• Baker, Ariana

    PARAdiGM Student: Ariana Baker
    Glutathione Peroxidase 2 is overexpressed in lung adenocarcinoma and predicts poor patient survival

    Lung carcinoma is the leading cause of cancer mortality worldwide including in the USA. It is the primary cause of cancer deaths in both men and women. While there has been some progress in early detection and treatment for lung adenocarcinoma patients based on molecular characterization, further studies are needed to identify new biomarkers forearly detection, disease prognosis, and therapeutic targets. In this study, we performed transcriptome sequencing analysis of lung adenocarcinoma patients from the Cancer Genome Atlas (TCGA) using publicly available data analysis tool “UALCAN” to identifypotential predictor of lung adenocarcinoma patient survival. Our analysis suggests that revealed Glutathione Peroxidase 2 (GPX2) is overexpressed in lung adenocarcinoma, with higher stage tumors showing highest expression. Additionally, individuals with lung adenocarcinoma that had an elevated GPX2 expression showed poor survival. Thus, our investigation identifies GPX2 as a potential biomarker of aggressive lung adenocarcinoma. Being an enzyme, GPX2 may serve as a therapeutic target.

  • Bibb, Jada

    PARAdiGM Student: Jada Bibb
    Vimentin intermediate filaments increase invasiveness of IPF fibroblasts

    Idiopathic pulmonary fibrosis (IPF) is a fatally progressive disease. A characteristic feature of pulmonary fibrosis is the presence of an invasive myofibroblast phenotype. The invasiveness of a fibroblasts is affected by the type III intermediate filamentand cytoskeletal protein, vimentin. The natural structure of Vimentin is a coiled-coil that contains α-helical regions with disordered head and tail domains. Vimentin has the ability to transform into soluble short squiggles. These squiggles can formulate into an elongated form of vimentin intermediate filaments that are nonpolar and insoluble. Soluble vimentin has been shown to localize in lamellipodia, which are important for cell migration. The versatility of vimentin as a soluble particle and formation of VimIFs assist in the cellular movement of fibroblasts. Fibroblast invasion is an important function in the deposition or remodeling process of the extracellular matrix and are hallmarks of IPF. Vimentin intermediate filaments and their active role in IPF fibroblasts invasion and remodeling in IPF was examined using a unique 3D ex-vivo model pulmospheres to determine their capacity for invasion. Our results show that increased expression of vimentin intermediate filaments cause increased fibroblast invasion and that Withaferin A decreases the assembly of VimIF by binding to Cys328; resulting in decreased fibroblast invasion.

  • Elam, Madisyn

    PARAdiGM Student: Madisyn Elam
    Characterization of Cardiac Function and Histology in MiR-486 Knockout and DMD Mouse Models

    Introduction: Duchenne Muscular Dystrophy, or DMD, is an X-linked, genetic disorder that results in progressive muscle degeneration over time. DMD results in numerous physical symptoms for those affected, including weakened muscles, breathing complications, decreased motor skills, loss of overall muscle function, and ultimately a decreased life expectancy. Recently, it has been demonstrated that microRNAs, or miRs, play a significant role in regulating the development and maintenance of muscle. Preliminary data from our lab has shown that in DMD patients and mouse models, miR-486 expression is low compared to non-diseased muscle.

    Objectives: The goalof this research was to understand the role of miR-486 in the progression of DMD. Because DMD results in muscle degeneration, which includes the degeneration of both skeletal and cardiac muscle, we wanted to understand the role of miR-486 in both tissues. The primary objective of this study was to characterize cardiac myofiber architecture and overall cardiac function in miR-486 -/-; mdx5cv-/ymice to better understand the role of miR-486 in the progression of muscle degeneration in DMD pathology.

    Methods: To investigate this, echocardiography was used to assess multiple parameters of cardiac function in both miR-486 KO and WT6-month-old male mice. In addition, H&E staining was used to quantify myocardial and ventricular area of the left ventricle to better understand the functional cardiac phenotypes observed.

    Results: Hematoxylin and eosin staining of 6-month-old male myocardium showed that miR-486 KO cardiac tissue demonstratesa predicted increase in myocardial thickness and a decrease in ventricular area. Additionally, miR-486 KO mice demonstrated a trending decrease in cardiac function measured by echocardiography, however, more tested mice will be needed to accurately conclude a statistical difference between groups.

    Conclusions: Overall, ablation of miR-486 resulted in disrupted cardiac function and histology. Based on these findings, additional experiments will be done to, 1) identify the mechanism of contribution of miR-486 to a degenerating cardiac phenotype and 2), to understand what gene targets may be overexpressed by the ablation of miR-486 that could be contributing to disease progression in DMD.

  • Holliday, Alexis

    PARAdiGM Student: Alexis Holliday
    Collecting duct NOS1 is critical for high salt-induced changes in glomerular filtration rate

    Introduction: Collecting duct nitric oxide synthase 1 (CDNOS1)is critical for maintaining fluid and electrolyte balance in male miceand regulatingintra-renal angiotensin II(angII). CDNOS1KO male mice have an attenuated glomerular filtration rate(GFR)to a high salt diet(HS). It is unknown whether the GFR response is due to activation of angIIpathway or if female CDNOS1KO mice have a similar phenotype as males.

    Objective–Aims: 1) determine GFR in male flox and CDNOS1KO miceonHSin the presence of vehicle or angII blockade, 2) determine female CDNOS1KOand floxmiceurine output.

    Methods–Aim 1: CDNOS1KO and floxmalemice were challenged with high salt diet (4.0% NaCl, 24h) in the presence of vehicle, candesartan (8mg/kg/day; angII blocker) in drinking water. GFR was monitoredby FITC-sinistrin. Aim 2: Female flox and CDNOS1KO mice were placed in metabolic cages to monitor urine output.

    Results–Aim 1: GFR was elevated in CDNOS1KO mice (vehicle:254.8± 7.5L/minvs. candesartan: 301.4 L/min, n = 1-5), similar to floxwith vehicle(332.4± 14.5L/min, n =5). The GFRremained unchanged in flox mice. Aim 2: Female CDNOS1KO and floxmice had similar urine output with HS. We initiated measurements of NOS3 activation by immunohistochemical analysis.

    Conclusion: These data indicate that CDNOS1regulatesGFR via modulation of the angII/AT1 signaling pathway in male mice and that female CDNOS1KO mice in response to a HSare able to compensate for the loss of NOS1 through an undetermined mechanism.

  • Lewis, April

    PARAdiGM Student: April Lewis
    Impact of Altering the Redox Chemistry and Environment of the Cell Cycle Regulator Cyclin E on its Localization and Abundance in Epithelial Ovarian Cancer Cells

    Cyclin E (CycE) is responsiblefor control and regulation of the cell cycle G1to S transition. CycE over-expression is a driver for certain ovarian tumors.In proliferating cells, mitochondria serve as the primary site of reactive oxygen species production. The Mitra lab previously described a novel interaction between CycE and mitochondria that connects mitochondrial function and the cell cycle. Previous studies and our unpublished data suggest CycE issensitive to redox chemistry. The mechanism of CycE association with the mitochondria might be due to redox chemistry at Cysteine residues in the CycE protein sequence. Mutatinga cysteine residueto Serine (C57S) in CycE via site directed mutagenesis will alter itsredox chemistry. We transfected epithelial ovarian cancer cells (EOCs) with WT and C57S CycE constructs tagged with a green fluorescent protein to determine the extent to which it can associate with mitochondria. We also manipulated the redox environment of WT and C57S CycE by introducing transfected EOC cells to reducing (DTT) and oxidizing (TBH) agents. We hypothesizedthat altering CycE redox chemistry will interrupt CycE’sinteraction with mitochondria. Confocal microscopy revealed that the correlation between nuclear and cytosolic CycEis dependent on environmental conditions. In reducing and oxidizing environments, EOC C57S mutated cells had a broader range of CycE abundance than WT. C57Salso had fewer cells with mitochondrial CycE compared to WT in control, reducing, and oxidizing environments. In WT cells, reducing environments are associated with decreased CycE abundance and oxidizing environments with increased abundance and more cellsexhibiting mitochondrial CycE.

  • Medina, Anaissa

    PARAdiGM Student: Anaissa Medina
    Glucose Mediated EZH2 in Heart Failure

    Diabetes is a complex metabolic disorder defined by hyperglycemia. The resulting chronic elevation in glucose may cause irreversible cellular damage. Previous studies have identified that glucose may directly contribute to heart disease by modifying DNA, gene expression, and proteins, referred to as epigenetics. This mechanism may be regulatied by the specific pathway of protein posttranslational modification via O-GlcNAcylation. Prior published data identified upregulation of enhancer of zeste 2, EZH2, an epigenetic regulator in ischemic versus nonischemic human heart failure, as a critical candidate for these mechanisms. EZH2 is part of the polycomb repressive complex 2 (PRC2), which contributes to the regulation of epigenetic maintenance of some genes via histone methylation and/or DNA methylation. Interestingly, when part of the PRC2, EZH2 has been shown to have increased stability when modified by protein O-GlcNAcylation. Our hypothesis is that genome wide DNA methylation encodes transcriptional reprogramming in diabetic cardiomyopathy. Specifically, EZH2 O-GlcNAcylation results in increased epigenetic silencing of critical genes. To examine the role of EZH2 in diabetic heart failure, we performed qPCR and western blot techniques. The results identified very low expression of EZH2 in control and diabetic mouse heart. However, ongoing studies will determine if its genomic localization is modified. Our work on epigenetic regulators, may lead to an understanding of how diabetes reprograms regulators of cardiac metabolism, leading to diabetic heart failure. This information may allow for development ofclinical treatments.

  • May, Micah

    PARAdiGM Student: Micah May
    Immunofluorescence Analysis of PINK1 Knockout Rats.

    Introduction/Background: Parkinson’s disease is the second most common neurodegenerative disease with a prevalence of over 1 million people and an incidence of 60,000 new cases per year in the United States. The cause of Parkinson’s disease is currently not known, however, loss of function mutations in the PINK1 gene cause a familial form of Parkinson’s disease. PINK1 knockout rats have a similar mutation in their PINK1 gene that results in difficulty with locomotion in the rats beginning about age 6 months. These rats experience hind limb paresis then recover a little over a month later.

    Objective: The purpose of this study is to further understand the cause of locomotor dysfunction in PINK1 knockout rats and ultimately contribute to further understanding of the cause of Parkinson’s disease inhumans.

    Procedure: Leg muscles will be harvested from PINK1 knockout and wild-type control rats euthanized before, during, and after onset of locomotor symptoms. Dissection of muscles from PINK1 and wild-type rats, and sectioning of muscles will be performed. Muscle sections will be analyzed by immunofluorescence using antibodies to detect abnormalities in mitochondria and neuromuscular junctions.

    Results: The results we wish to find in this experiment will hopefully support our hypothesis that the reason for the locomotion dysfunction in PINK1 knockout rats is found in the neuromuscular junction, and not in the brain or spinal cord. If these results are found, hopefully this will to lead to new understanding of the potential cause of Parkinson’s in humans.

