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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Control of Early Postnatal Brain Homeostasis by IL-33Signaling and Microglia
Drew Bookman, Dr. Shin-ichi Kano, Destynie Medeiros
The University of Alabama at Birmingham, Departmentof Psychiatry and Neurobiology

Microglia, members of neuroglia derived from yolk-sacprimitive myeloid progenitor cells, act asthe primary form of active immune defense in the CNSand play a central role in braindevelopment, homeostasis, and disease. Microglia recognizebrain tissue damage by sensingdamage-associated molecular patterns (DAMPs), secretinginflammatory mediators, andphagocytosing dead cells or debris. In particular,microglia are known to remove debris madefrom cell death during early brain development. Interleukin(IL) -33 is a cytokine called “alarmin”that is secreted from damaged cells and alarms surroundingcells. Microglia express thereceptor for IL-33 (ST2) and are the primary cellsthat respond to IL-33 in the brain. Previousstudies in the lab have found that microglia activationin IL -33 -/- mice are sustained duringearly postnatal development. Preliminary findingshave also suggested that microglia increasein IL- 33 -/- mice. Based on this data and the above-mentionedrole of microglia, I hypothesizethat a loss of IL-33 in early postnatal developmentreduces microglial phagocytic activities,resulting in parenchymal damage with uncleared debris.Thus, within this study, I willcharacterize microglial abnormalities and relatedneuronal damage in the brains of IL-33 -/- miceduring the early postnatal period by immunohistochemistry.The findings of the proposed studywould reveal how IL-33 signaling affects microglialphagocytosis and keeps neurons from beingdamaged during early brain development.

Haploinsufficient expression ofSON in ZTTK syndrome leads to aberrant splicing of proline-rich transmembrane protein 2 (PRRT2)
Deanine Dilworth, Lana Vukadin, Inga Tytler, Hanna Taglinao, Huashi Lee, Erin Ahn

A novel multisystem developmental disorder, named the ZTTK syndrome, has recently been identified by our lab and others. The ZTTK syndrome is caused by loss-of-function (LoF) mutations in the SONgene. Although the SON protein has been described as a splicing regulator and transcriptional repressor, its effect on development and its contributions to the ZTTK syndrome still remain vastly unknown. The ZTTK syndrome patients suffer from several disabilities, including delayed organ development, hematopoietic disorders, and severe intellectual disabilities that are often accompanied with seizures and epileptic episodes. We examined genes that are abnormally expressed in ZTTK syndrome patients to try to uncover potential mechanisms that could explain the occurrences of seizures and epilepsyand .Iinterestingly, we saw a markedly decreased expression of proline-rich transmembrane protein 2 (PRRT2). PRRT2 is mostly associated with paroxysmal kinesigenic choreoathetosis (PDC), a hyperkinetic movement disorder, and benign infantile epilepsy. To observe the relationship between SON and PRRT2, we depleted SON in U87MG and SH-SY5Y neuronal cell lines. PRRT2 expression is markedly decreased upon SON depletion, giving us the idea that PRRT2 is directly regulated by SON. Moreover, we initially observed thatSON depletion leads to PRRT2 intron 2 retention. Therefore, we hypothesize that the depletion of SON leads to aberrant splicing of PRRT2 mRNA, leading to decreased expression of the PRRT2 protein, subsequently contributing to seizures and epilepsy in ZTTKsyndrome patients. To test this hypothesis, we will deplete SON using small interference RNA (siRNA) methods in our neuronal cell lines, and evaluate the expression and splicing of PRRT2. This data will provide closer insight into mechanisms that could help alleviate ZTTK syndrome patient suffering.

Alternative Splicing Assessment of Developmentally Regulated Neuronal Microexons Associated with Schizophrenia and Autism

Introduction: Schizophrenia and autism are neuropsychiatric disorders whose pathways are still greatly unknown. While there has been some progress in the development of treatments for schizophrenia and autism, there is still much left to be discovered about the illnesses, current drug therapies for them do not eradicate their progression completely. In this study, we utilize zebrafish larvae to analyze 96 microexon genes associated with schizophrenia and autism and perform a phenotypic functional analysis of the microexons, some of which have shown to alter synaptic plasticity and behavior in mice.

Methods: To acquire the mutant lines, we microinjected wild type zebrafish with CRISPR/Cas9 protein and guide RNAs to knock-out each of the 96 genes. Screening is conducted to assess if the gene mutated the genome of the wild type. The new mutant will be crossed with another mutant zebrafish to see if the desired gene is mutated in the next generation. Through polymerase chain reaction (PCR) tests and subsequent gel electrophoresis, embryos are collected and sent off to sequencing to confirm the knockout gene’s location. With a positive outcross, the parent mutant is bred with another wild type zebrafish to produce heterozygous offspring who are incrossed to proceed with blind behavior testing and imaging. Whole-brain activity and imaging is performed through antibody staining for extracellular signal-related kinase and its phosphorylated form. Brain imaging for activity and anatomy is compared against that of wildtype or heterozygous mutant siblings for abnormalities.

Conclusion: Although this study is ongoing, our end goal is to identify any mutations associated with the alternative splicing of schizophrenia and autism related genes.

