Meet the Scholars
Learn more about the scholars and their research abstracts.
Presenter: Borcyk, Madeline
Major: Microbiology & Cell Science
Author(s): Madeline G. Borcyk, Julie K. Bray, Thomas D. Schmittgen
Faculty Mentor: Thomas Schmittgen, Ph.D.
Investigating the Role of REST Activity as a Novel Mechanism Governing Pancreatic Cell Identity
Pancreatic acinar and ductal cells have a remarkable plasticity for cellular transdifferentiation upon injury, thereby allowing recovery. This plasticity is key to maintaining pancreas viability; however, aberrant regulation of pancreatic cell identity can cause complications, such as predisposition to cancer. The transcriptional activator Ptf1a, promotes acinar differentiation, making regulation of Ptf1a critical for cell identity. Preliminary data suggest an inverse relationship between Ptf1a and RE-1 Silencing Transcription Factor (REST). REST binds to RE-1 sequences on DNA and represses downstream gene expression. In acinar cells, overexpression of REST causes a decrease in Ptf1a while knockout of REST increases Ptf1a. We hypothesize that REST represses Ptf1a expression by binding to a RE-1 sequence upstream of the Ptf1a gene. To investigate the regulation of Ptf1a expression by REST, we used CRISPR to knockout the RE-1 sequence in the human pancreatic cancer cell line PaTu-T. Five knockout and three parental clones have been successfully developed for evaluation of proliferation, morphology, and gene expression. We predict loss of REST binding will increase Ptf1a and acinar-like differentiation in the ductal PaTu-T cells. In conclusion, REST may play a critical role in regulating appropriate pancreatic cell identity, which may have implications in cancer prevention and treatment.
Presenter: Cassidy, John
Major: Biomedical Engineering
Author(s): John Cassidy, Vrunda Trivedi, Duane Mitchell
Faculty Mentor: Duane Mitchell, M.D., Ph.D.
Optimizing Lentivirus Based Human T-Cells Engineering Approaches
Glioblastoma multiforme (GBM) is a common and detrimental brain tumor, accounting for 15% of all cases and with a median survival rate of 11-16 months while receiving standard treatment. The disease is currently incurable, but new treatment approaches are attempting to engage the immune system to attack GBM. Advances in cell engineering have enabled gene transfer and the development of adoptive T cell therapies for tumor antigen-specific T cell responses. Our team’s laboratory work focuses on developing novel T cell engineering based immunotherapy approaches for treating GBM. One limitation is to engineer primary human T cells with higher gene transfer efficiency. I studied the effects of different concentration of retronectin, different concentrations virus, and varying amounts of time, on the transduction efficiency of T cells using a lentivirus carrying green fluorescent protein (GFP). Results were evaluated by measuring GFP-positive T cells using confocal microscopy, fluorescent microscopy, and flow cytometry. I found that the concentration of virus, not retronectin, played a role in transduction efficiency. Moreover, I was able to show an increase, though not significant, in the efficiency of virus transduction using multiple rounds of virus infection.
Presenter: Heflin, Cheyenne
Author(s): Cheyenne Heflin, Haya Ghannouma, Lei Zhou
Faculty Mentor: Lei Zhou, Ph.D., BMed
Role of a Specific P53 Binding Site in Limiting Tissue Overgrowth
The p53 protein is an important transcription factor known for maintaining tissue homeostasis by activating genes that have anti-proliferative function, such as pro-apoptotic genes and cytostatic genes. Transcriptional activation of pro-apoptotic genes has displayed a fundamental role in mediating apoptosis during Drosophila development. Using ChIP-Seq and RNA -Seq methods, we have identified p53 binding sites potentially responsible for p53-mediated induction of pro-apoptotic genes following DNA damage. We have since generated fly lines with the p53 binding site deleted by CRISPR-Cas9-mediated genome editing. To study the effects of the p53 binding site knockout (p53BSKO) on tissue homeostasis, wings of the knockout fly line were dissected, mounted, and then compared against WT wings. Results show P53BSKO animals had an increase in wing size compared to that of the WT. FijiWings 2.2 macros software was used to measure wing hair (trichome) densities, which is directly proportional to cell numbers. This analysis showed that P53BSKO led to hyperplasia of the wing as compared to the wt. Our study indicated that this single P53BS is required for ensuring the right number of cells in a given tissue, likely through mediating over-proliferationinduced apoptosis.