  • Nicole Hsiao-Sánchez

    PARAdiGM Student: Nicole Hsiao-Sánchez
    Mitochondrial Dysfunction in Skeletal Muscle Associated with Sarcopenia among Chronic Obstructive Pulmonary Disease Patients

    Introduction: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and is characterized by irreversible airway obstruction. Sarcopenia, age-associated decline in muscle mass and function, can manifest in COPD patients and is poorly understood. However, mitochondrial dysfunction is known to play a role in muscle degeneration in COPD and sarcopenia.

    Objective: To provide insight into COPD sarcopenia pathogenesis by assessing the relationship between mitochondrial function measures and COPD sarcopenia.

    Methods: Muscle biopsies were obtained from the vastus lateralis of sarcopenic (N=10 cases) and non-sarcopenic (N=10 controls) COPD patients. COPD was diagnosed using lung function. Sarcopenia was diagnosed as low fat-free mass index (FFMI) (<7.25 kg/m2 men, <5.67 kg/m2 females) and low handgrip strength (HGS) (<30 kg men, HGS<20 kg women). Mitochondrial function was characterized using citrate synthase and complex IV activity, mitochondrial DNA (mtDNA) damage and mtDNA copy number. Regressionmodels were fit and adjusted for smoking status to test the association between mitochondrial function and sarcopenia, using R version 3.5.3.

    Results: Mitochondrial enzymatic activity assays showed no association with sarcopenia, except complex IV total homogenate kinetics (OR=0.33; 95% CI=[0.00–61.54]). Similarly, mtDNA assays, excluding the long to short ratio of mtDNA assay (OR=0.21; 95% CI=[0.00–24.38]), were not associated with sarcopenia.

    Conclusion: Both complex IV total homogenate kinetics andthe long to short mtDNA ratio were reduced in sarcopenic patients, suggesting an increase in specific enzymatic activity and mtDNA damage could play a role in the pathogenesis of sarcopenia in COPD patients.

  • Vann, Carlee

    PARAdiGM Student: Carlee Vann
    BRD2 isareprogramming barrier for cellular reprogramming

    Introduction

    Induced pluripotent stem cells(iPSCs) are molecularly and functionally equivalent to embryonic stem cells. iPSCs are generated from somatic cells, most commonly from fibroblasts, by ectopic expression of four transcriptional factors, OCT4, SOX2, KLF4, and MYC. The production of induced pluripotent stem cells from human fibroblasts is a novel technologyfor stem cell research and regenerative medicine; however, this process is inefficient. To convert fibroblasts to iPSCs, we have to establish the pluripotent transcriptional program and erase the original fibroblast-specific transcriptional program. The latter is not studied well and remains largely elusive. This projectwill study the transcriptional barrier during iPSC reprogramming.

    Objectives

    This research is an extension of previous findings that chemical inhibition of BET bromodomains enhancedthe process of cellular reprogramming. Weare investigating which BET protein, among the three expressed in fibroblasts, could possibly be the targets of the BET chemical inhibitors that improves cellular reprogramming when included in the reprogramming media. We hypothesizethat the BET protein,BRD2, is a reprogramming barrier and that by its inhibition cellular reprogramming will be enhanced.

    Methods

    Two identical experimentswere performed. Human fibroblast cells were transduced with the conventional reprogramming factors as well as shRNA for each BET protein (BRD2, BRD3, and BRD4), to inhibitthe expression of each member of the BET genes. We measured reprogramming efficiency by the number of colonies positive for pluripotency surface markers alkaline phosphatase and TRA-1-60, in comparison with the control shRNA.

    Results
    Infection with two BRD2 shRNA constructsaveraged 294 colonies (#1) and 323 colonies (#2). Two constructsof BRD3 shRNA averaged 1.3 colonies (#1) and 10.6 colonies (#2). The two strains of BRD4 averaged 3 colonies (#1) and 4.3 colonies (#2).The control (shLUC) averaged 164 colonies.

    Conclusions

    The inhibition of BRD2 has enhanced iPSC reprogramming, indicating that it is a reprogramming barrier. Down regulation of BRD3 and BRD4, on the other hand, impairs iPSC reprogramming.

  • Westbrook, Ericka

    PARAdiGM Student: Ericka Westbrook
    Characterization of Tomosyn-1 Knockout Mice

    Introduction: Preliminary data from our lab has shown that tomosyn-1, a syntaxin-binding protein, negatively regulates insulin secretion in pancreatic beta-cells. Interestingly, Tomosyn-1 was shown to associate with insulin granules and acts as a brake to inhibit granule fusion with the plasma membrane. To further investigate the sub-molecular mechanism how Tomosyn-1 regulate insulin secretion, Tomosyn-1 knockout mice have been generated.
    Aim: In this project, I have characterized the Tomosyn-1 transgenic mice to confirmwild type (Tomosyn-1+/+) and heterozygous (Tomosyn-1+/-) mice.
    Methods: Polymerase Chain Reaction (PCR) was performed on tail DNA using Tomosyn-1 specific oligonucleotide primers.
    Results: SpecificPCR products were generated using wild type and heterozygous primers.
    Conclusion: This project successfully standardized the PCR protocol for identifying Tomosyn-1 wild type and heterozygous mice.

  • Bibb, Jada

    PARAdiGM Student: Jada Bibb
    Age Dependent Variability of Invasiveness of Lung Fibroblast Spheroids

    Fibroblast invasion is a biological phenomenon quantitatively and qualitatively specific to an individual and ultimately is linked tonormal wound repair ordisease progression. The invasiveness of fibroblasts is affected by the different growth factors placed in their environment. Most of the studies are limited to two-dimensional (2D) culture, but invasion is a three-dimensional (3D) phenomenon where the cells invade through extra cellular matrix (ECM). In the present study, 3D culture system (spheroids) for human lung fibroblasts will be developed. 3D culture systems, in contrast to the 2D culture systems, represent more accurately the actual microenvironment where cells reside in the tissues.Few studies have been done for age related responses to the invasiveness of fibroblasts in the absence or presence of growth factors.We haveutilized different ages of primaryhuman fibroblastsfrom the lung to preparespheroids andevaluate the invasive response of fibroblastsin the presence of various growth factors.Re-epithelizing growth factors such as Fibroblast growth factor -10 (FGF-10) , Keratinocyte growth factor (KGF), Hepatocyte growth factor (HGF) and pro-inflammatory cytokine Macrophage colony growth factor (M-CSF) were tested in fibroblast spheroids from three age groups.By understanding the effects of different growth factors on the invasiveness, we can stipulate the role of growth factors in wound healing.We overall have evaluated if the responsesto invasiveness are variable in an age-dependent manner.

  • Garcia, Alex

    PARAdiGM Student: Alex Garcia
    Pancreatic Islet Encapsulation with Peptide Amphiphile Nanomatrix for Pancreatic Islet Transplantation

    Pancreatic islet transplantation (PIT) has been validatedas a possible treatment of type 1 diabetes. The omentum, a fold of the peritoneum connecting the stomach to other abdominal organs, is considered a site of particular interest for PIT due to limited immunologic activity, size oftheimplantation site, and ease of surgical access and manipulation. However, somedifficulties regarding the innate immune response as well as revascularization and leakage of islets from the implantation site have decreased the efficacy of current approaches to PIT within the omentum. In order to improve the outcomes of PIT within the omentum, a method of islet encapsulation within a peptide-based (peptide amphiphile (PA) nanomatrix gel) hydrogelwas developed. Theself-assembled PAnanomatrix gel enables the encapsulation of islets within a nurturing microenvironment that will promote islet survival within the omentum. For greater mechanical stability,an electrospun poly (ε-caprolactone) (ePCL) nanofiber sheetwith porous crater-like structureswill be used in conjunction withfibroblast growth factor-2(FGF-2)tofacilitateretention andrevascularization of the implanted islets.The ePCL nanofiber sheet will be used synergisticallywith PA encapsulation and FGF-2 to form ahybrid nanosackfor transplantation of pancreatic islets. Angiogenesis within the ePCL nanofiber sheetwill be analyzedthroughimmunohistochemical analysis and micro CTand histological analysis by hematoxylin and eosin (H&E) staining. This analysis will characterize the revascularization of the hybrid nanosack in vivo. Functionality and viability of the encapsulated islets will be assessed preliminarily in an in vitro setting using glucose stimulated insulin release ELISA and Interleukin-1βELISA.

  • Elam, Madisyn

    PARAdiGM Student: Madisyn Elam
    Characterization of Cardiac Function and Histology in MiR-486 Knockout and DMD Mouse Models

    Introduction: Duchenne Muscular Dystrophy, or DMD, is an X-linked, genetic disorder that results in progressive muscle degeneration over time. DMD results in numerous clinical symptoms for those affected, including generalized muscle weakness, breathing complications, decreased motor skills, loss of overall muscle function, and ultimately a decreased life expectancy. Recently it has been demonstrated that microRNAs, or miRs, play a significant role in regulating many muscle-specific processes, including muscle development and structural maintenance of myofibers. Preliminary data from our lab has shown that in DMD patients and mouse models, miR-486 expression is low compared to non-diseased muscle.

    Objectives: Because DMD results in muscle degeneration, which includes thedegeneration of cardiac muscle, we want to understand the role of miR-486 specifically in the heart. The primary objective of this study was to characterize cardiac myofiber architecture and overall cardiac function in miR-486 -/-; mdx5cv-/ymice to better understand the role of miR-486 in the progression of muscle degeneration in DMD pathology.

    Methods: To investigate this, echocardiography and histology were used to characterize cardiac function and cardiac myofiber architecture in miR-486 -/-; mdx5cv-/ymice compared to WT mice. Muscles were sectioned and stained with hematoxylin and eosin to observe myonuclei location, myofiber architecture, and fibrosis.

    Results: Hematoxylin and eosin staining of 6-month-old male miR-486; mdx5cv-/ymyocardium showed adecrease in myofiber size, increased number of centralized myonuclei, and overall more fibrotic area than WT counterparts. Functionally,miR-486 -/-; mdx5cv-/ymales do not demonstrate altered cardiac function at6 months of age.

    Conclusions: Based on histology and echocardiography, miR-486-/-;mdx5cv-/ycardiac myofibers demonstrate an abundance of centralized myonuclei and an abnormal heterogeneous fiber architecture. MiR-486 -/-; mdx5cv-/ymale mice do not demonstrate a statistically significant difference in cardiac output, ejection fraction, or fractional shortening at 6 months of age. Moving forward, histological characterization using nuclear fast red staining can be used to further characterize myofiber nuclei. In addition, various older age groups will be tested to assess the age of onset cardiac function decline in miR-486 -/-; mdx5cv-/ymale mice.

