2010 Projects
"Assessment of DNA damage and DNA repair in clinical samples is hindered by a dearth of reliable high-throughput assays"
Dr. Fernando Cardozo-Pelaez
The main objective of this project will be to develop assays for DNA damage and DNA repair that can easily be adapted into high-throughput systems. Most of the work proposed will use measurement of the damaged DNA base 8-hydroxy-2’-deoxyguanosine (oxo8dG) and the enzyme 8- hydroxyglycosylase, Ogg1, required for the repair of oxo8dG. Although well established protocols are available for analysis of this modified base and its repair system, these approaches require state of the art equipment (HPLC-ECD or HPLC-MS/MS), are labor and time intensive, require the use of high levels of radioactive materials, and involve a myriad of steps that make them difficult to establish in most laboratory settings. The objective of this project would be to implement systematic approaches that relay in the specificity of nucleic acid pairing, the low level of detection of fluorescent probes, and the versatility of multi-well plates fluorescent analysis to develop analytical approaches that will allow us to determine DNA repair capacity (ogg1) in tissues and specimens in a fast, high-throughput, accurate, and reproducible way. These approaches can easily then be adapted for assessment of other DNA damage and DNA repair enzymes.
The specific objective of the project will be to develop of a high throughput analysis for the kinetic activity of the DNA repair enzyme Ogg1. This project will be advanced by:
- Design of double stranded oligonucleotides that carry a fluorescent:quencher couple and a modified guanine base (oxo8dG) to develop a fluorescent system to measure ogg1 activity.
- Validated fluorescent:quencher system will be applied to a 96-well plate format for development of a high-throughput quantitation of ogg1.
“Nanoparticles”
Dr. Andrij Holian
Research involving developing engineered nanoparticles (particles smaller than 100 microns in one dimension) has progressed rapidly based on anticipated usage of nanoparticles over a wide variety of fields including manufacturing, electronics, and drug delivery. However, only recently has research begun to give information about possible adverse effects on human health. Nanoparticles are unique due to their composition, structure, and small size. While research has shown a variety of these particles to be toxic, the mechanisms behind potential adverse effects are unknown. Furthermore, not all nanoparticles are bioactive, but the characteristics that define safe from bioactive nanoparticles remains to be determined.
Our laboratory is currently investigating a particular subset of nanoparticles called multi-walled carbon nanotubes. We are currently working with 24 different multi-walled carbon nanotubes that differ in structure, size, as well amounts and types of metal catalysts used to prepare them. Our research has shown that the particles differ in their capacity to cause inflammation, cell activation, and cytotoxicity in a human macrophage cell line, THP-1 as well as primary murine alveolar macrophages.
The proposed study will use the THP-1 human macrophage cell line to elucidate how and why different multi-walled carbon nanotubes differ in their effects on cells. To do so, several different genetic markers that indicate inflammation and toxicity will be examined. The student will learn media preparation, cell culture technique, real-time PCR, data compilation, and data interpretation.
“Methamphetamine”
Drs. Andrij Holian and Melisa Schelvan
Concern regarding the use of methamphetamine (MA) has been rising in recent years. In 2006, it was estimated that nearly 6% of the U.S. population aged 12 years or older used MA at least once in their lifetime for non-medical reasons. Studies linking MA use with neurological, cardiovascular and pulmonary disorders have resulted in intense research in the field of MA addiction. However, the consequences and mechanisms of environmental exposure to MA have been largely unexplored. There is a significant potential for inadvertent exposure to MA through residues left from MA previously smoked as well as exposures through second-hand smoke. We are presently developing an exposure system that will enable us to study the second-hand effects of MA exposure, in collaboration with Dr. Sandra Wells from the University of Nebraska Medical Center. Current strategies include delineating the pulmonary and cardio-pulmonary consequences of second-hand exposure.
Our laboratory is primarily interested in the mechanisms of pulmonary toxicity from second-hand MA exposure. The neurological and cardiovascular effects of MA are well documented, however we have also demonstrated significant pulmonary toxicity and altered pulmonary function in mice following second-hand exposure to MA. Recent data in the laboratory of Dr. Wells suggests that the pulmonary effects of MA are associated with changes in proteins previously considered to be specific to the cardiovascular system. Therefore, we propose a cardio-pulmonary link that contributes to both the cardiovascular and pulmonary consequences of MA exposure. These studies will characterize novel and potentially critical pathways for understanding MA toxicity as well as cardio-pulmonary physiology.
For these experiments, the student will be involved in the development and validation of an exposure system that will enable us to conduct long-term studies on the second-hand effects of MA. In addition, the student will learn proper care and use of laboratory mice, as well as key laboratory techniques including in-situ hybridization, RT-PCR, histology, and confocal microscopy.
