2013 Projects

Evaluating the Responses of Murine Epithelial Cells Lines to Silver Nanoparticles
Andrij Holian and Ray Hamilton, Mentors

Engineered nanomaterials (ENM) are a new class of materials that are being developed for a wide variety of uses ranging from medical to engineering applications and even many personal products.  Recent studies with murine epithelial cell lines have indicated that citrate-stabilized silver (Ag) nanoparticles are more toxic at 20 nm as opposed to 110 nm.  In addition, there was a variable response to Ag nanoparticles depending on the cell line and the seeding density.  This project will focus on evaluating the potential confounding variable of Ag dissolution in differing serum concentrations, and expand the previous results to include PVP-stabilized Ag nanoparticles (20 and 110 nm) in these models.  This project will involve maintaining murine epithelial cell lines, toxicity assays, and TEM to determine Ag internalization and dissolution.  There is a current need to develop a suitable model of epithelial cell exposure that would serve a predictive role for human exposures to Ag nanoparticles.  This summer project will be to test a series of epithelial cells using ENM under investigation in our laboratory to determine which cell line is the most appropriate and what properties of ENM cause adverse responses.

The student working on this project will develop expertise in the emerging field of nanotoxicology, cell culture, assays for cytotoxicity (necrosis and apoptosis), and uptake of nanomaterials.

Foreign Body Inflammatory Response to Nanofibrous Biomaterials
Andrij Holian and Kevin Trout, Mentors

Millions of Americans are hospitalized annually due to tissue loss or damage caused by disease, aging, or trauma.  Traditional treatments involve surgical reconstruction, organ transplantation, or life support by mechanical devices.  New life science and engineering technologies in the field of regenerative medicine are leading to great improvements in patient care.  One of these therapies is tissue engineering, which involves the implantation of a biomaterial to serve as a bridge between healthy tissues.  Natural healthy tissue consists of cells surrounded by an extracellular matrix, which contains nanometer-sized fibrous proteins that provide physical cues to direct cell organization, function, and survival.  Biodegradable implants are designed to mimic this native environment and temporarily support cells until they replace the implant material with newly formed extracellular matrix.  The stiffness, porosity, topography, and specific surface area of the native environment can be emulated by nanofibrous biomaterials synthesized from biodegradable polymers.

Immunogenicity may cause rejection during traditional tissue transplantation from one individual to another.  Because synthetic nanofibrous materials are biocompatible and used with the patient’s own cells, they do not lead to immunogenicity.  However, the surgical implantation procedure will cause an inflammatory response.  This response is an important initiator of wound healing, but excessive inflammation can be detrimental.  An important mediator of inflammation is the macrophage cell.  In order to control the foreign body inflammatory response, we will study how various nanofibrous biomaterials modulate macrophage activity.

The student working on this project will gain experience in the rapidly growing field of regenerative medicine, while learning techniques in biomaterial production and characterization, cell culture, viability assays, and assessment of inflammatory response by cytokine ELISA.

Macrophage Phenotypes and Autophagy Activation
Christopher Migliaccio, Mentor

Macrophages are key immunoregulatory cells following environmental exposures.  In most situations macrophages constitute the initial response and dictate the inclusion/activity of other cells in order to generate a specific response.  In addition to this vital regulatory role is the observation that there are multiple macrophage phenotypes that are unique in both their mechanisms of activation and the type of responses they promote. These subsets are activated via distinct signaling pathways, and some of these pathways, Akt and PI3K, are associated with autophagy.  Autophagy is an intrinsic process of each cell that can have profound effects on the ability of macrophages to respond to environmental particulates.  This project will utilize an in vitro system for generating macrophage phenotypes.  Each phenotype will be evaluated on the role of autophagy to influence subsequent responses to a variety of particulate exposures.  These studies will assist in the current work on our laboratory focusing on the contribution of different macrophage phenotypes to particle-induced pulmonary pathologies.

In this project, the student will learn sterile technique, cell culturing, in vitro macrophage models, ELISAs, and molecular biological techniques.

Evaluating the Role of the PTEN Tumor Suppressor Gene in Mesothelioma Tumorigenesis
Mark Pershouse, Mentor

Most non-scientists and many scientists would be surprised to find out that asbestos is contained in thousands of products still on the market in the US. Asbestos exposures are commonplace, but few worry about the effects due to a cloud of misinformation. Approximately 10% of asbestos exposures will result in a deadly cancer known as malignant mesothelioma, involving lesions on the pleural linings of the lung or the peritoneal lining of the abdominal cavity. Therapy for these tumors usually involves removal of large sections of effected tissue and a poor prognosis. Little has improved in therapy in the last 40 years. As the genetics lesions that cause normal mesothelial cells to lose their ability to maintain a regulated growth or repair capacity in mesothelioma have been discovered, our understanding of possible targets of therapeutics has grown. After years of negative data looking for the involvement of the tumor suppressor PTEN in these tumors, several new publications have shown clear involvement of this gene and have postulated that it is effective at encouraging tumor growth through a block to apoptosis mediated through the PTEN/PI3K/AKT/mTORc pathway. This pathway has been implicated in other types of tumors and is clearly one of the newest targets of cancer therapeutics.

This project aims to confirm the linkage between PTEN loss and p53 dysregulation through AKT/MDM2 in a mesothelial model system. Our hypothesis is that PTEN suppression by shRNA constructs will result in a decrease in cells’ ability to respond to apoptosis-inducing signals (camptothecin, UV light), as well as dysregulation of the levels of critical p53 responsive genes. The project will take advantage of cell lines in which PTEN expression has been down-regulated, mimicking the status in a tumor. In these cell lines, we hope to characterize changes in the ability of normal mesothelial cells to evade apoptosis, dysregulate cell cycle, and generally behave in a more tumor-like fashion.

The student working on this project will develop expertise in cell culture, protein expression measurements, quantitative PCR, assays of cellular transformation (growth curves, soft agar growth, morphological assessment), and apoptosis detection.

The Control of Protein Production Related to Lung Fibrosis
Liz Putnam, Mentor

Asbestos fibers are toxic substances that can cause serious health problems including cancer, asbestosis, and mesothelioma when they become airborne and are inhaled.  We have identified SPARC, a matricellular protein involved in tissue repair, extracellular matrix (ECM) regulation, cellular proliferation, and cellular adhesion, as a candidate for involvement in the fibrosis that occurs after asbestos exposure. Through our studies with wild type and SPARC knockout mice we found that expression of both SPARC and collagen (a major component of the ECM) is increased in asbestos treated mice compared to control and that SPARC knockout mice did not produce as much collagen in response to asbestos.  We now want to see if we can control the expression of SPARC after asbestos exposure with RNA interference (RNAi) and thus decrease the amount of collagen produced.  After establishing primary lung fibroblast cell strains, these cells will be exposed to previously prepared viral particles containing shRNA complementary to SPARC to inhibit collagen expression after asbestos exposure.

The student on this project will learn how to culture cells and titer virus.  The student will also analyze the resulting changes in protein expression in test cell cultures treated with the inhibitory shRNA constructs.