorophores by linking them to the N- and C-peptide moieties via 1,2,3-triazine linkers.
    • Guidelines for Healthy Food Production in an Urban Brownfield: Is Aquatic Vegetation Safe for Composting?

      Barrera, Bryuanna; Greene, Rhiley; Mondeddu, Sheena; Department of Biological Sciences; Clinical and Digital Health Sciences; Wear, Donna; Department of Biological Sciences; Augusta University (2018-02-12)
      Sibley Mill, located in the community of Harrisburgnear Augusta University, is a designated brownfield. The property was the site of theConfederate Powder Works and later that ofa cotton mill.Soil contaminantsinclude arsenic, barium, cadmium, chromium,lead and mercury.There are documented cases of children in this community with elevated blood concentrations of lead. Risks associated with lead poisoning are damage to the brain and nervous system, slowed growth and development, and behavioral problems.The benefits of urban agricultureare wellestablished, but currently there are no guidelines for safe methods of food production in brownfields. Aquatic vegetation is removed weekly from the Augusta Canal to enablehydroelectric powergenerationat Sibley Mill. We are using this vegetationto implementa novel approach for the production of compostfor raised-bed gardening.We measured the concentrations of 14 heavy metals, prior to composting, to establishbaseline data. Concentrations of barium(253.3-962.4 ppm)and lead(4.1-16.5ppm)exceed the guidelines recommended for drinking waterand are the two metals of greatest concern for the productionof safe, usablecompost.
    • Role of Perivascular Adipose Tissue in Vasoreactivity

      Prasad, Rosaria; Department of Biological Sciences; Kim, Ha Won; Department of Medicine; Augusta University (2018-02-12)
      Perivascular adipose tissue (PVAT) surrounds most systemic vessels directly around the lamina adventitia, which has an anti-atherosclerotic effect. However, inflamed and dysfunctional PVAT induced by high fat diet (HFD) is associated with various cardiovascular disease. The aim of this study was to investigate the systemic effect of PVAT on vascular functions in the setting of diet-induced obesity. 50 mg of PVAT or subcutaneous adipose tissue (SQAT, as a control) from obese donor mice (fed with HFD) was transplanted into the abdominal aorta in recipient mice. We found that PVAT transplantation group showed significantly higher insulin resistance than the SQ group (p=0.095) whereas no differences were observed in body weight, fat composition, and glucose tolerance between these groups. Interestingly, PVAT transplantation, but not SQAT or sham group, showed the impaired vasoconstriction in thoracic aorta, as examined by wire myography. Furthermore, PVAT transplanted group promoted endothelial dysfunction as evaluated by endothelium-dependent relaxation curve analysis.PVAT transplantation into the abdominal aorta is associated with endothelial dysfunction of the thoracic aorta. Dysfunction of PVAT induced by high-fat feeding may negatively affect metabolic and vasoreactivity in an endocrine manner.
    • Expression and Treatment of Pain-Related Depression of Fixed-Ratio and Progressive-Ratio Food-Maintained Behavior in Rats

      Baker, Frederick; Frazier, Eric; Marshall, Laura; Sinclair, Sequoia; Thakkar, Parth; Department of Psychological Sciences; Miller, Laurence; Department of Psychological Sciences; Augusta University (2018-02-12)
      Increasingly, preclinical studies on the expression, mechanisms, and treatment of pain have been aimed at improving understanding of pain-related interference with behavior. Positively reinforced operant behaviors are sensitive to depression by physiologically relevant pain stimuli. Most studies using operant conditioning procedures to examine pain-related depression of behavior have used fixed-ratio schedules of reinforcement. The primary dependent variable in these studies is the rate of behavior. In contrast, the primary dependent variable in studies using progressive-ratio schedules of reinforcement is breakpoint. Breakpoint is defined as the total number of reinforcers earned, and is thought to be related to the subject's motivation to obtain the reinforcer. This study examined effects of pain and analgesic manipulations on behavior maintained under fixed-ratio and progressive-ratio schedules of behavior. Intraperitoneal injection of dilute lactic acid was more potent at depressing behavior under the fixed-ratio schedule compared to the progressive-ratio schedule. Ketoprofen was equipotent at blocking pain-related depression of behavior maintained under both schedules. These findings support the validity of operant procedures astools to examine candidateanalgesics for the treatment of pain-related depression of behavior. Moreover, the use of diverse schedules of reinforcement may yield important scientific information on the mechanisms underlying pain-related interference with behavior.
    • The Significance of the Study of Evolution: Development and Implementation of an Interactive Course Module: Phase I