Presenter: Hegeman, Sandra
Major: Nutritional Sciences
Author(s): Sandra Hegeman, Haya Ghannouma, Lei Zhou
Faculty Mentor: Lei Zhou, Ph.D., BMed
Role of p53 mediated apoptosis in limiting metastasis
p53 is a transcription factor known to play important roles in limiting tumorigenesis, including controlling the induction of pro-apoptotic genes and apoptosis in response to oncogenic stress. Using ChIP-Seq and RNA-Seq, our lab has identified p53 binding sites potentially responsible for p53-mediated induction of pro-apoptotic genes following DNA damage. Using CRISPR Cas9, we have generated fly strains with deletions or mutations within the p53 binding motif located in the experimentally verified p53 binding site. This deletion blocked DNA damage induced apoptosis. To study the functionality of the motif in limiting tumorigenesis, we introduced the deletion into a genetic tumorigenesis and metastasis model, where the tumor-suppressor cell polarity gene scribble was knocked down via shRNAi in the nonessential tissue compound eye and introduced together with strong oncogenic mutation that can lead to neoplasia and metastasis. We developed a scoring scale to quantify the severity of neoplasia and metastasis, where 0 indicates a wild type phenotype and 5 indicates the most severe metastatic phenotype. Results show that the p53BSKO predominantly displayed a score of 5 (39%). However, our preliminary results also suggested that the severity of this model may also subject to genetic background besides the p53 binding motif.
Presenter: Le, Tiffany
Major: Health Science
Author(s): Tiffany Le, Tanaka Mai, Samantha S. Dykes, Dietmar W. Siemann
Faculty Mentor: Dietmar Siemann, Ph.D.
Axl is a Novel Therapeutic Target to Inhibit Cancer Metastasis
Cancer is the second leading cause of death in the United States and approximately 90% of cancer-related deaths result from metastasis, the spread of cancer cells to secondary sites. In tumors, the activation of signaling pathways, such as the receptor tyrosine kinase Axl, promotes the metastatic phenotype of cancer cells characterized by enhanced invasion and migration. Our data show that Axl is expressed in aggressive human breast cancer cell lines. Genetic depletion of Axl decreases tumor cell migration and invasion. These findings suggest that Axl may be a novel therapeutic target to inhibit the progression and spread of cancer cells which would ultimately decrease cancer mortality rates. To test our hypothesis that Axl activation promotes tumorigenesis and metastasis, we performed site-directed mutagenesis to generate an inactive AXL protein. Ongoing experiments are evaluating the impact of wild-type or kinase-inactive Axl overexpression on the metastatic phenotypes of T47D cells, a breast cancer cell line that normally lacks AXL expression. T47D cells will be transduced with plasmid containing wild-type Axl or kinase-inactive Axl. By identifying whether Axl activation is significantly involved in promoting the metastatic phenotype, we can develop strategies to inhibit Axl activation as an anti-cancer therapeutic.
Tiffany also presented her work at the following conferences:
- Florida Undergraduate Research Conference
- National Conference on Undergraduate Research
Presenter: Miscik, Natalie
Major: Microbiology & Cell Science
Author(s): Natalie Miscik, Akbar Nawab, Maria Guijarro, Maria Zajac-Kaye
Faculty Mentor: Maria Zajac-Kaye, Ph.D.
Thymidylate Synthase Expression During Pancreatic Ductal Adenocarcinoma Progression
Thymidylate synthase (TS) is a rate limiting enzyme for DNA synthesis that is overexpressed in numerous tumor types. Intratumoral expression of TS has been associated with development of drug resistance and poor clinical outcome after chemotherapy, reducing the patient overall survival. The main goal of this project is 1) to study the changes in TS expression with age in murine pancreas; and 2) to detect TS expression in pancreatic tumors derived from a hTS/KrasG12D Pten +/- transgenic model. Immunohistochemistry (IHC) staining was used to qualitatively determine TS expression and localization in the cells. The protocol for IHC was optimized in wild type and tumoral murine pancreas by using antigen retrieval and 3% methanol in the blocking buffer. This reduced significantly the stromal background in the pancreatic parenchyma. Then, wild type pancreas were harvested at varied mice age and expression of TS was detected. We observed that TS expression decreased with age after 40 days of age. In addition, we observed that TS levels were higher in more advanced PDAC than in PanIN or normal ducts and that the expression remained unchanged with age. TS expression levels were confirmed by Western blot.