The Effect of Hyperoxia onHypothalamicCA1 GABAergic cells

Background: Neurodevelopmental-related complications are significantly associated with prematurity. Consequently, preterm infants may suffer from forms of cognitive deficiency. When comparedto term-born persons, individuals born preterm are also at a higher risk of falling into lower IQ classifications and developing a plethora of disorders like autism spectrum disorder and attention deficit and hyperactivity disorder (ADHD). One potential reason for the propensity of preterm infants to develop neurodevelopmental deficiencies is that they are often exposed to prolonged supra-physiologic concentrations of oxygen during the critical neonatal developmental period because of the supportive care they receive due to the immaturity of their lungs. In animal models, exposure to supraphysiological levels of oxygen in the newborn period results in cognitive dystrophy, hippocampal volume reduction, hippocampal mitochondrial dysfunctions and memory and spatial recognition deficits. Additionally, The CA1 region of the hippocampus, which is critical for long term memory and spatial learning, is very vulnerable to the resultant oxidative stress.GABAergic interneurons are the main sourceof inhibition within this brain region andGlutamic acid decarboxylase (GAD) is the enzyme which regulates the conversion of glutamic acid to gamma-aminobutyric acid (GABA) whichis a majorinhibitory transmitter. It is hypothesized that thispremature overexposure to oxygen which results in hippocampal mitochondrial dysfunction willfurther lead to a decrease in the number of GAD67positive interneurons in the CA1 region of the hippocampus as compared to normoxia exposed mice.

Objective:To determine whether preterm infants’ hyperoxicexposure induces structural or developmental changes in the interneurons of the CA1 area of the hippocampus.

Methods:Brain slides prepared from postnatal day 14 (P14) and 14-week-old mice that were exposed to normoxia or hyperoxia will be immunohistochemically stained using primary monoclonal GAD 67 antibodies. Via microscopy,photomicrographs of area CA1 of the hippocampus will be taken at 2x, and 10x objectives and the area stained for GAD 67 will be analyzed.

Ammar Elshikh, Dr. Andrabi

Introduction: A stroke is an incident where there is a blockage of blood supply through the brain. In the event of a stroke, the cells in the brain do not receive glucose or oxygen and they cease to function. The core of the brain, which is located in the cortex, is the area where cells are irreversibly damaged due to the stroke.

Objectives: The objective is to stimulate stroke conditions through oxygen-glucose deprivation (OGD), in the neurons of mice. Through this simulation, treatments can be tested for the recovery of cells around the core region of the stroke impact.

Methods: The brains of mice fetuses are obtained from pregnant mice. The cortexes of these mouse fetuses are isolated and neuron cultures are obtained from them. These neuron cultures are placed in media that does not contain oxygen or glucose (OGD), and there are (undecided) treatments that will be applied to determine if the cells can be restored.

Chorioamnionitis ImpairsBioenergetics of Lung Fibroblasts and Mesenchymal Stem Cells
LaNae Fay1*, Miranda Graves1*, Ahmed S. Abdelgawad2, Rui Li2, Jegen Kandasamy2, Kent A. Willis2, Namasivayam Ambalavanan2
*Co-First Authorship
1PARAdiGMProgram, The University of Alabama at Birmingham
2Division of Neonatology, Department of Pediatrics, School of Medicine, The University of Alabama at Birmingham

Background:Bronchopulmonary dysplasia(BPD)is chronic pulmonary disease of premature newborns that results from dysmorphic lung development arising from exposure to inflammation and supra-physiologic levels of oxygen. Intrauterine inflammation or infection, usually clinically diagnosed as chorioamnionitis, iscommonly associated with pretermbirth,and increases the risk to develop BPD.Lung fibroblasts and mesenchymal stem cells (MSC)may modulate hyperoxia-induced abnormal lung development.

Hypothesis: Exposure to chorioamnionitis depressesmitochondrial bioenergetics in pulmonary fibroblasts and MSC.

Methods: We modeled chorioamnionitis exposure withlipopolysaccharide (LPS), a bacterial endotoxin,in human pulmonary fibroblasts and MSC isolated from human infant samplesfromthe LungMAPConsortium. Cells were derived from three human infants for each exposure group. Cultured cells were exposed to graduated concentrations of LPS for 24 h(0.01 to 100 ng), then exposed to hyperoxia (FiO20.85)for 24 h to model BPD. Controls were exposed to vehicle andhyperoxia or room air. After exposure, we assessed transcription and translation of cellular targetspotentially involved in BPD pathogenesis using qPCR and Western blotting. Mitochondrial bioenergetics were analyzed using a Seahorse XF96 Analyzer.

Results: We analyzed gene and protein expression in doubly exposed isolated human fibroblasts and mesenchymal stem cells. Using a Seahorse analyzer,we measuredthe oxygen consumption rate and extracellular acidification rate of the mitochondria.

Conclusions: Our results suggest that altered mitochondrial bioenergetics in pulmonary interstitial cells may augment hyperoxia induced impairment of lung development.

Emmanuel Fortuchang
Dr. Volpicelli-Daley

Introduction: Parkinson’s disease (PD) is a neurodegenerative disease that is characterized by the formation of Lewy body (LB) inclusions and the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Lewy bodies are primarily composed of phosphorylated alpha-synuclein, which in its native form, localizes to the presynaptic terminal. While the motor symptoms most commonly associated with PD are attributed to LB-induced cell death and subsequent reduction of dopaminergic terminals in the SNpc, some 80%of Parkinson’s patients present with non-motor symptoms. These symptoms do not correspond well with pathology within the SNpc and are thought to result from inclusion formation in other parts of the brain, such as the cortex or amygdala. Pathology in the amygdala may better explain some non-motor symptoms including depression and anxiety, apathy and psychosis. Interestingly, while alpha-synuclein aggregation in the SNpc results in cell death, the same level of neuronal death is not observed from amygdaloid aggregates. What we think is occurring instead is that aggregate formation in the amygdala is inducing a chronic neuroinflammatory response which results in the opsonization and microglial clearance of synapses containing these aggregates.