      Wilson, Cynthia Lynn; Sanyal, Nilabhra; Wise, Alisha; Department of Biological Sciences; Hull College of Business; Mukhopadhyay, Soma; Department of Biological Sciences; Augusta University (2018-02-12)
      The goal of this project is to create an interactive, one week coursemoduleto supplement teaching students about the connections between molecular evolution, macroevolution, microevolution and how they pertain tothehuman body and health. Thisinteractive course module is beingdeveloped using resources from the Internet that will allow the students to better understand the content. The main purpose of this course modulewill be to show that evolution is an evidence based science that affects public health and all fields of biology. Those who believe that evolution is antithetical to their beliefs, their concerns and the controversies that surround the study and teaching evolution will be addressed to ease any problemsthatthey may have. Surveys will be given at the beginning and end of the course to gauge the students' current and learned knowledge of evolutionand to get feedback for further improvement. This pedagogical research will be used to show that evolution is based on empirical evidence and is necessary to learn as it serves as the foundation of phylogenetic studies in biology. This knowledge can be applied to better understand individual human health and then to the wider field ofpublic health.
    • Structural Affinity of CAP1 and AC isoforms

      Mehrotra, Simran; Department of Biological Sciences; Sabbatini, Maria Eugenia; Department of Biological Sciences; Augusta University (2018-02-12)
      The major cause of death of pancreatic cancer is metastases. For that reason, it is of interestto study the mechanism through which the pancreaticcancercells migrate as itcould help with future medicine and prolong survivalrate. Adenylyl cyclase-associated protein 1 (CAP1)is involved in the regulation of actin microfilament formation, which ultimately regulatescell migration and invasion.CAP1 binds to G-actin,inhibiting polymerization. In previousresults,we found CAP1 interactswith a number ofadenylyl cyclase (AC) isoforms: AC1, AC3, AC4 and AC7. The goal for this project wasto study therelative affinityof CAP1 for each AC isoform. Using sequential co-immunoprecipitation, we found that AC1 is the isoform that interacts more firmly with CAP1 in HPAC cells. Further studies will be done usingthe homology modeling.
    • Expression of Bcl-2 in MCF-7 cells treated with PFOA

      Glenn, Manderrious; Glenn, Manderrious; Gaw, Tori; Cannon, Jennifer; Department of Biological Sciences; Cannon, Jennifer; Department of Biological Sciences; Augusta University (2018-02-12)
      Perfluorooctanoic acid (PFOA), also known as C8, is a man-made chemical that has the ability to repel oil and water. For that reason, it is used to manufacture a number of consumer goods like cookware and clothing. PFOA is an endocrine disruptor as it interferes with normal hormonal processes and proposes a health concern in high concentrations due to its high stability and its persistence in the environment and in our bodies. Previous research in our lab has shown that MCF-7 breast cancer cells treated with 50?M and100?M PFOA for 48h show a 25% decrease in cell viability. A significant decrease in estrogen receptor alpha (ER?) mRNA and protein and the reduction of peroxisome-proliferator-activated receptor gamma (PPAR ?) mRNA levels are thought to be associated with apoptosis in these cells. Experiments using the Caspase-Glo® 3/7 Assay (Promega) were used to determine that MCF-7 decreased viability in response to PFOA is in fact due to apoptosis. It is hypothesized that this PFOA-induced apoptosis may be caused by decreased levels of anti-apoptotic factors such as Bcl-2. We are using the Qiagen® RT2Profiler PCR Apoptosis Array and western blotting to determine the levels of Bcl-2 in control and PFOA-treated MCF-7 cells.
    • Production of a NF-¿B Deficient Microglial Animal Model