  • Hunter, Melodie

    PARAdiGM Student: Melodie Hunter
    Disparities in Consent Rates by Clinical Care Setting in a Chronic Hypertension Trial

    To help determine the best treatment for mild chronic hypertension in pregnant women, the Chronic Hypertension and Pregnancy (CHAP) project was designed. At the University of Alabama at Birmingham (UAB), patients are approached at the Obstetric Complications Clinic (OBCC), a resident-led clinic, and the Women and Infants Center (WIC) with faculty-led Maternal-FetalMedicine (MFM) and general ObGyn (PC) clinics. There appeared to be a disparity in participation in the CHAP project based on clinic setting and demographics; however this has not been objectively studied. We aimed to describe whether there was a disparity inconsent rates and analyze the specific reason(s) for nonparticipation by care setting. Women who were eligible for CHAP were approached for participation. Those that consented were randomized to treatment vs. no treatment. Data extraction from screening logs and study records provided the demographics including: clinical setting, age, race/ethnicity, parity, educational status, consent status and reason for participation refusal. Comparison groups were care setting at OBCC vs. WIC. We also compared MFM and PC settings each to OBCC. Using a descriptive and cross-sectional analysis, we evaluated whether care setting or differences in characteristics of patients seen at these settings explain the findings. We found that clinical care setting and the differences in demographics of patients at the settings were not associated with statistically significant differences in consentrates. Consentrates at OBCC and WIC clinics were high > 75%; a larger sample is needed to determine whether the lower rate at PC clinic is significantly different.

  • May, Micah

    PARAdiGM Student: Micah May
    What Causes Familial Parkinson’s Disease? An Immunohistochemical and Immunofluores-cence Analysis of PINK1 Knockout Rats.

    Introduction/Background: Parkinson’s disease is the second most common neurodegenerative disease with a prevalence of over 1 million people and an incidence of 60,000 new cases per year in the United States. Parkinson’s disease symptoms include: tremor, slowmovement, stiffness, and overall difficulty with locomotion. The cause of Parkinson’s disease is currently not known, however, loss of function mutations in the PINK1 gene are linked to a familial form of Par-kinson’s disease. PINK1 knockout rats have a similar mutation in their PINK1 gene that results in difficulty with locomotion in the rats beginning about age 6months.

    Objective: The purpose of this study is to further understand the cause of locomotor dysfunction in PINK1 knockout rats and ultimately contribute to further understanding of the cause of Par-kinson’s disease inhumans.

    Procedure: Leg muscles will be harvested from PINK1 knockout and wild-type control rats eu-thanized before, during, and after onset of locomotor symptoms. Dissection of muscles from PINK1 and wild-type rats, and sectioning of muscles will be performed. Muscle sections will be analyzed by immunohistochemistry and immunofluorescence using antibodies to detect abnormalities in mitochondria and neuromuscular junctions.

    Results: The results that were yielded from this experiment helped to conclude that in PINK1 Knockout rats during the locomotor dysfunction symptomatic period, the muscle mass of hind limbs is decreased when compared to Pre and Post symptomatic. Also, during the symptomatic period Immuno reactivity decreases in Knockouts from pre symptomatic to the symptomatic period. As far as determining the cause of Locomotor dysfunction, the data was inconclusive.

  • Medina, Anaissa

    PARAdiGM Student: Anaissa Medina
    Epigenetic Regulation of Gfpt2Gene Expression Occurs in Diabetic Heart Failure

    Introduction: Diabetes affects a large population of the United States. As Diabetes continues to be a primary concern so is the possible consequences that may come from the disease, such as cardiovascular disease. Diabetic patients are more likely to die from heart failure as a result of metabolic dysfunction. Additionally, race appears to play a role in increasing the susceptibility to the development of diabetes and its complications, but little is understood about this issue.

    Objective: Previous studies have identified that uncontrolled glucose levels directly contributes to cardiovascular disease by modifying DNA, gene expression, and proteins. The modifications in gene expression are a link to an emerging field of study called epigenetics, which studies the long-lasting changes in gene expression without directly changing the DNA sequence. The long-lasting changes in DNA expression happen through many changes including histone acetylation and DNA methylation. We and others have specifically examined the modification of DNA via DNA methylation and gene expression, which is often inversely changed. The link between glucose and epigenetics has identified the specific pathway leading to protein O-GlcNAcylation, a protein posttranslational regulation, that may affect DNA methylation. Our hypothesis is that fluctuations in glucose levels directly reprograms gene expression increasing susceptibility to heart failure.

    Methods: To test whether glucose levels directly reprogram gene expression increasing the susceptibility to heart failure I interrogated data sets of RNA changes and DNA methylation changes in both human and mouse heart.The targets were confirmed by qPCR and further analyzed by Western blotting.

    Results: From themouse and human RNA sequencing and DNA methylation arraysI identified GFPT2 as a target for further examination.There was a significant induction of GFPT2 in the diabetic mouse heart compared to the non-diabetic mouse heart and a significant induction of GFPT2 in African Americans with diabetic heart failure compared to Caucasians with diabetic heart failure. (p < 0.05) Experiments to validate the DNA sequencing and DNA methylation array results supported that GFPT2 is increased in the diabetic mouse heart.

    Conclusions: Diabetes appears to show GFPT2 induction. Larger studies need to be performed to confirm the significance of GFPT2 in the mouse and human diabetic heart compared to a nondiabetic heart. The results identifya new mechanismto define diabetic cardiomyopathy which will be furtherinvestigated by promoter cloning and siRNA knockdown of GFPT2.

  • Rivero, Marco-Jacob

    PARAdiGM Student: Marco-Jacob Rivero
    Transfusion of Red Blood Cells Enhances Platelet Adhesion and Aggregation on a Collagen Surface Under Arterial Flow

    INTRODUCTION: Primary hemostasis is largely mediated by adhesion of platelets to a collagen surface at a site of endothelial injury. Over the past decades, there has been increasing evidence that red blood cells (RBCs) further enhance hemostasis by increasing blood viscosity and pushing platelets towards the blood vessel walls. It follows that anemia (low RBC count) and thrombocytopenia (low platelet count) are linked to excessive bleeding, which can be ameliorated by RBC transfusions. However, research on the precise relation between hematocrit (HCT) and platelet aggregation remains limited.

    OBJECTIVES: The primary objective of this study was to observe how incremental HCT elevations affect platelet adhesion and aggregation.

    METHODS: Whole blood was obtained from healthy human subjects and diluted with platelet-poor plasma to induce anemia (HCT: 20%) and thrombocytopenia (platelet count: <50,000/μL). The reconstituted blood was supplemented with varying amounts of RBCs to generate experimental samples with elevated HCT values. Samples were assayed with a microfluidic system that simulates arterial shear flow.

    RESULTS: Shear flow assays revealed that HCT elevation resulted in higher levels of platelet adhesion and aggregation. Moreover, larger supplements of RBCs resulted in progressively larger signals from fluorescently labeled platelets.

    CONCLUSION: Given anemic and thrombocytopenic arterial shear conditions, RBC transfusion enhances platelet adhesion and aggregation in a dose-dependent manner. Our results suggest thatRBC transfusions are indeed effective in restoring hemostasis in patients with anemia and thrombocytopenia.

  • Shavers, Alexis

    PARAdiGM Student:
    Increasingoxidative stress with menadione can enhance T cell responses in Type One Diabetes

    Type one diabetes (T1D) is an autoimmune disease that plagues many people. In T1D autoreactive T cells attack pancreatic beta cells, which are required to produce insulin necessary for glucose homeostasis. While patients can manage T1D through exogenous insulin injection, ultimately there is no cure, patients must depend on insulin for the rest of their life, and the risk of secondary complications remains. This problem continues because there is little known about the dysregulation of T cells when it comesto the different signaling responses. The responses are the antigen (signal 1), costimulatory signals (signal 2), and the reactive oxidative species (ROS) and cytokines (signal 3). Recent studies have found evidence that the third signal for efficient T cell maturation is important in helping the immune system. The purpose of this study is to focus on the third signal, oxidative stress, and T cell responses in T1D. The non obese diabetic (NOD) model of the mouse will be used due to its spontaneity of acquiring diabetes, and similarities to human T1D. We hypothesize that if cells are treated in Menadione, a ROS donor that can lead to oxidative stress, it can boost the third signal and that T cell response will increase. Implications for research include thatantioxidants play a role in the signaling of T-cells and their response in T1D. In conclusion, Menadione did not negatively affect T cell viability. With the higher concentrations of Menadione, there were significant decreases in IL-2 and IFN-g with 1.0 mM treatment. There wereno significant changes in T cell activation markers CD25, CCD44, and CD69.

  • Weupe, Mason

    PARAdiGM Student: Mason Weupe
    μ-Optical Coherence Tomography to Characterize Pharmacologic Induction of Muc5B in WT and Muc5b KO Primary Ferret Bronchial Epithelial Air-Liquid-Interface Cultures

    Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with median-survival ranging from 3-5 years after diagnosis. The greatest risk factor for developing IPF is a gain-of-function promoter variant in the mucin MUC5B; which accountsfor ~30% of the risk for developing IPF.Our lab has generated a novel bleomycin-induced ferret model of pulmonary fibrosis which better recapitulates human IPF. Our lab’s long-term objective is to understand fibrotic mechanisms in altered mucin microenvironments in aferret model. Myshort-term goal was to identify pharmacologic agents that are capable of inducing preferential Muc5b overexpression in an in vitro ferret system.

    Methods: Primary wild type (WT) and Muc5B knockout (KO) ferret bronchial epithelial cells (FBEs) were cultured at air liquid interface (ALI) in Pneumacult media. FBE filters were induced basolaterally with either pyocyanin (PCN), phorbol myristate (PMA), or prostaglandin D2 (PGD2). Micro-optical coherence tomography (μOCT) was used to assess functional microanatomy of the FBEs including the following parameters: air surface liquid (ASL)depth, periciliary liquid (PCL)depth, ciliary beat frequency (CBF), and mucociliary transport (MCT). Ongoing studies include Muc5band Muc5acmucin protein expression and gene expression by Western blot and q-PCR.

    Results: In vitroevaluation of the mucociliary transport apparatus in FBEs 24-hourspost drug induction, μOCT demonstrated that only PCN significantly increased mucus depth (i.e. ASL)in WT relative to control PBS treatment (mean increase23.6 μm, P=0.015, N=3 filters/group x 3 ROI per filter),but not in Muc5b KO ferret ALI filters, indicating that PCN increased mucusin a Muc5b-specific manner. PMA and PGD2 significantly increased CBF in WT relative to control (mean difference for PMA 3.4 Hz, P=0.0018; mean difference for PGD2 4.8 Hz, P<0.0001).Confirmatory PCN induction studies in human bronchial epithelial cells (HBEs)demonstrated 1.6-fold increase in MUC5BmRNA levels.

    Conclusions: PCN significantly increases mucuslevelin WT, but not in Muc5b KO primary FBEALI cells, indicatingPCN increases ASL in a Muc5B-dependent fashion.In vivo studies of PCN-induced Muc5b-overexpression and responseto injury are ongoing.

  • Williams, Donald

    PARAdiGM Student: Donald Williams
    Elucidating the role of MALAT1 and mascRNA in Breast Cancer

    Background: Every two minutes a woman in America is diagnosed with breast cancer, and every thirteen minutes a woman dies from breast cancer. Breast cancer is the second leading cause of death for women in the United States. Breast cancer is a major concern in the United States, but recent scientific discoveries have uncovered a long noncoding RNA, that is found in most tissues of the body, including cancerous tissues such as lung, colon, pancreas, and even breast. Long noncoding RNA are a class of transcribed RNA molecules, which are longer than200 nucleotides. Long noncoding RNA donot encode proteins, and often promotethe metastasis of cancer. Metastasis is the secondary growth of cancer at sites that are distant from the original site of the cancer. The specific long noncoding RNA we are working with is called Metastasis Adenocarcinoma Lung Transcript1 (MALAT1). MALAT1 was discovered in 1997 and is overexpressed in the nuclei of many tissues around the body. Its highest expression can be found in the lung where it serves as an oncogene. MALAT1 is still fairly unknown in the world of science and it takes research such as this to determine the role it plays in our body.