Objectives:Neuroinflammation is associated with the amygdala pathology within Parkinison’s patients. We think that the complement cascades activate microglia to prune damaged synapses. We hypothesise that synapses with inclusions are marked for microglial clearance with C1q, which is thought to be the initiator of the classical complement system. We want to induce aggregation within the amygdalae of 3 month old mice and measure changes in microglia at six and twelve weeks to see if inclusion formation induced microglial activation within the amygdala.

Methods: We will be inducing inclusion formation within the amygdala of 3 month and 4 month old C57BL/6J mice using the preformed fibril (PFF) model. In this fragmented model, toxic alpha-synuclein is injected directlyinto mouse brains whereby the misfolded alpha-synuclein recruits endogenous synuclein to further misfold and form larger inclusions. We will inject mice into the dorsal striatum to induce inclusions in the amygdala. The mice will be profused at 6 weeks, 12 weeks, and at 6 months and analyzed using immunohistochemistry. We will quantify changes in synapse markers and microglial activation. The synaptic markers we will be employing include: Homer 1, VGLUT1 for excitatory glutamatergic synapses and VGAT and VGAT and Gephyrin for inhibitory GABAergic synapses. The markers of microglial activation we will be using are C1q, CD68, p-syn.

The Role of Cell Cycle in Enterovirus Production
Yenifer Garcia Hernandez, Jason Needham, Sunnie R. Thompson

Non-polio enteroviruses (NPEV), A, B, C, and D cause over ten million infections every year and are responsible for tens of thousands of hospitalizations in the United States (CDC, About Non-Polio Enteroviruses). Symptoms can range from mild to severe respiratory illnesses and,insome cases, acute flaccid myelitis (AFM);a serious neurological condition that affects the nervous system and causes paralysis in young children. Currently, there is no antiviral treatment. To develop effective broad-spectrum antivirals a better understanding of shared mechanisms used by NPEVs is needed. Our studies have focused an understudied area of how these RNA viruses arrest the cell cycle for viral productionand concluded whether NPEVs require cell cycle arrest in biologically relevant primary cells for efficient viral production.

Identification of genetic links between insomnia and cardiovascular diseaseusing drosophila as a model system
Muhammad Shaufe Hassan1, Farah Abou Daya2, Girish Melkani2
1.College of Science, George Mason University, Fairfax, Virginia
2.Division of Molecular and Cellular Pathology, Department of Pathology, School of Medicine, University of Alabama at Birmingham, Birmingham, AL

Insomnia is a common disorder defined by constant difficulty in falling and/or staying asleep. Recent studieshave shown the association between insomnia andcardiovascular disease (CVD) where insomnia confers a >2-fold increased risk of incident CVD.Our collaborators at MGH have found 57 genome-wide significant genetic loci for insomnia and established robust causal links with CVD. However, the understanding of specific shared causal pathways and mechanistic links to identify new therapeutic targets for insomnia that ameliorate CVD risks is still unknown. UsingDrosophila melanogaster, which is a well-established model system for sleep and CVD studies,we investigatedthe effects of knocking-down Drosophila orthologs of causal human insomnia geneson sleep and cardiovascular function.To test cardiac functionof 74 genes associated with the identified loci, we knocked-down these genes using the cardiac-specific Hand-Gal4driver. Middle-aged Drosophilaprogeny were then collected for semi-intact microscopic heart preparation followed byhigh-speed videography for measuring heart parameters. Our initial resultsshow that out of ten lines tested, cardiac-specific knock-down of two insomnia related genes ledto significantly compromised cardiac contractility, where one led to a dilated heart phenotype, while the other led to a constricted heart phenotype. Measurements of cardiac parameters and identification of mechanistic links to insomnia for all genetic lines arestill in progress. This work will reveal why insomnia adversely influences cardiovascular disease and help developtherapeutic strategies for both insomnia and CVD.

Johnson, E; Smith, A

Cardiomegaly is defined as the enlargement of the heart. The enlargement of the heartisusually a symptom of another underlying problem e.g.,cardiomyopathies. Cardiomegaly is often asymptomatic, but it can lead to complications like shortness of breath, dizziness, irregular heartbeat, heart palpitations,heart attacks, congestive heart failure,decompensated heart failure, sudden cardiac death, and malignant ventricular arrhythmias, as well as thromboembolism secondary to mural thrombi. Cardiomegaly is traditionally determined when the transverse diameter of the cardiac silhouette is greater than or equal to 50% of the transverse diameter of the chest inner wall or cardiothoracic ratio (CTR). The measurements are made on either radiograph (X-ray) or Computed Tomography (CT) imaging. We hypothesize that an AI, artificial intelligence,algorithm can more accurately detect cardiovascular events than traditional methods of CTR. We are training an AI to recognize cardiomegaly by giving examples of CTR measurements from axial CT images of patients that came in during March of 2016. Using imaging software, we measured the hearts, in millimeters, from epicardium to epicardium, while avoiding pericardial fluid, parallel to the patients’ back. The inner chest wall is also measured parallel in the same manner as the heart was measured. Results of the measurement were stored. TheCTR was calculated and a note was made as to whetheror notthe entire heart could be visualized. Once completed, we compiled a list of patients with a CTR greater than .57 and did a retrospective analysis. We first looked at if the patient's radiology reports had any mention of cardiomegaly, enlarged heart, or hypertrophic ventricles to detect if the radiologist noted the cardiomegaly in the report. We then checked if patients had an EKG or ECHO 12 months prior to when they had the CT scan we measured. Also, we checked to see if the patient had a pacemaker/ defibrillator or valve repair/replacement at the time of the CT image. This data will be used to see if patients were correctly managed for their cardiomegaly.