      Goodall, Michael; Soni, Karan; Department of Biological Sciences; Bradford, Jennifer; Department of Biological Sciences; Augusta University (2018-02-12)
      Our goal is to determine how the nuclear factor-kappaB (NF-?B) signaling pathway is used in the communication between microglia and the progression of glioblastoma (GBM) cancer cells. The NF-?B signaling pathway is very important in normal immune system function and has been implicated in various types of cancers, including, GBM. GBM is the most common type of adult brain cancer, has altered NF-?B signaling, and is also characterized by a large population of microglia, the immune cell of the central nervous system. Based on our recent studies, we hypothesize that deleting the major transcription factor (p65) of the canonical NF-?B pathway in microglia would slow the progression of GBM. To test this hypothesis, we have developed a p65fl/fl/CX3CR1CreERtransgenic animal, which should lack microglial p65 after exposure to tamoxifen. We currently have heterozygous animals and will soon begin characterizing them to determine p65 deletion efficiency.
    • Isolation and Culture of Microglia

      Doughty, Deanna; Venugopal, Natasha; Department of Biological Sciences; Bradford, Jennifer; Department of Biological Sciences; Augusta University (2018-02-12)
      Glioblastoma (GBM) is the most aggressive and common adult brain tumor subtype, with the majority of patients surviving less than one year. The GBM microenvironment is composed of tumor cells as well as non-cancerous cells, such as microglia, a component of the immune system in the brain. To better understand the role of microglia in GBM, we have optimized in vitroculture conditions for primary microglia. Growing microglia in culture is challenging, but this technique is needed for planned future experiments. Microglia were isolated from mouse neuronal tissue by magnetic bead antibody cell separation using the cellular marker CX3CR1. Isolated microglia were then cultured in various culture conditions, and cellular morphology by light microscopy was used to determine cell health, viability, and activation status. It was determined that the primary microglia grow best in neurobasal media in wells coated with poly-D lysine. Future studies aim to isolate a larger number of cells to allow forco-culture of the inactivated microglia with GBM cells. These results will allow us to better understand the role that microglia play in GBM progression.
    • Patient Education of Acid Reducing Medication and Duration of Use

      Lewis, Allison; Autry, Prentiss; Spell, Dan; Rao, Amy; Hampton, Sarah; Department of Biological Sciences; Sein, Michael; Department of Family Medicine; Augusta University; University of Georgia; et al. (2018-02-12)
      Gastroesophageal reflux disease (GERD) is a physical condition in which acid from the stomach flows into the esophagus. One of the most common symptoms of GERD is heartburn. Morethan 60 million Americans experience heartburn at least once a month, and some studies suggest that more than 15 million Americans experience heartburn symptoms every day1. With Proton Pump Inhibitors (PPIs) being one of the most commonly prescribed medications in the United States2, it is important to understand how patients are using these medications. Limited literature exists concerning a correlation between patient knowledge and duration of heartburn medication usage. This research identifies the demographic characteristics associated with the improper usage of acid reducing medications, specifically in the Southwest Georgia population. The participants of this study are representative of that area, with a majority of population residing in the medically underserved, low-income region of Albany, Georgia3. This information aims to help healthcare providers identify patients that are at a higher risk to misuse heartburn medication and be able to address these concerns.
    • Energy Transfer Phenomenon with Nanoluciferase as Energy Donor

      Bailey, Joseph; Department of Chemistry and Physics; Spencer, Angela; Department of Chemistry and Physics; Augusta University (2018-02-12)
      Bioluminescence is a phenomenon that occurs where light is emitted from a chemical reaction that occurs in an organism.An enzyme called a luciferase assists in the catalysis of a reaction that releases the energy of broken bonds as light at a certain wavelength. Bioluminescence Resonance Energy Transfer (BRET) is a technique that utilizes a luciferase as an energy donor, whichemits energy from catalysis of a substrate. Fluorescence by Unbound Excitation from Luminescence (FUEL) deviates fromBRET slightly. FUEL involves an energy donor exciting an energy accepting particle; however, the acceptor and donor are unattached to each other. The luciferase being used as an energy donor is Nanoluciferase (Nluc)that emits light at 454 nm. Nluc beenmodified to expressa His-6 tag. The energy acceptors being used are Alexa 488, 555, and 647 and reemit light at the corresponding wavelength. Each of the Alexa dyes has been modified to include an anti-His antibody, causing attachment between Nluc and the respective Alexa dye.Energy transfer in the form of BRET did occur between Nluc and the Alexa 488 and minimally with Alexa 555. No energy transfer was noted with Alexa 647. Energy transfer was quantified using a fluorometer.
    • Role of Aging in The Expression of Pain-related Depression of Nesting in Mice