“Molecular Mechanisms of Wood Smoke-Induced Alterations in Macrophage Functions”
Dr. Christopher Migliaccio
Exposure to wood smoke particulate matter (WS-PM) can be from either periodic or chronic exposures. Epidemiological data has shown a link between exposures to biomass smoke and increased incidence in respiratory infections. Recent data in our laboratory has shown an increase in bacterial deposition in WS-exposed mice. The main immune cell of the alveolar spaces, the alveolar macrophage, is key to responses to inhaled particles and antimicrobial immunity. Both WS inhalation and WS-PM instillation studies have resulted in the activation of the non-canonical NFkB member, RelB. RelB has been associated with a PAH induced decreases in macrophage functions in other systems. The present study proposes to assess changes to macrophages following exposure to wood smoke particles with respect to RelB activation. The results of these studies will elucidate the mechanism of an increased susceptibility to respiratory infection following exposure to wood smoke.
In this project, the student will learn animal exposure models, flow cytometry, immunohistochemistry, and sterile techniques, including tissue culture of primary cells.
“Pharmacogenomics”
Dr. Mark Pershouse
Pharmacogenomics can be defined as the study of genetic factors (genotype) that determine if a patient is likely to benefit from, or be adversely affected by, a particular medication. A great deal has been learned from studies of polymorphisms in a small set of genes that determine or predict the outcome of frontline cancer therapeutics. These polymorphisms confer differential responses in patients through altered forms of key drug-metabolizing enzymes, drug transporters or drug targets (Bosch et al., 2006). Knowledge of population frequencies of specific gene polymorphisms (i.e. variants) associated with having an adverse drug reaction is typically based on studies in predominantly non-American Indian populations.
A number of population genetic factors, such as admixture (intermarriage), genetic drift and migration, can skew the distribution of allele frequencies in any one ethnic or cultural group, causing a skewed proportion of individuals with the risk genotype. Our overall hypothesis is that allele frequencies for key gene polymorphisms in understudied American Indian populations are distinctly different from those in previously studied groups. In other words, American Indians can be at higher risk of having an adverse drug reaction, or conversely, they can be at much lower risk.
Allele frequencies for functional polymorphisms in drug metabolizing genes have not been studied in American Indian population for a variety of reasons. Yet this population has a spectrum of cancer incidence similar to other ethnic groups. Our studies attempt to measure for the first time the allele frequencies for several key gene polymorphisms to predict whether the tribal population (Confederated Salish & Kootenai Tribes) is at higher or lower risk for adverse reactions to frontline cancer chemotherapy.
A summer project in our labs would involve genotyping 50-100 American Indian DNA samples for specific polymorphisms in glutathione S-transferase M1 (GSTM1), GSTP1, or other key metabolic genes. A student would be taught the basics of DNA isolation, characterization of the purity and yield of these isolations, polymerase chain reaction, operation of automated fragment analyzers such as the Bioanalyzer 2100 from Agilent. The student would also be introduced to basic genetic concepts; Hardy-Weinburg equilibrium, the concept of race and ethnicity at the genetic level, inheritance patterns, sensitivity to privacy issues, and specificity, sensitivity of genetic data.
“The control of protein production related to lung fibrosis”
Dr. Elizabeth Putnam
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. By inserting inhibitory sequences into Adeno-associated viral vectors and packaging them into virions, we will generate the reagents to treat mice already developing fibrosis after asbestos exposure.
The student on this project will learn how to isolate DNA, clone DNA fragments into vectors, culture cells, and titer virus. The student may also be able to analyze the resulting changes in protein expression in test cell cultures treated with the inhibitory AAV constructs.
“Regulation of inflammation and immunity by the Aryl hydrocarbon receptor”
Dr. David Shepherd
The Aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that is differentially expressed in many cells of the immune system. The AhR binds to many pollutants such as dioxins and PCBs that contaminate the global environment and have been linked to many adverse effects on humans such as cancer and chronic inflammatory diseases. Recently, a novel role for the AhR has been discovered that indicates that this cytosolic receptor plays an integral role in regulating the immune and inflammatory responses by macrophages and dendritic cells. Because both of these immune cell populations contribute to the generation and regulation of both innate and adaptive immune responses, it is critical to understand how the AhR functions in them. To this end, we are studying the effects of AhR activation in both macrophages (Macs) and dendritic cells (DCs) by in vitro cell culture approaches as well as in vivo animal modeling. The goals of these studies are to define the consequences of AhR activation on inflammation and immunity and on diseases such as colitis and silicosis that can be significantly affected by environmental pollutants.
This summer project will provide the student researcher with an opportunity to learn both the theory and techniques behind the immunotoxic effects of environmental pollutants. It will also give the student hands-on experience related to the design and execution of novel experiments intended to discover how the AhR regulates immune and inflammatory responses. Technically, you will learn how to culture bone marrow cells into primary cultures of Macs and DCs as well as transformed macrophage and dendritic cell lines. You will learn cutting-edge techniques such as flow cytometry and real-time RT-PCR and apply those to defining the role of the AhR. In addition, you will be exposed to ongoing animal experiments to evaluate the effects of environmental pollutants on chronic inflammatory diseases in the gut and lungs. Collectively, it is expected that the student will not only learn how to set up and perform biomedical research but will also generate novel data that will improve our understanding of environmentally-induced diseases.
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