    Objective: The purpose of this research is to evaluate the role of a specific long noncoding RNA named Metastasis Adenocarcinoma Lung Transcript 1 (MALAT1)in breast cancerand to determine if possible treatment options for breast cancer are viable for the future.

    Methods: We used SYBR Green real-time Polymerase Chain Reactionto determine the expression levels of MALAT1 in the breast cancer tissue and cell lines. SYBR Green PCR is a method of PCR that allows a SYBR Green dye to detect DNA generated during the PCR process. The dye intercalates and bindswith the target gene, which was MALAT1 in this case.After binding with the target, the dye fluoresces at different intensities allowing you to locate and determine expression levelsof the gene. Through SYBR Green Polymerase Chain Reaction (PCR), we copied the genetic material of breast tissuesand cell lines to compare MALAT1 expression levels in normal breast tissues/cells and breast cancerous tissues/cells.

    Conclusion: We’ve identified that MALAT1 plays a role in cell proliferation, migration, and invasion.Our results determined that MALAT1 is expressed more highly in breast cancer cells and tissues compared to normal breast tissues and cells. The higher expression of MALAT1 in breast cancer cells and tissue promotes the growth of breast cancer and the metastasis of cancer to other parts of the body. With further research and study, MALAT1 could possibly serve as a therapeutic target for breast cancertreatment.

  • Ayokanmbi, Adetokunbo

    PARAdiGM Student: Adetokunbo Ayokanmbi
    Mentor: Farruk Lutful Kabir, William Tom Harris

    Background: Cystic Fibrosis (CF) is a common genetic disorder most prevalent in Caucasians where it affects 1/2500 individuals. The geneticmutation involvesthe CF transmembrane conductance regulator (CFTR) gene, resulting in a defect in ion (Cl-andHCO3-) transportation. The most common mutation is a deletion of phenylalanine at position 508 (F508del-CFTR). Currently, there is no cure for CF. However, pharmacological treatments directly targeting the mutated CFTR protein have recently received FDA approval. We have recently identified microRNA (miRNA; noncodingRNAs that regulate gene expression)thatspecifically target CFTR transcription and translation and alter F508del CFTR response to corrector strategies.
    Hypothesis: miRNA-145 is increased in CF biospecimens in association with lung disease progressionand response to 508del CFTR correction.
    Methods: Exosomes isolated from CF/non-CF plasma/serum specimens and from cell culture supernatant will be analyzed and compared for the presence of miRNA including miRNA-145, a TGF-beta dependent miRNA that suppresses CFTR expression. miRNA isolated from purified exosomes will be quantified by real-time qPCR.
    Anticipated Results: We expect miR-145 content in exosomes will be increased in CF compared to non-CF subjects. In CF patients, we hypothesize that miRNA-145 expression is elevated in association with disease progression.
    Conclusions: Our studies will identify miRNA as a potential biomarker of CFand suggest that altered miRNA expressionmay contribute to CF pathobiologyand response to therapeutic intervention. These results will complement previous in vitrowork in primary airway epitheliaand underscore the significanceof miRNA to CF pulmonary disease.

    Keywords: cystic fibrosis, CFTR, F508del, miRNA-145, biomarker

  • Garcia, Jacob

    PARAdiGM Student: Jacob Garcia
    Enhancement of pancreatic islet transplantation via islet encapsulation with a peptide amphiphile nanomatrix gel

    Pancreatic islet transplantation(PIT)is a viable treatment for Type-1 Diabetes. However,substantial pancreatic islet β-cell loss during theperitransplant period is detrimental to the efficacy of PIT.A large contributing factor to islet death is the immediate inflammatory response after transplantation. Thus, to protect the transplanted islets from the immune response, an immunoisolation strategy has been applied to encapsulate islets within a semi-permeable immune barrier. Aself-assembled peptide amphiphile (PA) nanomatrix gel enables the encapsulation of islets, while providing an immune barrier and extracellular matrix (ECM)-mimicking,islet nurturing microenvironment. In this study, the islet protective effect of the PAnanomatrix gel was assessed by culturing encapsulated isletswith differentiated inflammatory cells (U937 cells; human monocyte cell-line)for 3 and 7 days.PA nanomatrix gel encapsulated islets did not stimulate pro-inflammatory cytokine (IL-1β and TNF-α) secretionand maintained excellent islet viabilityup to 7 days. Additionally, dithizone (DTZ) staining analysis showed that PA nanomatrix gel encapsulated isletsexhibita greater number ofinsulin producingβ-cellsthanthat ofthe bare islets group. Further evaluation of islet functionality was assessed by performing a glucose stimulated insulin release testat 3and 7days.PA nanomatrix gel encapsulated islets demonstrated greater glucose stimulated insulin release than bare islets. Based on these results, the PA nanomatrix gel shows great potential for protection ofimplanted islets against inflammatory responses and increasedislet survival, which will lead to enhancedislet engraftment during the early post-transplant period.

  • Garcia-Perez, Daniela

    PARAdiGM Student: Daniela Garcia-Perez
    Understanding the Phenotypic Variability in Zebrafish Models of Limb-Girdle Muscular Dystrophy

    Muscular dystrophies are a group of diseases characterized by muscle degeneration. One of these muscular dystrophies, Limb Girdle muscular dystrophy, is a group of dystrophies that affect hip and shoulder muscles. Different mutations in the FKRP gene result in different severities of this disease. FKRP protein works by modifying the membrane protein α-dystroglycan through O-mannosylation, thus allowing α-dystroglycan to latch the myofiber cytoskeleton to the extracellular matrix, allowing for the stabilization of muscle fibers. Zebrafish are an ideal model for studying muscular dystrophies because of their transparent embryos and ability to quickly produce offspring. We have generated human FKRP pathogenic mutations in zebrafish, thus allowing us to observe zebrafish development. This technique enables us to understand the consequences of the different severities of individual FKRP mutations. Zebrafish were injected with a wildtype human FKRP gene (hFKRP), hFKRP with a mild mutation (L276I), and hFKRP with a severe mutation (C318Y). Using heat shock promoter to express these proteins, the Prestwick drug library was then used to screen for beneficial effects in the developmental phenotype of each mutant. We compared phenotypic variability of each mutation and demonstrated that a mutation in C318Y caused greater severity. Using the drug screen, therapeutic compounds that decrease abnormalities will be identified. Any abnormal phenotypic characteristics were accounted for and rated from minor, moderate, to severe. These drug groups will be further tested, in order to pinpoint which specific drug was effective and could lead to new therapies.

  • Hunter, Melodie

    PARAdiGM Student: Melodie Hunter
    SIN3A is a Key Regulator of Breast Cancer Metastasisbr>

    Metastasis occurs when cancer cells travel to and grow in distant parts of the body, away from the original site of the tumor. The study of metastasis is critical because most cancer deaths are caused by metastasis and there is a lack of treatment optionsfor patients with metastatic disease. SIN3 chromatin modification complexes have been demonstrated to play important roles regulating breast cancer metastasis and are being pursued as targets for therapy. There are two paralogs of SIN3 (SIN3A and SIN3B) that have different functions in regulating the process of breast cancer metastasis. Based on our previous work that shows an increase in cancer cell invasion and metastasis when SIN3A is decreased with shRNA and evidence for lower levels of SIN3A expression in triple negative breast cancers, we hypothesize that SIN3A is a metastasis suppressor. To test this, SIN3A was ectopically expressed in order for us to observe if higher levels of SIN3A inhibit metastatic growth. A retrovirus was used to transduce metastatic breast cancer cell lines. In vitro assays of metastatic potential including migration, invasion, and 3D growth assays were utilized. These studies will further distinguish the divergent functions between the two SIN3 paralogs so that we can better understand how to effectively target these complexes for cancer therapy. The results from this study will be especially useful for patients with triple negative breast cancer for which few targeted therapies exist.

  • Otiji, Ogechukwu

    PARAdiGM Student: Ogechukwu Otiji
    Mentor: Ganesh V. Halade PhD Role of Arachidonate 5-Lipoxygenase in Angiogenesis Post-Myocardial Infarction in Mice

    Introduction: Myocardial infarction (MI) also known as a heart attack is characterized by the lack of oxygen supply to the heart muscle which leads to necrosis of the infarcted heart muscle. The formation of new blood vessels from preexisting ones, a process also known as angiogenesis play a vital role in preventing as well as healing the damage caused by MI. Angiogenesis expedites healing of damaged heart muscle by allowing reperfusion to the infarcted area or area at risk, increasing and restoring blood flow to the damaged area and preventing hypoxia. The 5-lipoxygenase enzyme has been closely linked to angiogenesis and cell proliferation, but its specific role in dithering heart failure after myocardial infarction is not well known.5-Lox catalyzes the oxygenation of arachidonic acid which ultimately forms the bioactive compound leukotrienes.Leukotriene’s main function is in the pathogenesis of inflammation.

    Objective: The study will test the hypothesis that the arachidonate 5-lipoxygenase, will facilitate angiogenesis, and its contribution in preventing and resolving heart failure pathology post MI.

    Method: To measure angiogenesis, we analyzed 3-4 marked areas on the left ventricle using histological analysis. Dr. Halade lab provided the samples for histology after myocardial infarction surgery to mice.

    Results: the infarcted area as well as the non-infarct area were examined under a microscope at 40X magnification.

    Conclusion: GLS-1 increases post-MI in infarcted area of LV.

  • Rivero, Marco-Jose

    PARAdiGM Student: Marco-Jose Rivero
    Mentor:

    Von Willebrand factor (VWF) is a large, multimeric glycoprotein that plays an essential role in hemostasis. VWF dysfunction can lead to von Willebrand’sdisease or thrombotic thrombocytopenic purpura. Current methods of obtaining VWF from plasma remain costly and time-consuming. Recent studies have shown that DNA aptamer ARC1172 binds the A1 domain of VWF with high affinity and stability. In the present work, we employ VWF-ARC1172 binding in the development of a novel, single-step method of vWF purification. Cryoprecipitate from normal human plasma was supplemented with ristocetin and loaded onto a column packed with streptavidin agarose and biotinylated ARC1172. After washing steps, the column mixture was treated with restriction enzymes DNase I and EcoR I, and eluted with physiological buffer. EcoR I and DNase I were removed by centrifugal filtration using a 100-kDa cut-off membrane. The resultant VWF displayed high purity, appearing as a single band on SDS-PAGE gel visualized by Coomassie blue staining. However, Western blots, while confirming the presence of VWF, revealed a significant loss of larger VWF multimers. The purified VWF also demonstrated a diminished ability to adhere platelets under shear flow. These results indicate that further refinements (e.g. protease inhibitor and EDTA) are warranted to ensure that VWF retains its structure and function following purification.