      McPherson, Sarah; Patton, Tadd; Hunter, Lance; Department of Psychological Sciences; Miller, Laurence; Department of Psychological Sciences; Augusta University (2018-02-12)
      Pain stimulates some behaviors (e.g. flinching, vocalization), and depresses others (e.g. locomotor activity, social interactions). Pain-related depression of behavior is a key diagnostic criteria and treatment target in clinical settings, but preclinical research has primarily focused on pain-related stimulation of behavior. The present study aims to improve understanding of the impact of aging on pain-related depression of behavior by examining pain-related depression of nesting behavior in male ICR mice. The mice are placed in a cage containing nesting material, and the rate of consolidation of that material is determinedwith a schedule of data collection intervals. The impact of pain stimuli and analgesic drugs on nesting behavior are then determined. Previous studies have shown that physiologically-relevant pain stimuli depress nesting behavior, and clinically-relevant analgesics block pain-related depression of nesting. The present study will examine the role of aging as a determinant of the expression of pain-related depression of behavior by comparing pain-related depression of nesting by three age groups.
    • High glucose treated cells may lead to cellular senescence effecting function of bladder

      Vincent, Julie; Klee, Nicole; Webb, Clinton; Department of Physiology; College of Education; Klee, Nicole; Department of Physiology; Webb, Clinton; Augusta University (2018-02-12)
      Introduction: Diabetic bladder dysfunctioneffects 30-50% of all diabetespatientsand is characterized by symptoms of overactive and underactive bladder, which greatly effects quality of life.Diabetes is correlated with increased cellular senescence. Senescence is a physiologic phenomenon; however, chronic high levels can lead to tissue dysfunction. Multiple in vitrostudies have shown that high glucose exposure results in an increase in cellular senescent cells.The smooth muscle layer of the bladderis responsible for contraction and relaxation of the bladder; therefore, we hypothesize that primary bladder smooth muscle cells exposed to a high glucose environment will result in an increased number of cellular senescent cells.Methods:Rat primary BSMcells were incubated in normal glucose (4mM), high glucose (22mM), high mannitol (22mM), and bleomycin(+ control). Abeta-galactoside assay was utilized to visualize the presence of senescent cells.Results: Cells treated with high glucose exhibited increased cellular senescent cellscompared to both normal and high mannitol control. Conclusion: We conclude that high glucose exposure increases cellular senescence in primary bladder smooth muscle cells. An increased amount of cellular senescence within the smooth muscle layer of the bladder could contribute to bladder dysfunction as seen with diabetes.
    • EDUCATION AND FITNESS BENEFITS COGNITIVE PERFORMANCE IN AN OLDER AGE

      Dojack, Amanda; Schulte, Megahn; Meyers, Amos; Curry-McCoy, Tiana; Department of Kinesiology and Health Science; Department of Radiology; Holland, Angelia; Department of Kinesiology and Health Science; Augusta University (2018-02-12)
      Cognitive function and cardiovascular health often decline with age. Purpose: The relationship between cognitive performance and cardiovascular health in older versus younger men and women was examined. Methods: This cross-sectional study included 13 younger(18-35years old)and 10 older (55-75 years old) individuals. Participants visited the lab fasted and the following occurred in order: informed consent and questionnaires filled out, blood pressure and resting heart rate recorded, triglyceride and cholesterol measured via a fingerprick, anthropometric measures recorded, cognitive performance assessed via tests from the Automated Neuropsychological Assessment Metrics, and a modified YMCA 3-minute step test assessed recovery heart rate. Results: No differences between male and female between six different cognitive tests. The older group demonstrated significantly greater scores on five of the six cognitive tests (P<0.01-0.05) and had a higher education level (P<0.001). The younger group had lower systolic (P<0.01) and diastolic (P<0.05) blood pressure while the older group demonstrated a lower resting heart rate (P<0.05). Females demonstrated a greater recovery heart rate (P<0.01) and total cholesterol (P<0.05) than males. There were no differences in age groups for BMI, fitness level, or glucose, triglyceride, and cholesterol levels. Conclusion: Higher education and fitness may negate age-related cognitive declines.
    • LOCALIZATION AND FUNCTION OF AN EXTRACELLULAR MATRIX PROTEIN, TINAGL1, IN RENAL EPITHELIAL CELLS