Presenter: Nair, Shalini
Major: Public Health
Author(s): Shalini Nair, Lacey Mead, Debra Lynch Kelly, Wendy Joanne Dahl, Nosha Farhadfar
Faculty Mentor: Nosha Farhadfar, M.D.
Quality of Diet of Adult Hematopoietic Stem Cell Transplant Survivors
Advances in the field of hematopoietic stem cell transplant (HSCT) have markedly increased the number of long-term HSCT survivors. Higher rates of comorbidity among survivors supports the need for lifestyle interventions targeting this vulnerable population. Nutrition is one of the modifiable factors that may prevent or delay the onset of chronic diseases. This study aimed to assess dietary intake of allogeneic and autologous HSCT survivors in order to provide adequate information for development of a targeted nutritional intervention. Nutrient intake and diet quality was evaluated in 102 HSCT survivors using the Block 2014 food frequency questionnaire (FFQ). Diet quality was quantified by calculating Healthy Eating Index- 2010 (HEI 2010) scores. Analysis of covariance was used to assess whether diet quality was affected by demographics or lifestyle factors. The mean HEI-2010 was 62.2 ± 11.6 (age 18-65) and 61.2 ± 8.6 (age>65)(maximum score of 100). Referenced to the recommendations of the Dietary Guidelines for Americans, HSCT survivors consumed excessive amount of saturated fat, sodium, added sugars, and phosphorus and inadequate fiber, calcium, vitamin D, and potassium. This emphasizes the need for nutritional guidance in HSCT survivors’ care to improve quality of diet and mitigate long-term complications.
Presenter: Recker, Kristin
Major: Microbiology and Behavioral & Cognitive Neuroscience
Author(s): Kristin Recker, Sharon Norton, Amin Sobh, Alberto Riva, Kenneth Chang, Jeffim Kuznetsov, Michael Durante, Fernanda Flores, Richard L. Bennett, Christopher Vakoc, Kieran Smalley, William Harbour, Jonathan D. Licht
Faculty Mentor: Richard Bennett, Ph.D., Jonathan Licht, M.D.
Synthetic Lethal Screen to Identify Molecular Mechanisms that Drive Uveal Melanoma
Melanoma that strikes the uveal cells of the eye represents about 5% of all melanoma cases. Uveal melanoma is a highly aggressive form of cancer that exhibits a propensity to metastasize to the liver. At this time there are no effective treatments for metastatic uveal melanoma and new therapeutic strategies are urgently needed. The majority of uveal melanomas are derived from early activating mutations in the G-proteins GNA11 or GNAQ. In order to further explore and identify molecular mechanisms that uveal melanoma cell lines rely upon for proliferation and survival, we will perform CRISPR/Cas9 mediated synthetic lethality screening. To accomplish this, we will generate uveal melanoma cell lines Mel202, 92.1 and Mel270 that express the endonuclease Cas9 and introduce a guide RNA library into them that targets the functional domains of epigenetic regulator proteins. The guide RNAs that drop out likely disrupt essential genes that the cell relies on for growth and proliferation. Cells carrying these gRNAs are consequently depleted from the culture. This will indicate that Cas9 mediated disruption of the gene targeted by that guide RNA causes cell death and will therefore identify key dependencies of the uveal melanoma cell lines. We expect that the results of these experiments will reveal novel genetic mechanisms characteristic of GNAQ mutant uveal melanoma cells which could be therapeutically targeted in the future.
Presenter: Sandoval, Zachary
Major: Biology & Psychology
Author(s): Zachary Emmanuel Sandoval, Matthew Cascio, Marc Salute, Bikash Sahay, Galaxia Cortés-Hinojosa, Rowan Milner
Faculty Mentor: Matthew Cascio, D.O., Rowan Milner BCVSc, MMedVet
Comparative Expression of GD2 and GD3 as Translational Osteosarcoma Immunotherapy Targets
Prognosis of human osteosarcoma has not improved in the last forty years. Immunotherapy has been promising in treating other cancers, offering potential to osteosarcoma. Disialyl gangliosides GD2 and GD3 are surface antigens that have been targeted for immunotherapy in human cancers. Because canine osteosarcoma is very similar to human osteosarcoma, we sought to evaluate the expression of GD2 and GD3 in both to weigh translational capacity. Several cell lines were stained for flow cytometry to determine expression on the cells. Our findings showed that canine and human osteosarcoma cells displayed significant expression of GD2 and/or GD3. The canine cells expressed more GD3 than GD2, as expected from past data. One human osteosarcoma cell line showed higher expression of GD2 while another human line showed higher GD3. These findings added to literature that emphasizes targeting GD2 only in humans. This showed that human osteosarcomas have variable expression of gangliosides, supporting plausibility for treatments targeting GD2 and GD3. A canine soft tissue sarcoma cell line showed positivity for both, implying that GD2/GD3 therapies may be applicable to other sarcomas. This lays the foundation for future projects to examine immunotherapies in canine osteosarcoma patients to serve as translational models for humans.