  • Roberts, Steven

    PARAdiGM Student: Steven Roberts
    In vitro studies of new anticancer natural compounds in neuroendocrine tumor (NET) cell lines

    Neuroendocrine (NE) cancer is acollective name of tumors that are derived from neural crest cells, and its incidence has increased more than six-fold since 1973.Surgery represents themost viable treatment optionfor localized cancer. Unfortunately, many patients present with distant metastases making surgery ineffective,and there are limited medical therapies available. We are working in collaboration with the UAB Department of Chemistry to identify novel compounds that can effectively inhibit proliferation of neuroendocrine tumor (NET)cell lines. The objective of our study is toidentifynoveltherapeutic compounds for the treatment of NE cancers. NET cell lines, specificallypulmonary carcinoid cellswill be treated with two naturalcompounds and then assessed for cell cytotoxicity by 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect on apoptosis was assessed with flow cytometry. Possible mechanisms of growth inhibition wereanalyzed by detectingcell cycle and apoptotic protein markersby Western blot.Moreover, wetestedthe ability of these compounds toreduce NETmarkers correlated with malignant phenotypes and poor prognosessuch as achaete-scute homolog 1 (ASCL1) and chromograninA (CgA) using Western blot.We concluded that novel natural compounds haveantiproliferative and cytotoxic effects on NET.The flow cytometryrevealedpossible mechanism of cytotoxicity. Western blotting allowed the identification ofspecific proteins involved in this processand confirmedthe reduction of the highly expressed NETmarkers.

  • Thomas, Shantasia

    PARAdiGM Student: Shantasia Thomas
    The Role of Calreticulin on Extracellular Matrix Production in Diabetic Nephropathy

    Background: Diabetes is a major cause of end stage renal disease.Diabetes is characterized by hyperglycemia, which can induce endoplasmic reticulum (ER)stress and enhanceextracellular matrix (ECM)production via activation of TGF-beta signaling. Activation of TGF-betahas been shown to stimulate ECM production which is a hallmark of fibrotic diseases such as diabetic nephropathy. Our preliminary data demonstratesthat theERcalcium regulatory protein, calreticulin (CRT), modulates the expressionand processing of collagenI as well as ECM deposition. Initial studies have alsoshownthat CRT is required for glucoseandTGF-beta stimulation of ECM protein expressionin thehuman proximal tubular cell line (HK-2 cells),making it an important regulator of ECM productionin fibrotic diseases, such as diabetic nephropathy.

    Objective: The primary objective of this study is to determine whether calreticulin regulates high glucose-mediated ECM production in HK-2 cells.

    Methods: CRT will be knocked down in HK-2 cells by transfection with siRNA specific for CRT, and as a control, non-targeting(NT)siRNA. Cells will then be stimulatedwith glucosefor 72 hours. CRT knockdown andits effects on the ECM proteins,fibronectin, collagen I,and collagenIV, will be determined by Immunobloting. mRNA levels will be assessed by Quantitative Real-Time PCR.

    Anticipated Results: It is expected that downregulation of CRT will decreasethe production of ECM proteins by HK-2 cells under high glucose environment.

    Outcome of Research: This study will investigate anovel molecular mechanism underlying glucose-mediated ECM production and potentially identify CRT as a therapeutic target for treatment of fibrotic diseases, specifically diabetic nephropathy.

  • Tirado, Brian

    PARAdiGM Student: Brian Tirado
    Mentor:

    Background: Living organisms experience peaks and troughs of biological processes over a 24 hour cycle. These oscillations are essential for maintaining homeostasis in the human body and are known as circadian rhythms. Circadian rhythms are observable in all cells including cardiomyocytes (the muscle cells of the heart). These rhythms allow cardiomyocytes to anticipate sleep/wake and feeding/fasting cycles by regulating metabolism over the course of the day. For example, glycogen levels oscillate in the heart over thecourse of the day, peaking during the active/fed state. Currently, the mechanisms that drive daily rhythms of glycogen turnover are unknown. Additionally, it is unknown whether feeding or fasting cycles or circadian rhythms are the main driving force of glycogen levels.

    Objective: The objective is to investigate whether daily rhythms in glycogen turnover in the heart are secondary to feeding/fasting cycles.

    Methods: The mice were randomly divided into one of two experimental groups: ad libitum (normally)fed versus fasted. For fasted mice, food was removed four hours into the light phase. This protocol was chosen because mice exhibit a natural trough in food intake at this time of the day.Hearts were isolated from fed and fasted mice at four hour intervals over a 24-hour period. Glycogen levels were assessed using a spectrophotometric assay.

    Results: In ad libitum fed mice, glycogen levels oscillate approximately 2.8 fold throughout the day. In fasted mice, oscillations were attenuated in the heart.

    Discussion: This data suggest that feeding/fasting cycles contribute toward cardiac glycogen level oscillations throughout the day.

  • Weupe, Mason

    PARAdiGM Student: Mason Weupe
    Mentor:

    Yersinia pestis is the causative agent of plague which has devastated mankind throughout history, including Europe in the mid-1300s as the Bubonic Plague. Today Y. pestiscontinues as a threat in bioterrorism yet many aspects of its biology remain poorly understood. An important process for Y. pestis to survive during infection is transition metal homeostasis, which is the influenced by the ability of Y. pestis to sequester limited metal nutrient from the host. YfeA is a periplasmic substrate binding protein (SBP) in Y. pestis that is the only known polyspecific metal chaperone capable of binding zinc, manganese, and iron atoms, and is crucial for infection. YfeA delivers these metal nutrients that have been sequestered from the host to the YfeBCD transporter for import into the cytoplasm. To understand this process, we crystallized YfeA with (holo) and without (apo) a metal bound. To obtain apo YfeA, we engineered a laboratory strain of E. coli to express the full YfeABCD transporter and fractionated the E. coli periplasm to specifically extract YfeA that had already delivered its substrate to YfeBCD. To generate holo YfeA, we reintroduced metal substrate to apo YfeA and reconstituted the metal-bound state. X-ray scattering data from apo and holo YfeA crystals indicates metal transfer between YfeA and YfeBCD requires the unfolding and refolding of a flexible lobe in YfeA. These results reveal a novel mechanism for metal transfer that can be targeted for future antibiotic drug discovery, as well as, defense against this potential agent of bioterrorism. Results also revealed a significant binding affinity of YfeA towards zinc, as opposed to, manganese and iron. A second polyspecific binding site was observed when YfeA was co-crystallized with manganese and zinc present. The secondary binding site showed, in the presence of manganese, zinc binds.

  • Ayokanmbi, Ayo

    PARAdiGM Student: Adetokunbo Ayokanmbi
    Effectsof Tumor Microenvironments on Glioblastoma Growth

    BirminghamABSTRACTGlioblastoma (GBM) is a primary malignant and deadly brain tumor due to genetic and epigenetic differences between tumor cells. Chromodomain-helicase-DNA-binding protein 7 (CHD7)was identified as an epigenetic factor consistently repressed by acidic stress that binds to SMAD transcription factors which are downstream mediators for Transforming Growth Factor (TGF) signaling, regulating self-renewal, cell proliferation, differentiation, and migration. Also, recent studies have shown that CHD7 may act as animportant epigenetic regulator that can cause CHARGE syndrome when mutated.The BTICs and 293T cells infected with the shRNAs in the pGIPZ vector were imaged before and after puromycin selection. Brain tumor initiating cells (BTICs) and human kidney cells transfected with different shRNAs in the pGIPZ lentiviral vector were subjected to real-time quantitative PCR and western blot. The results demonstrate the expression of CHD7 decreased in the shRNAs chosen for potential testing. These results suggest thatshRNA containing the pGIPZ vector targeted the CHD7 and decreased the mRNA and protein expression of CHD7.

  • Chavez, Jennifer

    PARAdiGM Student: Jennifer Chavez
    What Role Does NreB Play in the Stress Response toHypochlorous Acid in Lactobacillus reuteri?

    Hypochlorous acid (HOCl), the active ingredient of household bleach, is one of many antimicrobial oxidants that play an important role in the innate immune system. Our interest focuses on how Lactobacillus reuteri, a Gram positive probiotic lactic acid bacterium with anti-inflammatory properties, detects HOCl and whether it is able to signal to the host to reduce inflammation in response to HOCl. Recent transcriptome data from L. reuteri exposed to HOCl demonstrated up-regulation of a gene homologous to NreB of Staphylococcus carnosus. NreB is a sensor protein involved in a two-component system that stimulates the expression of genes in nitrate respiration under anaerobic conditions. NreB regulates nitrate reductase, potentially producing nitrite and nitric oxide, which could contribute to host HOCl defenses. It is possible that this is a novel mechanism by which L. reuteri senses inflammation and signals the host to stop producing toxic oxidants, thereby reducing inflammation. We aimed to construct a mutant of L. reuteri that lacks NreB using a CRISPR Cas-9 genome editing system. We plan to test the ability of this mutant to grow when exposed to oxidants, nitrate, and other stresses. To determine conservation of the mechanisms we characterized in L. reuteri across different bacteria, bioinformatic analyses were used to compare NreB and genes potentially controlled by NreB to similar genes in other organisms. By discovering the mechanism by which L. reuteri reduces inflammation we hope to understand the symbiotic relationship between such bacteria with the human intestinal tract.

  • Moronta, Shaidy

    PARAdiGM Student: Shaidy Moronta
    Cell and Gene Therapies for Rescue of Cooley's Anemia Mouse Model

    The Ryan laboratory has developed a humanized mouse model of CA by replacing the murine adult α-and β-globin genes of hemoglobin with human α-, γ-, and non-functional β⁰-globin genes. These humanized CA mice survive on 100% human fetal hemoglobin (HbF) at birth, but succumb to lethal anemia upon completing the fetal-to-adult hemoglobin switch. The Ryan lab has developed several therapies to rescue these CA mice from lethal anemia which include regular blood transfusions, non-cyto-reductive allogenic bone marrow transplants, andtargeted gene therapy. Several genome editing approaches aimed at reactivating the silenced γ-globin gene are under active investigation. The transcription factor Bcl11a, which strongly represses γ-globin expression, has been targetedby the Ryan lab using CRISPR/Cas9 microinjection in developing embryos isolated from humanized hemoglobin switching mice. Founder mice derived from these microinjected embryos displayed a mosaic mixture of Bcl11aenhancer mutations. Droplet digital PCR (ddPCR) was employed to quantify the overall efficiency of indel formation in the Bcl11aenhancer sitewhich is important for considering BMT of gene edited bone marrow cells. Better understanding of gene editing frequency and non-cyto-reductive bone marrow transplantation could yield an effective therapy that reduces the lethality of this disease. These findings are important first steps for potential clinical applications of novel therapies for hemoglobinopathies.