      Kalra, Aarushi; Department of Cellular Biology and Anatomy; LeMosy, Ellen; Department of Cellular Biology and Anatomy; Augusta University (2018-02-12)
      Theextracellular matrix (ECM)playsimportant rolesin cell adhesion and communication. We arestudying the localization and function of a small ECMprotein, tubulo-interstitial nephritis antigen-like protein 1 (Tinagl1),in mammalian cell lines. Previous studies conducted in zebrafish suggest that Tinagl1is imperativefor function of motile cilia; however, it is still uncertainas to how this presumed basement membrane protein could regulate cilia, and whether thisfunction has relevance inmammalian cells havingonly primary, non-motile cilia.We will useimmunostaining ofTinagl1 in mouse renal collecting duct epithelial (IMCD3) cellsto determine if it is only on basal side or if it can also be detected on cilia on the apical side. We will establishthe knockdown of tinagl1expression using siRNA methodswith measurements of mRNA andprotein levels. Using appropriate assays, we will examine iftheknockdown of Tinagl1 causes theloss of primary cilia, changes in cell viability and programmed cell death, and/or the detachment of cell-cell or cell-matrix adhesions. Thisproject will showbasic functionsof Tinagl1 in renal epithelial cell behavior, which will eventually give us moreinformation about itsanticipated rolesin cell adhesion, cell survival, and primary cilia homeostasis.
    • Does the JNK/Jun Signaling Node Regulate Autophagy in Breast Cancer Cells?

      Joseph, Carol; Department of Cellular Biology and Anatomy; Schoenlein, Patricia V.; Department of Cellular Biology and Anatomy; Augusta University (2018-02-12)
      A common treatment for estrogen receptor positivebreast cancers is the use of selective estrogen receptor modulators such as Tamoxifen.1Unfortunately, 30-40% of patients experience relapse due tothe development ofantiestrogen resistance. Autophagy, a process that is typically seen in cells that are exposed to a variety of stresses, is critical to development of antiestrogen resistanceand may play a key role in metastatic progression.2,3 To further combat antiestrogen resistance, a potential target for breast cancers is JNK (c-Jun N-terminal kinase), a member of the mitogen-activated protein kinase (MAPK) family. The mechanismsby which JNK inhibition affects breast cancer cell growthare not fully characterized.Our hypothesis is that JNK is a key regulator of autophagy and the emergence of autophagy-dependent antiestrogen resistant breast cancer. Our aims are todetermine the effect of JNK inhibition on autophagy, cell number, and cell viability under conditions of antiestrogen treatment.By utilizingMCF-7breast cancercells andthe irreversible JNK-IN-8 inhibitor our current data provides strong evidence that JNK inhibition blocks autophagy. Data supporting a role for JNK in the regulation of antiestrogen-mediated autophagy have the potential to identify JNK as a molecular target for the improved treatment of breast cancer.
    • Disease Gene Discovery for Microcephaly in Consanguineous Pakistani Families

      Sivised, Vattika; College of Science and Mathematics; Kim, Hyung-Goo; Department of Obstetrics & Gynecology; Augusta University (2018-02-12)
      Microcephaly is a genetic condition where the brain does not develop normally resulting in a reduced head circumference, and the disorder can be non-syndromicor syndromic.There are two types of microcephaly: primaryand secondary. Microcephaly is passed down through family lines as an X-linked recessive autosomal dominant or recessive disorder.Microcephaly can be segregated in families through consanguineous marriages. These interfamilial marriages can lead to the child inheriting identical defective copies of genes from both parents, which results in an autosomal recessive disease.This paper will explore four Pakistani families, each practicing consanguineous marriages that have resulted in individuals displaying syndromic and non-syndromic microcephaly.A list of chromosomal regions obtained from genotyping methods allowed for the narrowing down of potential causative genes in each family. Using online search engines, including Endeavour and the Human Genome Browser, four lists of candidate genes were obtained, one for each family.For two families, a defect in the ASPMgene, a gene that is reported to cause primary microcephaly,has beendiscovered. Potential candidate genes, including SCN7AandPKIB, present in chromosomal regions found through homozygosity mapping of the remaining two families could be the cause of the phenotype and will be discussed.
    • Epigenetic modifications in rat pancreas following ethanol abuse.