Title: Exercise-induced angiogenesis in skeletal muscle is blunted by heparanase inhibition
Author(s): Leign-DeVito Johnson, Drue Benefield, Dr. Glenn C. Rowe

Regular physical exercise (resistance and aerobic) can improve both muscle strength and metabolic function and is used as a treatment for various pathological conditions. Aerobic exercise, specifically, remodels muscle by increasing mitochondrial number and functionality. Mitochondria are the powerhouse of the cell and use oxygen for cellular respiration. To maintain normal mitochondrial function a constant supply of oxygen is required for normal metabolic function, and angiogenesis, the formation of new blood vessels iscritical for this process. Heparanase is an enzyme that is released into the extracellular matrix by endothelial cells to promote angiogenesis. We therefore sought to determine whether disruption of angiogenesis by targeting the heparanase pathway affected muscle mitochondrial metabolism. To test this hypothesis, animals were divided into four groups (sedentary+no drug, sedentary+drug, wheel+no drug, and wheel+drug). The animals in the wheel groups were allowed free access to a voluntary running wheel and the animals that were given drug, were given daily intraperitoneal injections for four weeks. The animals were euthanized, and muscle harvested. RNA was isolated and subjected to reverse transcriptase reactions and real time PCR to measure gene expression. We observed that heparanase inhibition successfully blunted the expression of genes involved in exercise-induced angiogenesis. However, the drug did not affect the expression of mitochondrial metabolism gene expression. Taken together thesedata suggest that heparanase inhibition negatively affects exercise-induced angiogenesis butdoesnotaffectmetabolism. However, further studies are still needed to directly measure the effect on mitochondrial function and new vessel formation.

The effect of circadian rhythm disruption on cardiac and skeletal muscle function
Aaryana Jones1, Yiming Livelo2and Girish Melkani2
1 Department of Chemistry, Louisiana State University
2 Division of Molecular and Cellular Pathology, Department of Pathology, School of Medicine, University of Alabama at Birmingham

Circadian rhythm is a near 24-hour cycle that is driven by a self-sustained transcriptional-translational feedback loop and synchronized to external cues, with light being the most important. Genetic and environmental disruptionsof circadian rhythm have been associated with increased cardiac and skeletal muscle diseases, however, the mechanistic basis of this is still unexplored. Moreover, the effect of circadian rhythm disruption on cardiac and skeletal muscle development is yet to be evaluated.We hypothesize that disrupted circadian rhythm during development will lead to abnormalities in morphogenesis due to the lack of diurnal regulation, resulting in deleterious effects on cardiac and skeletal muscle function. In this study, using a model organism,Drosophila melanogaster, we are investigatingthe impact of circadian disruptions with constant light exposure and genetic silencing of clock genes in a tissue-specific manner using the UAS-Gal4 system. Cardiac and skeletal muscle functions will be evaluated during development,and at 1, 3,and 5 weeks of age. Consistent with our previous data, environmental disruptions of circadian rhythms with constant light resulted in striated muscle dysfunction, demonstrated by deformationof the conical chamber, reduced flight,and climbing ability. Overall, this study provides a novel opportunity to understand the impact of circadian disruptionin vivoand the importance of circadian rhythm for the development/maintenance of striated muscle.

Title: Role of CTSD in mitochondrial function of SHSY-5Y cells
Quintera Knight Andrabi Lab
Key words: mitochondria, stroke, ctsd, lysosome, mitophapy

In the cell the process of mitophagy, known as the degradation of mitochondria by autophagy, is achieved through lysosomes. In neurodegenerative diseases, lysosomal function is compromised. The lysosomal dysfunction leads to the accumulation of damaged organelles and proteins in the cells, which play a critical role in the pathology of neurological diseases like Alzheimers and Parkinsons. The lysosomal protease, Cathepsin D (CTSD), has been previously found to improve lysosomal dysfunction (Hossain et al.).Based on this finding I would like to assess the role of CTSD in maintaining mitochondrial function. For this we use CTSD overexpression and knockdown system in SH-SY5Y cells. After using western blots to verify CTSD knockdown in SHSY-5Y cells, we will utilize mitochondrial assays like seahorse extracellular flux analyzer and TMRE in order to assess mitochondria function in CTSD knockdown cells. We expect to see a decrease in mitochondrial function in CTSD knockdown cells. Results and discussion for the experiment are forthcoming.Restoration of CTSD (cathepsin D) and lysosomal function in stroke is neuroprotective

M. Iqbal Hossain, Joshua M. Marcus, Jun Hee Lee, Patrick L. Garcia, VinodKumar Singh, John J. Shacka, Jianhua Zhang, Toby I. Gropen, Charles N. Falany & Shaida A. Andrabi (2021) Restoration of CTSD (cathepsin D) and lysosomal function in stroke is neuroprotective, Autophagy, 17:6, 1330-1348, DOI: 10.1080/15548627.2020.1761219

PARAdiGM Student: Amy Lee
Disruption of Muc5b Expression Leads to Chaotic Mucus Flow