  • Onatunde, Maria

    PARAdiGM Student: Maria Onatunde
    Determining whether 14-3-3theta inhibition plays a role in the regulation of a-syn release and the toxicity found in Parkinsons disease

    Parkinson’s disease(PD) is one of the most prevalent neurodegenerative diseases. One of the pathological indicators of PD are Lewy bodies that form due to the misfolding and aggregation of alpha-synuclein (a-syn). Although the cause of this disease is still unknown, evidence supports that the protein a-syn plays a large role in this disease. Misfolded alpha-syn can be released by neurons and spread to other areas of the brain by infecting other neurons and further inducing further a-syn misfolding in a prion-like manner. Colocalized with a-syn in the Lewy bodies of Parkinson’s disease is a family of seven proteins with chaperone-like functions called 14-3-3s(Kawamoto et al 2002). Out of the seven isoforms, 14-3-3theta has shown to be protective in multiple models (Yacoubian etal 2010). We have developed a paracrine culture model in which a-syn is overexpressed in cultured neuroblastoma cells (isyn cells) that releases excess, toxic a-syn to separately cultured neurons. Using difopein (pan inhibitor of 14-3-3) on the cells, we find that there is a decrease in a-syn release but an increase in toxicity. When the isoform 14-3-3theta is overexpressed in these isyn cells, we find that there is an increase in a-syn release, but a decrease in toxicity. Using the culture model which a-syn is over expressed, we used the shRNA lentivirus directed against 14-3-3theta. We successfully knocked down 14-3-3theta and we plan on using these 14-3-3θ KD isyn cells to analyze a-syn release by western blot and measure the corresponding toxicity. We will test if the absence of this isoform alone will alter release and toxicity or if the other six isoforms will compensate in its absence.

  • Pulliam, Alexis

    PARAdiGM Student: Alexis Pulliam

    The understanding of memory loss in epileptic patients is still incomplete. Many patients suffer from traumatic loss of memory, and pharmaceutical treatments are often ineffective. To better understand epilepsy, we are studying the epigenetic changes associated with the disorder. Epigenetics is the study of the modification of gene expression. One of the most extensively studied mechanisms is DNA methylation. DNA methylation is the process of silencing genes by adding a methyl group directly to the DNAat specific sites on the nucleotide residues. In epileptic rodents, the Bdnf gene expression is increased. Previous studies used methyl supplements and treatments to manipulate DNA methylation on Bdnf on a global scale. DNA methylation can decrease the expression of the gene. However, no one has used a targeted method to increase DNA methylation. Therefore, the objective of the project is to target DNA methylation at the Bdnf exon IV using a CRISPR effect system. Plasmids were grown and the endonuclease restriction sites, BstXI and XhoI, cleaved the sequence creating a fragment with sticky ends. The new target specific sequence was ligated into the plasmid. This will be used in conjunction with the dcas9-DNMT3a to mediate site-specific methylation. Inthe cell, the Bdnf gene expression will decrease and methylation will increase, potentially impacting memory in rodent models.

  • Roberts, Steven

    PARAdiGM Student: Steven Roberts
    Three Dimensional Cell Culture Model for Drug Efficacy Testing

    Cystic fibrosis (CF) is a chronic disorder generated by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel. CFTR has over 2,000 mutations causing abnormalities in many organs, predominately in the lungs. Recent studies havedeveloped CFTR modulators, which increase the activity of CFTR. However, these drugs are not applicable to all CF mutations. Our research is directed towards optimizing ex vivo assays for responses to different drugs, which will advance CF therapy toward a more individualized approach when applied in a clinical setting. The study is designed to develop 3-D organoid models of human nasal epithelium (HNE). Two sphere formations were created through cell culturing, “apical membrane out” (AMO) and “apical membrane in” (AMI). AMO spheres shrink in response to CFTR activation, and AMI spheres swell in response. Both models were sectioned for H&E staining allowing cross sectional analysis and imaging. 3-D models were also fixed and immunofluorescence (IF) was performed. Photomicrographs were taken of spheres displaying their morphology in relation to that of a human nasal epithelium. We obtained AMO H&E stained spheres that are ciliated and AMI spheres with mucus formation, displaying differentiation. We are currently developing IF microscopy protocol to observe other proteins that indicate differentiation. Future directions would be to continue adjusting culturing conditions to optimize differentiation, to evaluate other markers of differentiation, and to test drugs to stratify the response of the spheres and determine which is most discriminating to judge effectiveness.

  • Thomas, Shantasia

    PARAdiGM Student: Shantasia Thomas
    Inhibition of HGF activation, a: Novel Approach to Overcome Resistance to MET Targeted Therapy

    Aberrant HGF/MET signaling supportssurvival,proliferation, invasion, and metastasis of cancer cells. Lung cancer cells with amplified MET are sensitive to MET kinase inhibitors. Our preliminary data demonstrated that hepatocyte growth factor (HGF) inhibits the responsiveness of MET amplified cancercells to the MET tyrosine kinase inhibitor (TKI), JNJ-38877605. HGF is secreted in an inactive form called pro-HGF. Proteolytic processing of the single-chain pro-HGF to the active (mature) heterodimeric form by one of the serine proteases, hepatocyte growth factor activator (HGFA), hepsin, or matriptase is required for HGF/MET signaling. We developed a novel inhibitor of HGF activation, SRI 31215, and tested its ability to overcome HGF-mediated resistance to JNJ-38877605 in lung cancer cells with amplified MET. The primary objective of this study was to show that concurrent inhibition of the ligand (HGF) and its receptor (MET) is required to inhibit proliferation of MET-amplified lung cancer cells. We showed that both recombinant HGF and HGF-producing fibroblasts inhibit the sensitivity of MET-amplified lung cancer cells to JNJ-38877605. Inhibition of HGF activation by SRI 31215 overcomes fibroblast-mediated resistance to JNJ-38877605. Taken together, we have demonstrated that small-molecule inhibitors of HGF activation, such as SRI 31215, would greatly improve the outcome of lung cancer patients that display amplification of the MET receptor.

  • Torres, César

    PARAdiGM Student: César Torres
    Cytomegalovirus Strain Diversity in Infants with Confirmed Congenital Infection

    Introduction: Cytomegalovirus (CMV) is the most common form of congenital viral infection, affecting tens of thousands of newborns each year. Because of its prevalence and role in causing hearing loss and other disabilities in children, CMV has been extensively studiedover the years(1). Through these investigations, it has been shown that the virus displays a wide range of genetic mutations and polymorphisms that lend to its unique strain diversity(2).
    Objectives: Theprimary objective of this research is to fully characterize the strain variability of CMV seen in infants with confirmed congenital infection (cCMV)born to mothers with primary CMV infection that were treated with CMV hyperimmune globulin.
    Methods: Urine and saliva samples were collected from infants within the first few days of life. Infants were born to mothers with primary CMV infection during pregnancy. The mothers we re-enrolled in an efficacy study for hyperimmune globulin (HIG) as a treatment to inhibit the intrauterine transmission of the virus tothe fetus. The samples obtained from these infants were then evaluated for strain diversity of three selected glycoprotein genes –gN, gB, gH. gN genotyping was conducted through PCR of viral strains followed by culture and sequencing. Whereas gB and gH genotyping was performed by real-time PCR assays.
    Results: Of the 23 infants, mixed infection with more than one CMV strain was found in all 23. Genotyping of gN clones shows that strains gN3a and gN3b predominate in both saliva and urine compartments. Only6% of clones showed genotypes other than these two primary genotypes observed (gN1, gN4a, gN4b). gN3a represented 64% of clones genotyped, whereas gN3b represented 30%. As for gB genotyping, only 21% (5/23) of samples examined showed single strain infection in the saliva group, and about 17% (4/23) for urine. Also, genotyping of gH strains showed that 100% of both compartment samples displayed infection by both gH strains.
    Conclusion: This study demonstrated the commonality of multiple strain infectionin infants born to mothers with primary maternal CMV infection. In addition,variability ofCMVstrain infectionisseen in the salivary and urinary compartments. It is unknown whether or not the diversity of strains observed with CMV infection is associated withsymptomatic or asymptomatic infection.

  • Veizaga-Zurita, Melanie

    PARAdiGM Student: Melanie Veizaga-Zurita
    The Role of miR-214, miR-219, & miR-876 in Bronchopulmonary Dysplasia

    Introduction: Bronchopulmonary dysplasia (BPD) is the chronic lung disease of prematurity characterized by inadequate alveolar septation and lung inflammation. MicroRNAs (miRs) are small non-coding RNAs that down regulate the gene expression of target mRNAs. The role ofepigenetic regulation of alveolar septation by miRs is not well studied. We hypothesized that the expression of miR-214, miR-219, miR-876 and their corresponding predicted target genes (Cox-2, PDGFRα, MCL-1, RBBP6) would be dysregulated with hyperoxia exposure, in vitro. Methods: Human bronchial epithelial cells were grown via cell cultures and exposure to normoxia (21% O2) and hyperoxia (85% O2) conditions for both 12 and 24 hours. After total RNA was isolated, miRNA and mRNA analysis was performed via reverse transcription and qPCR. Results: miR-214 and miR-219 both increased at 24 hrs under hyperoxia (fold change= 1.7 and 1.3 respectively) while miR-876 decreased in expression (0.6 fold change). All the mRNAs (Cox-2, PDGFRα, MCL-1, RBBP6) showed an increase in expression at 24 hr hyperoxia with Cox-2 having the most drastic increase. For RBBP6, there was great variability in data at 12 hr hyperoxia with no clear outlier. Discussion: An inverse miR-mRNA relationship has been observed between miR-214 and Cox-2 as well as miR-219 and PDGFRα in previous studies. Hence there is a possibility that our result are due to a random error, and require further testing. Alternatively, these miRNA-mRNA regulation may be specific to non-epithelial cells, which we haven’t tested. The down regulation of miR 876 with a corresponding up regulation of MCL-1 with hyperoxia exposure, may explain the lung inflammation seen in BPD. RBBP6 expression under hyperoxia requires further testing due to the high variability of data seen.

  • Washington, Joshua

    PARAdiGM Student: Joshua Washington
    Effects of Ghrelin and Restricted Time Feeding on Circadian Clock Rhythms

    The suprachiasmatic nucleus (SCN), the primary pacemaker of the circadian clock, coordinates 24-hour oscillations throughout the brain and periphery that prepare the body for internal and external changes. When these central and peripheral clocks desynchronize with one another and/or with the environment, negative health effects may ensue, such as increased adiposity and cognitive decline. When external synchronizing signals (e.g., light and food availability) are in conflict with each other, the SCN is primarily reset by light exposure, but other pacemakers within the brain (e.g., hippocampus) and periphery (e.g., liver) are synchronized by food intake. The hunger hormone ghrelin is released in a 24-h rhythm, in anti-phase to food intake; however, it is not known how food intake, lighting conditions, and ghrelin receptor activation interact to synchronize central and peripheral clocks. Therefore, this project will test whether ghrelin receptor activation that is mis-timed with food intake will differentially entrain circadian clocks in the hippocampus, liver, and SCN. Using a computerized time feeder, two groups of mice (N = 10/group) will be fed pellets containing a ghrelin agonistat the beginning of lights on or lights off. These groups will be further divided into groups that are fed on either a light-restricted or dark-restricted 12-h availability schedule. We will measure the peak expression of the clock gene, Period2(Per2), using a knock inreporter mouse model that rhythmically produces PER2-Luciferase protein. Chronic bioluminescence monitoring of organotypic cultures of the SCN, hippocampus and liver will be used to determine circadian clock phase. It is expected that the phase of the clock gene Per2 in the SCN will be unaffected by food or ghrelin, but that the restricted feeding during the day will shift the clock earlier in the liver. In groups in which the ghrelin agonist is delivered at the end of the 12-h feeding period (i.e., mis-timed), the effect of day-restricted feeding will be dampened.