      Liao, Kristie; Nancy Jhanji; Pruitt, Allison; Patton, Tadd; Hernandez, Caterina; Department of Biological Sciences; College of Nursing; Department of Psychological Sciences; Department of Pharmaceutical Sciences; Sabbatini, Maria Eugenia; et al. (2018-02-12)
      Chronic consumption of alcohol can lead to pancreatitis, which can predispose to pancreatic cancer. Because combinations of histone modifications have been implicated in pancreatic tumorigenesis, our goal is to find histone modifications in pancreatic acinar cell nuclei following by ethanol abuse. As an animal model of alcoholism, alcohol preferring (P) rats and alcohol non-preferring rats (NP) were used. Histones were extracted from rat pancreatic nuclear fractions using 0.4 N sulfuric acid and dialysis. Histone modifications were studied by Western-blotting analysis using the following antibodies: anti-trimethyl-histone H3 at Lys9 (H3K9me3), anti-dimethyl-histone H3 at Lys9, anti-phospho-histone H3 at Ser10, anti-acetyl-histone H3 atLys14, anti-histone H3, anti-acetyl-histone H4, anti-histone H4. We found that alcohol abuse caused a decrease in the phosphorylation of histone H3. This result was observed in pancreatic tumor specimens.Further studies are needed to determine the extentto which both modifications are related and which gene expression is affected.
    • Examining the Effects of Aetokthonos hydrillicola extract on Oxidative Stress in C6 Cells

      Ward, Kayla; College of Science and Mathematics; Wiley, Faith; College of Science and Mathematics; Augusta University (2018-02-12)
      Aetokthonos hydrillicola,a species ofcyanobacteria,has colonized an invasive species of hydrilla in the lakes of the Southeastern United States. This cyanobacterium is suspected to cause Avian Vacuolar Myclinopathy (AVM). AVM is a neurological disease that affect birds. Bald eagles and American coots havebeen primarily studied and known to be affected by AVM. Symptoms of AVM consist of brain lesions, loss of basic motor skills, and the disease often leads to death. Extracts of A. hydrillicolaare toxic to C6 cells, and this cell line is used as a model toexamine the mechanism of toxicity. The aim of this research project is to understand the role of oxidative stress in A. hydrillicolacytotoxicity and determine if antioxidant compounds may protect the cells. Common oxidative stress inhibitors, Gingko bilobaextracts and selenium, have been tested in different concentrations in order to determine if oxidative stress is present and preventable. These compounds did not prevent toxicity in the C6 cells exposed to the cyanobacterial extracts. The presence of oxidative stress in cytotoxicity will be further tested using oxidative stress analysis tests as well as other antioxidant compounds. Various measures of oxidative stress will be used to assess if oxidative damage occurs following exposure to the cyanobacterial extract.
    • CHARACTERIZATION OF NF-¿B DEFICIENT BONE-MARROW MACROPHAGES

      Pepper, Anthony; Fischer, Jeffrey; Department of Biological Sciences; Bradford, Jennifer; Department of Biological Sciences; Augusta University (2018-02-12)
      The aim of this study was tocharacterize bone-marrow derived macrophages (BMDMs) that lack canonical nuclear factor-kappaB (NF-?B) signaling. The macrophages for the study were obtained by harvesting the bone-marrow fromp65LysMCre (KO) mice and LysMCrecontrolmice.To determine NF-?B deletion efficiency, p65 (a transcription factor in the canonical pathway) protein levels were evaluated by fluorescent microscopyin bothKOand control BMDMs that had been stimulated withlipopolysaccharide(LPS).The induction ofiNOSwasmonitoredin KO and control BMDMswhenactivated by NF-?B stimulatorsIFN-?andLPS.The regulation of iNOSwas assessedby comparing macrophages that had been treated withLPS, IFN-?, or both to a control treatment under fluorescent microscopy. In addition to staining, a nitric oxide assay was employed to help determine the extent of iNOS activity.The macrophages were also visualized under light microcopyby comparing macrophagesthat were stimulated with LPS andIFN-?tounstimulated cellsusing fluorescence microscopy.Currently,a caspase assay is in progress to help further evaluate the effects of p65 losswithin macrophages.