Amy P. Lee, Courtney M. Fernandez-Petty, Farah Abou Daya, Steven M. Rowe
Cystic Fibrosis Research Center and Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL
Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene(CFTR), which is responsible for proper movement of chloride and bicarbonate ions across the apical cell surface. In the respiratory tract, abnormal mucus clearance contributes to chronic infection and progressive organ decline. There is little mucus movement in CF subjects, whereas in non-CF subjects, mucus moves with a higher velocity and in a laminar flow pattern.
Recent evidence suggests bicarbonate transport may have significant impact on the biogenesis of Muc5b, a principalconstituent ofa gel-forming mucuslayer. In CF, where bicarbonate transport is deficient, Muc5bmucins form non-linear and globular domains that adhere tothe epithelial surfaceand block the normal laminar flow of mucus.
We identified chaotic flow in the absence of CFTR-dependent bicarbonate. In this project, I will determine how chaotic flow is precipitated inthe CF airway microenvironment using a ferret trachea knockout model of Muc5b. We hypothesize thatthe absence of bicarbonate induces chaotic mucus flow.Using, micro-optical coherence topography we canvisualize the microanatomy and functional aspects of the airway to analyze the chaotic flowby measuring the velocitiesof the mucus transport. To visualize the mucus flow,velocity vs. distance from the cilia wasplotted.We observed that all points were positive in the non-chaotic data sets while both positive and negative in the chaotic data sets. Therapies that restore the microenvironment to non-CF conditions will be sufficient to correct these mucus transport abnormalities.

Keywords: Cystic Fibrosis, Mucus, Muc5b, CFTR, Ferret

Effect of acute versus chronicexposure tostress Catecholamines onovarian cancercell viabilityand endothelial cell permeability
Mosquera-Reinoso. Kenneth, Quintero. Liz Macias,.Karthikeyan,Mythreye
The University of Alabama at Birmingham, Departmentof Pathology

INTRODUCTION: Ovarian cancer(OVCA)is a leading cause of gynecologic-related deaths in women worldwide.Recent studies have reported that prolongedexposure to stress could disrupt homeostasis and release stress hormones known as catecholamines –epinephrine and norepinephrine.These important biomolecules are known for regulating the body fight-or-flight response and have an effect on blood vessels. These hormones have been recently reported to impact tumor progression and affect therapeutic efficacy of cancer treatment, thereby altering cancer celland patientsurvival. Thus, I aim to investigate the specific effects of stress exposure on viability of ovarian cancercellsandendothelial cellsand permeability of the vasculature as it may relate to tumorigenesisMETHOD: I will first initiate a screenin a panel of OVCA cell lines, tumortissues, host tissues and endothelial cellsto determine the mRNA expression levels of -adrenergic receptors via qRT-PCR. Next, I will test the effect of Epinephrine and Norepinephrine on cell viability via SRB. Furthermore, I will examine the role of Epinephrine and Norepinephrine on endothelial cell permeability.

CONCLUSION:Conclusion of this investigation should yield strong initial findings on the effects of stress on the cells that contribute to ovarian cancer.

Cigarette smoke induces coronary artery endothelial CXC Chemokine Receptor 2 expression

Coronary artery disease (CAD)develops when arteries supplyingblood to the heart become hardened and narroweddue to atherosclerosis and plaque formation. The resulting impaired blood flowultimately leads tomillions of health-related deathsworldwideeach year.Cigarette smoking is a well-established risk factor for CAD. This is due in part to the ability of smoke to disrupt normal function of the arterial endothelium, a critical regulator ofblood pressure and inflammation. Previously, we have observed that expression of CXC Chemokine Receptor 2 (CXCR2)on endothelial cells contributes to smoke related vascular injury. Specifically, in vitro experiments with cultured human coronary endothelial cells exposed to cigarette smoke extract (CSE, 2% in media) have observed increased cxcr2gene and CXCR2 protein expression. Furthermore, mice exposed to whole-body cigarette smoke for 6 months were shown to have increased endothelial CXCR2 expression within the aortaby immunohistochemistry, while endothelial-specific deletion of cxcr2reduced smoke-induced dysfunction. Despite these discoveries, questions as to how endothelial CXCR2expression increases remain. We hypothesize that the transcription factor heat shock protein 90 (HSP90)and its client protein aryl hydrocarbon (AhR)mediate endothelial cxcr2 expressionin response to cigarette smoke.Our assertion is due to previous reports linkingAhR with inflammationand environmental pollution. Additionally, cxcr2gene regulation has been linked with AhR activation. Nevertheless, no study to date has linked AhR or HSP90 with smoke-induced vascular disease. Hence, the major goal of our researchwill be to explore the roleof AhR and HSP90 as a possible mediatorof endothelial CXCR2 induced with CSE.

Mathematical modeling of Simultaneous Two-Wavelength Axial Ratiometry microscopy as a tool for studying endocytic vesicle formation.

Clathrin-mediated endocytosis (CME) facilitates the internalization of nutrients and pathogens from the outside of the cell surface. Nevertheless, the understanding of how the formation of cargo transporting clathrin-coated vesicles (CCVs) occurs in living cells remains unclear due to the limited resolution of live-cell fluorescent microscopy techniques and the need for sample fixation for electron microscopy (EM) studies. To bridge this gap, our lab leverages the wavelength-dependent illumination properties of Total Internal Reflection Fluorescence (TIRF) microscopy and developed a new microscopy technique called Simultaneous Two-wavelength Axial Ratiometry (STAR). Although the exponential decay of the evanescent wave is critical for our approach, it results in an uneven excitation of fluorescently tagged molecules, which might weigh down STAR measurements. To address that, we used mathematical modeling and represented CCV as a monodisperse sphere. Then, using the STAR equations and calculations, we measured vesicleheight (∆z) and theoretical Center of Mass while modulating vesicle radius, the number of contributing molecules, the distance of the formed vesicle from the plasma membrane, and TIRF’s incidence angle. The ∆z value is gradually less than the center of mass of the sphere, especially as the radius or elevation are being increased. This accomplishment has allowed us to assess how the z-position measured by STAR compares to the ground truth. Currently, using Monte Carlo simulation, we are investigating the influence of shot (Poisson) noise on STAR measurements. In the future, we will use these theoretical findings to improve STAR microscopy and to record a 3D model of the clathrin-coated vesicle formation during the entire process of endocytosis.