  • Arguello, Alexandra

    PARAdiGM Student: Alexandra Arguello
    Dynamin-2 is a negative regulator of NOS1βin the collecting duct

    Nitric oxide synthase 1 (NOS1)/NO pathway in the inner medullary collecting duct (IMCD) promotes natriuresis. Rat IMCD express NOS1α and NOS1β splice variants, while mouse and human IMCD expresses predominantly NOS1β. We reported dynamin 2 (DNM2) activatesNO production via direct interaction with NOS1α; thus we further hypothesized thatDNM2 also activates NOS1β. To test this hypothesis, we used COS7 cells, which lack endogenous NOS, and mIMCD-3 immortalized cells that express NOS1β. By immunoprecipitationand western blot, we confirmed that NOS1β interacts with DNM2. Nitrite production (an index of NO production) was assessed by HPLC, and we determined that overexpression of DNM2 in mIMCD-3 cells resulted in a significant reduction in nitrite production (323 ± 25 pmol/mg pr/h vs. 1734 ± 35, N = 3-4, P = 0.016). Next, we used siRNA toinhibit the expressionof DNM2. DMN2siRNA resulted in a 95% reduction in mIMCD-3 DNM2 expression and produced a significant increase in nitrite production (scramble siRNA 112 ± 15 vs DNM2 siRNA 249 ± 21 pmol/ mg pr/ h). Toconfirm the cell culture experiments, in vivo mice experiments were performed. Freshly isolated mouse IMCD’s were treated with dynasore (a DNM2 inhibitor), which resulted in a significant increase in nitrite production(530 ±32 vs 1118 ±59, p = 0.03). By immunoprecipitation and western blot, we confirmed that NOS1βand DNM2 also interact in vivo. As opposed to our initial hypothesis, these data indicatethat DNM2 is a novel negative regulator of NOS1β.

  • Bailey, Thomas

    PARAdiGM Student: Thomas Bailey

    My research is on diabetic cardiomyopathy, specifically the epigenetics. Which is the alteration of gene expression that can be both transient and inherited.While working in Dr. Wende’s laboratory I plan to test the hypothesis that TET3 is regulated in the methylation of DNA. Specifically we will examine the glycemic memory, which is the genetic code to regulate the glucose being used in the heart, and how this modification in DNA creates a change in the mitochondrial function and metabolism of cardiac cells. The laboratory has previously mappedDNA methylation and hydroxymethylation epigenetic markers suggested to contribute to the maintenance of glycemic memory. These earlier studies have pointed to the TET (ten eleven translocation) family of enzymes, which have shown to regulate DNA methylation. My project will be to examine this regulation at a number of different levels including protein, DNA, and RNA. Specifically I will beusing Western Blots, Data Mining, PCR tests, and potential cloning the promoter regions to determine regulation incell cultures. Ultimately, I plan to study how TET is related to the methylation of DNA in the diabetic mouse modelsand how glucose might initiate thesechanges.

  • Jones, Alana

    PARAdiGM Student: Alana Jones

    Hyperoxia is the exposure of the lungs to excess O2. Prolonged exposure can lead to oxygen toxicity, alveolar death, and even chronic lung injury. Platelet-activating factor (PAF) is a phospholipid mediator with numerous functions, one of whichincludes playing a major role in the body's pro-inflammatory response. This summer, I will be working with neonatologists Dr. Ambalavanan and Dr. Jilling to study the role of PAF in hyperoxia-induced lung injury. My research will include in vitro and possibly in vivo studies on Dr. Jilling’s PAF mice. However, because of time constraints, mice work will be limited. Instead the majority, if not all, of the sample tissues will be previously collected, and those tissues, not the mice, will be exposed to hyperoxia. For example, we will put expose PAF-deficient, PAF-overexpressing, and Wild Type lung tissues to 100% O2 in the hyperoxia chamber and then study the gene expressions of the enzymes that synthesize PAF. Because PAF is synthesized from a series of enzymatic remodeling, studying the gene expression of these enzymes in conjunction with tissue culture studies will allow us to draw conclusions about the extent of PAF’s impact on the cells during hyperoxia. Results should give insight on platelet-activating factor’s role in chronic lung injury resulting from hyperoxia.

  • Jones, Keri

    PARAdiGM Student: Keri Jones
    Effects of a Protein-blend Nutritional Supplement on Resistance Exercise-induced Protein Synthesis Signaling within Skeletal Myofibers

    Muscle atrophy and reduced functional capacity are well-established, deleterious consequences of acute and chronic conditions. Skeletal muscle mass is determined largely by the rates of muscle protein synthesis (MPS) and degradation (MPD), each of which occur daily under regulation of cellular and molecular controls. The cellular protein synthesis pathways in skeletal muscle cells (myofibers) are acutely responsive to protein-based nutritional interventions; thus, there is an interest in assessing potential synergism when resistance exercise is accompaniedbyadjunctive nutritional supplementation.

    This study evaluated how a combined (exercise + protein) treatment affectsnet protein synthesis as indicated by MPS/MPDsignaling responses. Samples from 10 human subjects randomized to perform two bouts of knee extension resistance exercise with or without the nutritional supplementwere collectedThese de-identified muscle tissue samples were provided to UAB’s Core Muscle Research Laboratory for analysis.Acute (3hr) cell signaling responses were determinedby western blotting. Translation initiation signaling (the rate limiting step of MPS) was investigated by quantifying Akt/mTOR, S6K1/rpS6, GSK3/elF2B, and ERK 1/2activation. MPD pathway activation was assessed via NF𝜅Band ubiquitin proteasome signaling.

    This data may lead to further investigation of the role of protein-blend supplements in increasing the metabolic response to resistance exercise.

    Keywords: Resistance exercise, muscle atrophy, nutritional supplement

  • Moronta, Shaidy

    PARAdiGM Student: Shaidy Moronta
    Activation of γ-globin to Result in Adult Persistence of Fetal Hemoglobin Using CRISPR/dCas9-Ldb1 as a Therapy for Cooley’s Anemia

    β-thalassemia is a group of inherited blood disorders that results in defects in β-globin chain production. Cooley’s anemia (CA), or β-thalassemia major,is the most severe form of the disease and occurs when an individual has mutations in both copies of the adult β-globin gene. Patients with CA fail to make adult hemoglobin, have ineffective erythropoiesis, suffer from severe anemia, and are transfusion dependent for life. Based on the fact that individuals with persistent fetal hemoglobin expression in adulthood are protected from otherwise deleterious β-globin mutations, this project aims to reactivate endogenous fetal hemoglobin in erythroid cells.

    The first phase of this project is to engineer a lentiviral vector that contains the dCas9 gene fused to Ldb1 and a CRISPR single guide RNA. The construct will be verified by restriction digests and sequencing. The vector will encode proteins that direct Ldb1 to the promoter of the fetal globin genes in the hopes of producing a sustainable amount of γ-globin toreplace the function of the absent β-globin.

    The next phase will be to transfect the vector into a packaging cell line to produce a lentivirus capable of transducing hematopoietic stem cells. In the long run, genetically modifiedhematopoieticstem cells collected from bone marrowof CA mice will betransplantedinto recipient CA mice. If successful, the recipient mouse will produce γ-globin sustainably for the rest of its lifetime, thus providing a potential cure for Cooley’s anemia.

  • Onatunde, Maria

    PARAdiGM Student: Maria Onatunde
    Ten-week targeted training program to improve balance and walking post stroke: A Case Study

    One of the most common disabilities found in people post-stroke is a lack of motor control on one side of their body (i.e., the side contralateral to the brain damage). With research performed at the Locomotor Control and Rehabilitation Robotics Laboratory(www.uab.edu/locolab), we have identified five domains that determine the extent of a person’s walking disability: balance, speed, loco motor challenge, force, and endurance.When working to rehabilitate participants, the participant is tested oneach of these five domains and then performs exercises designed for the domain they performed the lowest on. In order to determine the actual effectiveness of this system, data is collected from sessions and analyzed to see the progression within a session (online learning) and between sessions (offline learning) for the duration of the two weeks in which the participant works on the particular domain. To date, we have collected data from one participant who was engaged ina ten week programinvolving sessions of endurance, speed generation, dynamic balance, and loco motor challenge. The participantwas 64 years old, 25 years post stroke, with left sided hemiparesis. We found that both online and offline learning vary from task to task and domain to domain.With this single case example of a participant involved in the 10 week training program, we see trends that show the effectiveness of the exercises within each domain. Further conductionof this study will answer important questions about the effectiveness on outcome measures related to activities and participation.

    Keywords: Physical Therapy, Stroke, Walking, Balance, Rehabilitation

  • Pettway, Yasminye

    PARAdiGM Student: Yasminye Pettway
    Digeranyl bisphosphonate attenuates pulmonary fibrosis by the modulation of mitochondrial dynamics

    Pulmonary fibrosis is a chronic, devastatingdisease, having an average life expectancy of 3-5 years after diagnosis in certain cases. In chronic disease, macrophages have apoptosis resistance, which is associated with disease progression through the secretion of growth factors that stimulate fibroblasts to differentiate and produce collagen. Two processes integral to apoptosis resistanceare mitophagy and mitochondrial biogenesis. Previous data shows that apoptosis resistance in macrophages is due to greater activation of the mevalonate pathwayvia the geranylgeranylation of the small GTPase Rac1. Moreover, mice with a conditional deletion of Rac1 in macrophages are protected from fibrosis development. Digeranyl biphosphonate (DGBP) attenuates pulmonary fibrosis by inhibiting geranylgeranyl diphosphate (GGPP) synthase, an enzyme that activates Rac1. This project hypothesizes that disruption of the mevalonate pathway by treatment with DGBP mediates apoptosis by modulation of mitophagy and mitochondrial biogenesis in alveolar macrophages. Macrophageswere exposed to chrysotile in the presence or absence of DGBP. Chrysotile increases mitochondrial ROS production, whereas DGBP-treated cells showed decreased Rac1 activity in the mitochondriaanddecreased ROS production. Mitophagy and mitochondrialbiogenesis were inhibited by DGBP treatment. DGBP induced apoptosis, as evidenced by increased caspase-3 activity in treated cells. Additionally, treatment with DGBP led to lower expression of TGF-in macrophages. The reduction in TGF-resulted ininhibition of fibroblast differentiation and significantly less fibronectin and collagen1A production in fibroblasts exposed to conditioned media. These results suggest that targeting the mevalonate pathway, specifically the inhibition of Rac1 activation, may be a therapeutic targetfor pulmonary fibrosis, as the disease currently has no treatment.