Zachary also presented his work at the following conferences:
- National Collegiate Research Conference at Harvard University
- Stanford Research Conference at Stanford University
- American Association for Cancer Research
- National Conference of Undergraduate Research at Kennesaw State University
- British Conference of Undergraduate Research at University of South Wales, Treforest
Presenter: Staton, Lindsey
Author(s): Lindsey Staton, Brenda W. Dyal, Khulud Abdudawood, Tasha M. Schoppee, Stacy Jean, Valandrea M. Smith, Amelia Greenlee, Laurie Duckworth, Molly W. Mandernach, L. Vandy Black, Coy D. Heldermon, Yingwei Yao, Diana J. Wilkie, Miriam O. Ezenwa
Faculty Mentor: Diana Wilkie, Ph.D., R.N., FAAN
Lived Experiences of African American Patients with Cancer or Sickle Cell Disease
The purpose of this study was to gain an understanding of the healthcare experiences that African American adult patients with cancer or sickle cell disease associate with their frequent healthcare utilization in acute care settings. We conducted a cross sectional, single session study of 30 adult African American patients diagnosed with cancer (n=15) or sickle cell disease (n=15). Consented subjects responded to open-ended questions from the Healthcare Experience Interview Guide. The Healthcare Experience Interview Guide was used to discover the patient’s experiences before and during the current and previous hospitalization and expectations after being discharged. The experiences identified included 1) reason for admission, 2) hospital experience, and 3) discharge expectations. Positive and negative comments emerged when asked about their present and previous admissions. Patients with sickle cell disease exclusively dealt with accusations of drug-seeking behavior upon admittance and during the hospital visit. Expectations after discharge for members of both groups were positive. In conclusion, in this race concordant study participants with sickle cell disease reported accusations of drug-seeking behavior and provider disbelief of their pain level. Their perceptions of mistreatment by providers illustrates the need for implementation of standards of clinical care for sickle cell disease in all clinical settings.
Lindsey also presented her work at the following conferences:
- UF Health Cancer Center Research Day
- UF College of Nursing Research Summit
Presenter: Winer, Aaron
Major: Pre-Professional Biology
Author(s): Aaron Winer, Vindhya Vijay, Amy Meacham, Jesse Terrell, Lauren Katzell, Leylah Drusbosky, and Christopher R. Cogle
Faculty Mentor: Christopher Cogle, M.D.
Heterogeneous Nuclear Ribonucleoprotein C As A Novel Therapeutic Target For Acute Myeloid Leukemia
Acute myeloid leukemia (AML) is a highly refractory cancer despite conventional treatment. Previously, we found that blood vessels are sanctuary sites for chemo-refractory AML cells. To target AML cells sequestered in the vascular niche, we developed an in vitro co-culture assay consisting of AML cells on bone marrow- derived endothelial cells (BMECs), mimicking the chemo-protective effect of the vascular niche in the bone marrow microenvironment. Using this assay, we performed high-throughput chemical-phenotypic screening of 31 million compounds and identified a novel polyamine sulfonamide (2470-51) that selectively killed AML cells, while sparing underlying BMECs. In vivo AML patient-derived xenograft modeling further validated the efficacy of 2470-51 as a selective anti-leukemic agent compared to cytarabine (conventional control) and vehicle control. Protein target identification experiments using label-free shotgun proteomic analysis revealed heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) as the covalent target of 2470-51. These findings were further validated by shRNA mediated knock-down of HNRNPC (HNRNPC-kd) in AML cell lines THP-1 and MV411. HNRNPC-kd significantly reduced AML cell proliferation and AML cell viability by inducing apoptosis, compared to scramble controls. Collectively, our data indicate that hnRNPC is an indispensable factor in AML and that inhibiting this regulatory splicing factor may be a new therapeutic strategy.
Aaron also presented his work at the following conferences:
- UF Health Cancer Center Research Day
- American Association for Cancer Research