Keywords: Clathrin, MATLAB, TIRF, STAR
Presentation Type: Poster Presentation

Cardiac desmosomal proteins in a mouse model of Huntington’s disease
Ahmad G. Wilson ab, Annesha C. King b, Lawela Enfinger b, Michelle Gray b
a Paradigm Summer Program, the University of Alabama at Birmingham, Birmingham, AL USA
b Department of Neurology Center for Neurodegeneration and Experimental Therapeutics, the University of Alabama at Birmingham, Birmingham, AL, USA

Huntington's disease (HD) is a rare, inherited neurodegenerative disease. This disease impacts motor, cognitive, and psychiatric performance in patients. Huntington's disease is caused by a CAG repeat expansion in the gene encoding the widely expressed huntingtin protein. Interestingly, epidemiological evidence suggests that cardiac dysfunction is one of Huntington's disease patients' leading causes of death. Given the nature of this debilitating disease with significant nervous system abnormalities, changes outside the nervous system are not studied in great detail. However, an increasing number of studies demonstrate that cardiac abnormalities are present in HD patients and mutant huntingtin (mHTT) expressing mouse models. One mHTT expressing mouse model is the BACHD model, which manifests phenotypes similar to HD patients, including motor abnormalities, psychiatric deficits, and cognitive dysfunction at an advanced age (12 months). Evidence from BACHD mice suggests that as the disease progresses, pathological changes are observed in the cardiac tissue and abnormal cardiac rhythms that could lead to sudden cardiac arrest. Mutations in desmosomal proteins cause abnormal rhythms, pathological changes in heart tissue, and sudden cardiac arrest. These phenotypes are similar to what we observe in BACHD mice. Therefore, we hypothesize that BACHD mice have changes in desmosomal proteins that contribute to their cardiac phenotypes. We performed a western blot analysis of proteins localized to the desmosome using protein extracted from the hearts of BACHD mice at an advanced age. Here we report our analyses of cardiac desmosomal protein levels to help determine if we should focus more research in this area.

PARAdiGM Student: Kassandra Arias-Arbul
Relation of Fission Levels in Mitochondria Towards Muscle Atrophy

Skeletal muscle atrophy is a loss of muscle mass from disease, nutritional deficiencies, or aging. Depending on its severity, it can lead to weakness, fatigue, and increased mortality and morbidity rates. The dysregulation of mitochondrial dynamics has been linked to this atrophy as cells are unable to generate sufficient energy. For mitochondria to maintain this regulation, they undergo fusion and fission through the usage of Opa1, mfn1, mfn2, and Drp-1 with Fis-1 to expand and regulate damage. While fusion levels via mfn1 and mfn2 have been shown to affect muscle atrophy, few investigations have been led to dictate therelation of fission. In this study, we evaluated the role of mitochondrial fission in muscle atrophy via Drp-1. Specifically by investigating whether these factors change with atrophy. We propose that during atrophy, Drp-1 levels will increase as the cells undergo a reduction in mitochondrial levels as apoptosisincreases. Differentiated C2C12 myoblast cells underwentstarvation tomodel muscle atrophy to assess the level of fission. We measured the level of fission activity via Drp-1and Fis-1 expression, which we thencompared to their fusion components of Opa1,mfn1, and mfn2.Preliminary findings show asignificant decrease in Fis-1 expression between starved and non-starved cells.It could be possiblethat this decrease will result in a decrease in fission leading to mitochondriabeing unable to regulate damage. This work could lead toproviding a greater understanding of the extent of dysregulation of fission on muscle atrophy.

1: Ji, Li Li, and Dongwook Yeo. "Mitochondrial dysregulation and muscle disuse atrophy."NIH, 11 Sept. 2019, doi:https://f1000research.com/articles/8-1621/v1. Accessed 23 June 2022.
2:Bell, Margaret B., et al. "Adult skeletal muscle deletion of Mitofusin 1 and 2 impedes exercise performance and training capacity."Journal of Applied Physiology, 27 Sept. 2018, doi:https://doi.org/10.1152%2Fjapplphysiol.00719.2018. Accessed 23 June 2022.

PARAdiGM Student: Kaleb Branch
Role of Pasteurization on Tetraspanin Expression in Breast-Milk Exosomes

Breast milk derived-exosomes have demonstrated the ability to improve immune cell functionand assist in cell-to-cell communication for many biological processes. To make these exosomeswidely available for use by the general population, breast milk must undergo a chemical processcalled pasteurization. Despite the necessity of pasteurization for public health concerns, theprocess may hinder the positive effects and characterisitcs of exosomes. A critical class ofprotein for cell-to-cell communication is tetraspanins. These are multifunctional families ofproteins residing on the membrane of exosomes that, for this study, will be used as biologicalmarkers, primarily proteins CD9, CD81, and CD63. Our objective was to determine the effect ofpasteurization on tetraspanin expression as it relates to the role they play as cellular signals forexosomes. We hypothesize that pasteurization does not cause a significant change in tetraspaninexpression. Methods include the Holder Technique for means of pasteurization, Western Blotsfor exosome and tetraspanin detection, and the comparisons of size and concentrations viananoparticle characterization. Our findings have indicated that future investigations should bedone to discover methods of pasteurization to use as a negative control for a minimumexpectation of tetraspanin expression. Furthermore, the difference in expression betweentetraspanins that were not used in this study could be helpful for the implications of this topic.