  • Tymes-Wilbekin, Kristina

    PARAdiGM Student: Kristina Tymes-Wilbekin

    The CDC has acknowledged the growing levels of obese children and with it the likelihood of an increasing percentage of obese adults in the coming years. This problem raises alarm because more people will be susceptible to heart disease, type 2 diabetes and other weight related illnesses. We are conducting an analysis of child obesity related research to examine the significance in different methods for improving body composition. We will review 14 studies that examined the effects of behavioral therapies on weight change (reduced weight gain while growing or reduced body fat percentage) in children. The studies collected are randomized control trials (RCTs) from a systematic review that documented weight change as a primary or secondary outcome. Using accepted meta-analytic methods,we will examine the studies collectively quantify effects to determine whether behavioral therapies are effective in improving body weight/body compositionamong children. The data collected will be used to statistically estimatemean values of weight loss and Body Mass Index (BMI) seen amongst all of the studies. From the data,we will develop forest plots to summarize the effectiveness of the behavioral interventions versus the control group and also assess the quality of the results by creating a risk of bias assessment. The results collected will analyzed to determine whether behavioral therapies have a statisticallysignificanteffect on improving body composition of children. The results can then be utilized by numerous organizations to inform treatment ofpatients, create policies and better inform the public.

  • Tshimbombu. Tshibambe

    PARAdiGM Student: Tshibambe Tshimbombu
    Intrinsic connectivity is related to antipsychotic treatment in schizophrenia

    For patients with schizophrenia, antipsychotic drug treatment is variable –patients respond differently to different drugs –and currently unpredictable. This may be due to the fact that functionally connected areas are intrinsically wired in a way that favor or do not favor treatment response. Inadequate treatment predicts poor long-term patient outcomes, and ~25% of all patients with schizophrenia switch medications each year in an effort to better control their symptoms. Switching medications is a lengthy and expensive process, which exposes patients to significant risk of relapse and hospitalization. However, by measuring the intrinsic connectivity, a measure of voxel to voxel connectivity (VVC), there may be a possibility to identify brain areas that respond positively to antipsychotics. Thus, adequate treatment can be developed to improve patient outcomes. These voxels connectivity are computed using the intrinsic connectivity contrast (ICC),which assesses the amount of disturbance in connectivity occurring in patients with schizophrenia compared to controls, the impact of antipsychotic medication on connectivity, and whether or not the pattern of connectivity before beginning antipsychotic therapy predicts treatment response after six weeks.

    We compared a group 34 non-medicated schizophrenics (SZ) and 46 matched healthy controls (HC). SZ were scanned while off medication, and thenthey were treated with risperidone, antipsychotic, for six weeks and re-scanned. Throughout the treatment period, mental status and symptom severity in SZ were assessedusing the Brief Psychiatric Rating Scale. Afterward, ICC was used to measure the strength of the total connectivity pattern between a specific voxel and the rest of the voxels in the brain.

    Prior to treatment there was a widespread differences in VVC between HC and SZ, with HC having greater cortical VVC and SZ having greater subcortical, particularly thalamic, VVC. After six weeks of treatment with risperidone, SZ appeared to significantly normalize but still presented aberrant VVC. These findings suggest that antipsychotics decrease psychotic symptoms significantly in brain areas that havestrongerVVC pattern.

  • Washington, Joshua

    PARAdiGM Student:
    Inducing Reactive Gliosis Usinga Pediatric Traumatic Brain Injury Model

    Traumatic brain injury (TBI) is the leading cause of death and disability in young children under the age of four. Once thought to be more resilient to brain trauma it is now appreciated that young children are more likely to experience cerebral edema and post-traumatic seizures relative to their adult TBI patients. Furthermore, they are at high risk for lasting injuries. The developing brain is inherently different from that of an adult inregard to protein expression, cellular packing density and water content. Young children are also more likely to experience diffuse brain injury. Despite these differences in anatomy and outcome, nearly all treatment strategies for children are extrapolated from adult TBI studies. This represents a major obstacle for developing appropriate treatment strategies for the pediatric population.

    Astrocytes are the most numerous cells in the brain and perform many essential homeostatic functions. In response to trauma, astrocytes in the brain become reactive, which appears to disrupt an astrocyte’s normal functions: regulation of glutamate in the extracellular fluid, maintenance of the blood brain barrier (BBB), and pH. This can lead to BBB breakdown, glutamate excitotoxicity, and edema, contributing to neuronal dysfunction following TBI. In the current study we aim to develop a clinically relevant model of diffuse brain injury in young rodents. We focus on aspects of reactive gliosis including BBB breach and edema to more accurately model TBI in pediatric patients.

    Keywords: Pediatric,TBI,Astrocyte,ReactiveGliosis

  • Bailey, Thomas

    PARAdiGM Student: Thomas Bailey
    Mentor: Adam Wende (arwende@uab.edu)

    My research is on diabetic cardiomyopathy, specifically the epigenetics. Which is the alteration of gene expression that can be both transient and inherited.While working in Dr. Wende’s laboratory I plan to test the hypothesis that TET3 is regulated in the methylation of DNA. Specifically we will examine the glycemic memory, which is the genetic code to regulate the glucose being used in the heart, and how this modification in DNA creates a change in the mitochondrial function and metabolism of cardiac cells. The laboratory has previously mappedDNA methylation and hydroxymethylation epigenetic markers suggested to contribute to the maintenance of glycemic memory. These earlier studies have pointed to the TET (ten eleven translocation) family of enzymes, which have shown to regulate DNA methylation. My project will be to examine this regulation at a number of different levels including protein, DNA, and RNA. Specifically I will beusing Western Blots, Data Mining, PCR tests, and potential cloning the promoter regions to determine regulation incell cultures. Ultimately, I plan to study how TET is related to the methylation of DNA in the diabetic mouse modelsand how glucose might initiate thesechanges.

  • Cochrun, Steven

    PARAdiGM Student: Steven Cochrun
    Mentor: Dr. Lesley Smythies, Dr. Philip Smith

    Crohn’s diseaseis achronic inflammatory disease of the intestinal and colonic mucosa. Genetics and environmental factors, particularly the microbiota, play predisposing roles in the inflammatory process. Adherent-invasive E. coli (AIEC,LF82) is a Gram-negative bacterium that reportedly drives inflammation in the terminal ileum of patients with Crohn’s disease(Darfeuille-Michaud, Inflammatory Bowel Disease, 2007).The role of intestinal macrophages in this inflammatory response has not been fully defined. Therefore, we investigated the responses of blood monocytes, the source of intestinal macrophages, and intestinal macrophages isolated from normal subjects and patients with Crohn’s diseaseto LF82 in vitro.

    LF82 stimulated monocyte,but not intestinal macrophage, activation, as evidenced by increased expression of surface receptors CD80 and CD86 and the differentiation molecule HLA-DR. Consistent with this activation profile, LF82 and Salmonella LPS induced blood monocytes, but not intestinal macrophages, to express high levels of the pro-inflammatory cytokinesIL-1β, IL-6 and TNF-α. Confirming the inducible cytokine production by monocytes but not intestinal macrophages, qRT-PCR analysis showed LF82 induced monocytes, but not intestinal macrophages, to express high levels of mRNA for IL-1β, IL-6, and TNF-α.

    In summary,blood monocytes display LF82-stimulated phenotypic and functional (pro-inflammatory) responses, which appear to beregulated at the transcriptional level, whereas intestinal macrophages do not. This inability of intestinal macrophages, which are derived from blood monocytes, to be activated by or respond to, inflammatory signals such as LF82 and LPS is consistent with the down-regulation of monocytes newly recruited to the mucosa.

  • Jones, Alana

    PARAdiGM Student: Alana Jones
    Mentor:Namasivayam Ambalavanan MD (ambal@uab.edu)

    Hyperoxia is the exposure of the lungs to excess O2. Prolonged exposure can lead to oxygen toxicity, alveolar death, and even chronic lung injury. Platelet-activating factor (PAF) is a phospholipid mediator with numerous functions, one of whichincludes playing a major role in the body's pro-inflammatory response. This summer, I will be working with neonatologists Dr. Ambalavanan and Dr. Jilling to study the role of PAF in hyperoxia-induced lung injury. My research will include in vitro and possibly in vivo studies on Dr. Jilling’s PAF mice. However, because of time constraints, mice work will be limited. Instead the majority, if not all, of the sample tissues will be previously collected, and those tissues, not the mice, will be exposed to hyperoxia. For example, we will put expose PAF-deficient, PAF-overexpressing, and Wild Type lung tissues to 100% O2 in the hyperoxia chamber and then study the gene expressions of the enzymes that synthesize PAF. Because PAF is synthesized from a series of enzymatic remodeling, studying the gene expression of these enzymes in conjunction with tissue culture studies will allow us to draw conclusions about the extent of PAF’s impact on the cells during hyperoxia. Results should give insight on platelet-activating factor’s role in chronic lung injury resulting from hyperoxia.

  • Mason, Zavier

    PARAdiGM Student: Zavier Mason
    Mentors: Tim Townes (ttownes@uab.edu)
    Brugada Syndrome

    Inherited Arrhythmia Syndromes are genetically inherited heart disorders that usually result in Sudden Cardiac Death. This syndrome affects from 1-2000 to 1-10,000 people with a higher prevalence in men. Brugada Syndrome, a form of inherited Arrhythmia, is caused by a mutation in theSCN5A gene, which codes forthe cardiac sodium ion channel. The mutation in SCN5A is an autosomal dominant inherited disorder and has proved to have 20-30% involvement in patients.This disease is usually not detected until after Sudden Cardiac Deathsinceit is particularly hard to detect on anECG. With death usually occurring at night it is the leading cause of death in young men residing in the countries of East and Southeast Asia. There is also proof that Brugada Syndrome is responsible for most of the Sudden Cardiac Deaths in young men seen around the world.Currently there is no successfultreatmentfor Brugada syndrome. Unlike other Inherited Arrhythmia Syndromes Brugada syndrome does not respond to Beta-blockers. We plan to produce induced Pluripotent Stem Cells (iPSC) from skin fibroblasts of Brugada Syndrome patients and to use CRISPR-Cas to correct theSCN5Agene. Corrected iPSCs will be differentiated into cardiomyocytes and analyzed for correct sodium ion channel function. These studies will provide a foundation for new therapies for this debilitating disorder.

  • Tymes-Wilbekin, Kristina

    PARAdiGM Student:
    Mentor: Kathryn Kaiser (kakaiser@uab.edu); David Allison (dallison@uab.edu)

    The CDC has acknowledged the growing levels of obese children and with it the likelihood of an increasing percentage of obese adults in the coming years. This problem raises alarm because more people will be susceptible to heart disease, type 2 diabetes and other weight related illnesses. We are conducting an analysis of child obesity related research to examine the significance in different methods for improving body composition. We will review 14 studies that examined the effects of behavioral therapies on weight change (reduced weight gain while growing or reduced body fat percentage) in children. The studies collected are randomized control trials (RCTs) from a systematic review that documented weight change as a primary or secondary outcome. Using accepted meta-analytic methods,we will examine the studies collectively quantify effects to determine whether behavioral therapies are effective in improving body weight/body compositionamong children. The data collected will be used to statistically estimatemean values of weight loss and Body Mass Index (BMI) seen amongst all of the studies. From the data,we will develop forest plots to summarize the effectiveness of the behavioral interventions versus the control group and also assess the quality of the results by creating a risk of bias assessment. The results collected will analyzed to determine whether behavioral therapies have a statisticallysignificanteffect on improving body composition of children. The results can then be utilized by numerous organizations to inform treatment ofpatients, create policies and better inform the public.