"Interactions of Carbon Nanoparticles with Cell Membranes"
Dr. Andrij Holian
The field of manufactured nanoparticles (particles less than 100 microns in one dimension) has become anintense field of research over the past few years. This is due to the projected high impact that nanoparticles will have in many fields including manufacturing, computers, and human use for imaging and drug delivery. However, while the manufacture of nanomaterials has progressed rapidly little is known about potential adverse human health impacts of these particles. Nanoparticles have a number of unusual properties that are due to their composition and small size. Carbon nanoparticles (CNP) have high strength, electrical conductivity, and are in general extremely hydrophobic. A few animal studies have demonstrated that inhalation of CNP results in lung injury including formation of granulomas. Additional, studies with isolated cells have suggested that certain CNP may distribute into cell membranes. We propose that the extremely high degree of membrane solubility will be responsible for their unusual biological activity. The purpose of this project will be to determine if CNP distribute into cell membranes and to determine what types of membrane changes CNP cause. In addition, a number of different CNP will be tested to determine what characteristics or properties of CNP are associated with membrane interactions.
These studies will use macrophage cell lines, cell culture, enzyme assays and fluorescent techniques to evaluate membrane properties. The student will learn how to do sterile cell culture, conduct some different enzymatic assays and learn how to use a fluorimeter, confocal microscope and observe flow cytometery for the fluorescence assays.
"Simulating Human Exposures to Particulates Using Animal Models"
Dr. Andrij Holian and Dr. Christopher Migliaccio
It is challenging to devise an animal model that accurately simulates human exposures to particulates such as silica. One of the challenges is that no method is a perfect replication of the human condition and few investigators have conducted good comparison studies to validate the various techniques. Therefore, in this study we will compare four different procedures to deliver different particulates (e.g. crystalline silica, fluorescent amorphous silica) to mice and compare outcomes such as effects of pulmonary function, lung injury and inflammation, and uniformity of dispersion in the lung. The results of this study will provide important data to many investigators and will be used by center investigators to determine procedures for future studies.
In this project, the student will learn different methods for particle delivery to mice, how to measure lung injury, and examine particle dispersion in the lungs of mice.
"Oxidative Stress in Cellular Models of Parkinson’s Disease (PD)"
Dr. Fernando Cardozo-Pelaez
The highest risk factor for the development of PD is aging. PD is manifest by motor behavior deficiency that is rooted in the loss of dopaminergic neurons located in the substantia nigra region of the brain. Sufferers of PD evidence specific biological characteristics that are associated with the onset of the disease, but little is known of the role these physiological differences play in the specific neuronal loss seen in PD.
PD patients, in addition to the neuronal loss, evidence alterations to mitochondrial energy metabolism at what is called complex I and have deficiencies in endogenous antioxidant systems. Despite the relationship in these two alterations, there are no studies aimed to identify a cause-effect association with neuronal loss.
In this project, the student will expose PC12 cells (neuronal tissue culture cells) to inhibitors of mitochondrial complex I and establish changes to endogenous antioxidants. In a reverse approach, the student will reduce endogenous antioxidants and evaluate the function of the mitochondrial complex I. These studies will allow discerning the relationship between these two markers of PD and the role in neuronal susceptibility. The student will learn to maintain a cell culture, extract cytosolic proteins and measure levels of antioxidants. Also, the student will learn how to quantify mitochondrial complex I activity.
"The Effects of Woodsmoke Particles on Macrophage Function"
Dr. Tony Ward and Dr. Christopher Migliaccio
Exposure to biomass smoke can be from either periodic or chronic exposures. Periodic exposures are typified by communities in proximity to forest fires, while chronic exposures are typified by home heating/cooking in developing countries where open fires are commonly utilized. Epidemiological data has shown a link between exposures to biomass smoke and increased incidence in respiratory infections. The main immune cell of the alveolar spaces that is key to responses to inhaled particles is the alveolar macrophage. The present study proposes to assess changes to macrophages following exposure to woodsmoke particles. The results of these studies will elucidate the mechanism of an increased susceptibility to respiratory infection following exposure to woodsmoke.
In this project, the student will learn animal exposure models, flow cytometry, and sterile techniques, including tissue culture of primary cells.
"Understanding human exposure to wood smoke"
Dr. Curtis Noonan
One of the measures used to characterize air quality in a community is particulate matter (PM). PM is the term used for particles found in the air, including dust, dirt, soot, smoke, and liquid droplets. PM exposure in communities has been associated with increases in death and sickness from respiratory and cardiovascular health conditions.
The sources of PM can be varied depending upon the community. The predominant source of PM in many Rocky Mountain communities is wood smoke. During cold temperature periods people in these communities are exposed to wood smoke because wood stoves are a common source of heating. During hot seasons people can be exposed to wood smoke from nearby forest fires. In order to conduct health studies of people that are exposed to PM in these communities it is necessary to develop tools to assess people’s exposure to wood smoke. Our lab is currently conducting studies to assess chemical markers of wood smoke exposure that can be assessed in biological samples. We will be conducting small pilot studies in humans that are exposed to both dietary and wood smoke sources of these chemicals.
In this project the student will learn how to conduct human studies, including collection and processing of biological samples, analysis of chemicals in these samples, and data analysis.
"Mechanisms of Elevated Asymmetric Dimethylarginine (ADMA) in Asthma"
Dr. Sandra Wells
Asthma affects 300 million people worldwide and is predicted to affect over 400 million people by the year 2025. Despite significant advances in the understanding and management of asthma, treatment strategies predominantly focus on alleviating symptoms rather than the underlying disease. A better understanding of the physiological mechanisms underlying the pathology of the disease will be critical in identifying new therapeutic approaches and improving clinical outcomes.