PARAdiGM Student:
Effects of Female Human Reproductive Exosomes on Neonatal Mouse Intestinal Morphology

Background: Previous studies have shown that psychological stress during pregnancy releases corticosterone where large levels can directly affect the offspring’sintestinal development.A probable result ofsuchan effectis neonatal intestinal dysfunction, which can be characterized by differentiating functions in sections ofthe colon, ileum, or jejunum. The purpose of the study is to view how exosomes from human breast milk can potentially lower intestinal stress, lowering the possibility of contracting neonatal intestinaldysfunction.

Methods:Controlled offspring’s colon, ileum, and jejunum were harvested to collect biopsies that were placed in media for 48 hrs. The media was observed through spectroscopy after mixture with Griess reagent to view cellular activity to determine where to inspect where exosomes can affect.

Results:Here the colon and ileum are relative to each other in cellular activity to which both sections can be measured in its affects after exosome introduction. Its assumed that stressed mice should have increased activity based on corticosterone.

PARAdiGM Student:
Comparative Analysis of Angiotensinogen, Vasorin, and Vestigial Like Family Member 1in Normal and Preeclampsia Human Placental Tissue

Preeclampsia (PE) is a common pregnancy disorder worldwide, characterized by hypertension,along with optional systemic manifestations such as proteinuria, elevated liver enzymes, lowplatelet count and/or lung edema. Without treatment, the mother and fetus are at risk of severecomplications that may lead to premature birth or death. The placenta plays a major role tomaintain PE, and the only way to end maternal symptoms of PE is the delivery of the placenta.Identification of placenta-derived mediators/biomarkers are sought after as diagnostic tools forthe early detection of PE. In this study, we sought to identify novel biomarkers for the earlydetection of preeclampsia. Using unbiased proteomics to analyze extracellular vesicles fromboth urine and plasma of women with severe PE (sPE) as compared to women withnormotensive pregnancy (NTP) we identified angiotensinogen (AGT), which is increased in sPEand Vasorin (VASN), which is decreased in sPE as compared to NTP, as potential biomarkers.We hypothesized that AGT and VASN originate in the placenta and that the change in theirquantities in plasma is reflected in the change in their quantities in the placenta. We usedimmunohistochemistry and microscopy to visualize AGT and VASN along with Vestigial LikeFamily Member 1 (VGLL1), a marker of trophoblasts in human placental tissue collected fromwomen with sPE and NTP. We used Mouse Anti-human AGT, Rabbit Anti-human VASN, RabbitAnti-human VGLL1, and Mouse Anti-human VGLL1 to determine protein levels of AGT andVASN, their localizations relative to VGLL1 and to test if there is a quantitative differencebetween sPE and NTP. We expected differential staining of Mouse Anti-AGT and RabbitAnti-VASN between preeclampsia placental tissues and controls and similar staining of bothRabbit and Mouse Anti-VGLL1. Immunohistochemistry analysis showed increased expressionof AGT, and decreased expression of VASN in preeclampsia placenta tissue samples.VGLL1stained similarly between PE and controls with slightly more staining of Rabbit Anti-VGLL1 inthe control tissue sample.Additional staining inmultiple specimens from each condition willallow us to determine whether changes in AGT and VASN expression in placenta tissue areresponsible for changes in their levels in the systemic circulation. These results will help usdetermine the potential utility of these molecules as biomarkers for preeclampsia.

Keywords:preeclampsia, placenta, immunohistochemistry,AGT, VASN, VGLL-1

PARAdiGM Student:

Oncolytic viruses(OVs)are a form of immunotherapeutic treatments that are implemented to lyse cancer cellsand can prompt immune responses from the body using released tumor antigens.Moreover, Herpes simplex viruses havethe capability to be genetically engineered to acquire oncolytic characteristics to replicate in, and release tumor antigens after killing cancer cells. A case of the innate immune response triggered by OVs is the anti-tumoral response by the body’s Natural Killer cells(NK).The innate lysis of virally infected cancer cells by activated NK cells could potentiallypromote a favorableresponsein the destruction of the cancer cells. In this experiment, the effect of oncolytic herpes simplex virus oHSVwas examined in vitro GSCstem cells model which resembledtumor assortmentin animmunosuppressive microenvironment in mice. Furthermore,it was investigated wether the viral infection by oHSV in the GSC stem cell model generated NK cell memory.

PARAdiGM Student: Amy Lee
ETIImproves CFTR Function and Mucus Hydration in Cystic Fibrosis Human Nasal Epithelia

>Amy P. Lee, Courtney M. Fernandez-Petty, Reny Joseph, Steven M. Rowe, George M. Solomon

Cystic Fibrosis Research Center and Department of Medicine, The University ofAlabama at Birmingham, Birmingham, AL

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is responsible for proper movement of chloride and bicarbonate ions across the apical cell surface. In the respiratory tract, abnormal mucus hydration and clearance contributes to chronic infection and progressive organ decline. Elexacaftor/tezacaftor/ivacaftor (ETI) is an approved therapyfor patients with certain CFTR mutations. It is a combination therapy composed of ivacaftor, a CFTR channel potentiator, and elexacaftor and tezacaftor, CFTR correctors that enhance trafficking of CFTR from the endoplasmic reticulum to the cell surface modulators.We hypothesize that ETI wouldimprove the mucus hydration and clearance for patients with CFand correlate to the level of rescue of CFTR by short-circuit current (Isc)in vitro. Differentiated monolayers from 20donors (11F508del/F508del, 9 F508del/minimal function) have been analyzed to date. Monolayers demonstrated a mean ETI-treated, forskolin-stimulated Iscof 7.1±6.2μA/cm2(vs 2.1±1.9μA/cm2for vehicle, P<0.01, representing 48% restoration of WT-CFTR Isc).Using Micro-optical coherence topography (μOCT),a real time imaging modality that visualizes the microanatomy and functional aspects of the airway,we observed a significant increase in airway surface liquid (ASL) in ETI-treated culturesof 9.64.3 m(vs5.32.3mfor vehicle, P<0.0001).We are currently evaluating how ETIaffectsother airway parameters, such as mucociliary transport using μOCT. Once complete we will correlate these data to key clinical outcomes measures in these subjects including FEV1.

PARAdiGM Student: Emmanuella "Ella" Sanvee
Mesenchymal Stem Cell Bioenergetic Function Predicts Risk for Bronchopulmonary Dysplasia

Extremely low birth weight (ELBW) infants are born with immature lungs and thereforerequire prolonged ventilation and supplemental oxygen exposure which leads to oxidant stress, and chronic lung disease referred to as bronchopulmonary dysplasia (BPD). Mesenchymal stem cells (MSCs) are responsible for differentiating into adipocytes, chondrocytes, and osteocytes, all of which aid in healthy lung development. Mitochondria, which are highly sensitive to oxidant stress play a significant role in the differentiation and tissue repair ability of MSCs. Therefore, oxidant stress-induced mitochondrial dysfunction could disrupt MSC differentiation and ability to repair lung injury. However no previous study has explored the association between endogenous MSC mitochondrial function and BPD. We hypothesized that MSC mitochondrial dysfunction is associated with increased BPD risk and severity in ELBW infants. To test this hypothesis, we isolated cells from umbilical cords of 39 infants, characterized them as MSCs and measured oxygen consumption rates which were then correlated with the severity of BPD inthese infants. Cells were verified as MSCs by conforming that they were able to differentiate into adipocytes, chondrocytes, and osteoblasts. Surface markers identified through flow cytometry analysis also revealed antigen expression typically expected ofMSCs. In addition, bioenergetic function of these MSCs assessed using basal oxygen consumption rate (OCR) that was measured using a Seahorse™ Extracellular Flux analyzer showed that infants with mild BPD had higher MSC basal OCR compared to MSCs from infants with moderate to severe BPD. We conclude that MSCs can be successfully isolated from umbilical cords of prematurely born infants and that MSC mitochondrial function is associated with their risk for BPD.

PARAdiGM Student:
Determine the Frequency of CMV Reinfections During Pregnancy in a Cohort of Seropositive Women Utilizing Strain-Specific Responses

Cytomegalovirus (CMV) is a frequent cause of congenital infection (cCMV) and the most frequent infectious cause of brain disease and hearing loss in children. CMV can be transmitted from the mother to the fetus in women with primary infection (acquiring CMV infection for the first time during pregnancy) or those with non-primary infection (mothers who have had CMV infection prior to pregnancy). Although, thereasons for the failure of preexisting immunity to prevent intrauterine transmission are not clear, we propose that CMV reinfections in seropositive women plays a role. Recent studiesdemonstratedextensive genetic diversity among CMV strains. The objective of the study is to determine whether CMV seropositive women with evidence of reinfection during pregnancy are at higher risk for delivering infants with cCMV. Recombinant proteins containing the strain-specific neutralizing epitopes within the CMV glycoproteinsH and B from AD169 and Towne strains of CMV were developed in the laboratory. By utilizing these antigens, sera obtained in early gestation and at delivery froma cohortof seropositive women (N=956) were tested to detect the appearance of new antibody specificities during pregnancy as indirect evidenceof CMV reinfection. Our preliminary data shows 4.5% CMV reinfection rate. We will compare the frequency of CMV reinfection between women whose infants are born with cCMV (transmitters) and those with uninfected infants (non-transmitters), as well as demographic characteristics and exposure to possible sources of CMV between the groups. This information could allow us to develop interventions to prevent CMV reinfections and subsequent intrauterine transmission.

PARAdiGM Student:

Understanding the process and specifics of the protein’s roles in the Ras/MapK pathwayleads to greater improvement in medicine and care for those with pancreatic cancer. Pancreaticcancer is an abnormal growth in the pancreas, the organ that secretes vital enzymes andhormones for the body to function. As pancreatic cancer has little response to immunology, thetreatments available are limited to surgery, chemotherapy, or targeted therapy. With this,pancreatic cancer is becoming one of the top deadliest cancers in the body with a 11% survivalrate of 5 years. Due to the poor prognosis indicating the cancer, those impacted do not recognizetheir condition until the growth reaches a severe level. Most patients face the symptoms at stage3 or 4, where treatment may likely only prolong their life. The primary driver for this cancer isthe mutation of KRAS as well as alterations of the TP53, SMAD4, and CDKN2A. As the chiefmutation that makes up 95% of pancreatic cancer patients and can exist within the body, unlikePD53 which gets deleted, current treatments aim to target the RAS pathway. By using a PanRASinhibitor, ADT-007, cancer proliferation and survival can be slowed. Focusing the study onmeasuring the effects of various concentrations of ADT-007 on G2M phase in the cell cycle andthe pERK and pAKT, two proteins crucial in increased cell cycles and survival, we candetermine and analyze the effects of the drug and the necessary dosage to limit the occurrence oftoxicity in other parts of the body.