ADMA is a naturally occurring protein breakdown product that is present in all cells. ADMA has been shown to be elevated in the blood of patients with various diseases including hypertension, chronic renal failure, and diabetes. Common mechanisms in the development of these diseases and diseases of the respiratory system including asthma suggest that ADMA may also play a role in asthma. Our laboratory was the first to demonstrate a link between elevated ADMA and key features of asthma utilizing mouse models. Our preliminary studies indicate that ADMA may also be elevated as a consequence of the disease, further supporting the notion that ADMA is an important molecule in asthma. The purpose of this project will be measure the levels of enzymes that play a role in regulating ADMA in the lung. The goal of this project is to identify which enzymes are most important in regulating ADMA in hopes of identifying potential future targets for treating asthma in humans.
In this project, the student will assess expression of the key enzymes regulating ADMA levels in mouse lung tissue. He/she will learn techniques including Western blot, immunohistochemistry, fluorescence microscopy, and laser scanning cytometry.
"Mechanisms of Cell Toxicity Following Exposure to Methamphetamine Smoke"
Dr. Sandra Wells
Methamphetamine (MA) is a highly addictive central nervous system stimulant that can be injected, snorted, smoked, or ingested orally. The abuse of MA is an extremely serious and growing problem. An estimated 10.4 million people age 12 or older (4.3 percent of the population) have tried methamphetamine at some time in their lives. Users are increasingly choosing smoking as their primary mode of administration which has surpassed all other forms of administration combined. According to the National Institute on Drug Abuse (NIDA), smoking MA may result in a more rapid addiction to the drug than snorting or injection since smoking causes a nearly instantaneous, intense, and longer-lasting high.
Currently, there are no published reports on the relationship between individuals exposed to MA smoke and resultant health consequences. There have been anecdotal reports of increased asthma, pulmonary fibrosis, and upper respiratory complaints, however no documented health statistics are available at this time. In our laboratory, we are utilizing mouse models of inhaled MA smoke exposure to determine the pulmonary consequences of these exposures. Our studies using mouse models have shown that inhalation of MA smoke results in lung injury and this may be mediated through oxidative stress. There are chemicals in MA smoke that may be toxic once they enter into cells. The purpose of this project is to determine whether these chemicals are toxic to cells in culture, and if so, explore the mechanisms of this toxicity. The goal of this work will be to better understand the effects of exposure to MA smoke with the hope of identifying a biomarker of exposure for use in future clinical studies.
In these studies, the student will utilize lung macrophage and epithelial cell lines to determine the toxicity of chemicals found in methamphetamine smoke. Through these studies, the student will learn sterile cell culture technique, how to conduct cell viability and death assays utilizing a fluorimeter and enzymatic assays, and how to assess oxidative stress utilizing various methods including confocal microscopy.
"Determining Whether the Length of Asbestos Fibers is Associated with Toxicity"
Dr. Andrij Holian and Dr. Tony Ward
Asbestos exposure is well known to cause a number of chronic lung diseases such as asbestosis and lung cancers, including mesothelioma. Although the association between asbestos exposure and lung disease has been known the physical characteristics of asbestos fibers responsible for disease causation remain unclear.
For many years, investigators have debated and tested whether there is an association between the length of asbestos fibers and causation of lung diseases. Smaller fibers are proposed to be cleared from lungs more rapidly, and therefore may be less important in disease causation. However, smaller fibers could move into the lung tissue faster, thereby becoming more important in disease causation. In this project the student will separate a heterogeneous mixture of asbestos fibers into different fractions using a process called elutriation. Once different size fractions are generated, our collaborator at the Wadsworth Center (Albany, New York) will conduct physical characterization of the fibers in each fraction. The different fractions will be tested in cell culture to determine the biological potency of each fraction. Different biological assays will be used to determine the correlation between fiber length and biological activity.
In this project the student will learn to fractionate asbestos fibers by elutriation. The student will also learn to conduct various in vitro toxicity and biological response assays. Using murine models assessment of initial markers of lung inflammation will also be conducted.
"Protein production related to lung fibrosis in models of asbestos-related disease"
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. Attention to asbestos-related diseases has re-emerged due to the exposure of Libby, Montana residents to asbestos-contaminated vermiculite. The distribution of this vermiculite to over 200 sites nationwide and the long latent period for disease development make asbestos-related diseases (ARD) a continuing public health issue.
We have used gene expression studies in a mouse model to identify potential candidate genes involved in asbestos response, one such gene being Sparc. Sparc (secreted protein acidic and rich in cysteine) is a matricellular protein involved in tissue repair, extracellular matrix (ECM) regulation, cellular proliferation, and cellular adhesion. The ability of Sparc to regulate the ECM makes it a novel gene candidate for involvement in the fibrosis that occurs after asbestos exposure. We have studied wild type and Sparc knockout mice exposed to crocidolite asbestos, Libby amphibole, or saline. Through Real-Time PCR and protein analysis we found that expression of both Sparc and collagen (a major component of the ECM) are increased in crocidolite and Libby amphibole treated mice compared to control. While the mouse studies continue, we are moving to a cell culture model to further examine the pathways controlling fibrosis development.
The student on this project will learn how to culture cells, extract RNA and protein from the cultures, and analyze the resulting changes in RNA and protein expression after exposure to different types of asbestos.
"The effect of prenatal arsenic exposure on placental integrity and
Dr. Howard Beall
The project focuses on arsenic mediated pregnancy complications. Environmental exposure to pregnant women causes premature deliveries and miscarriage. We conducted arsenic exposure experiments in mice and found that arsenic causes defects in placental vascular development. Although we have conducted cellular and molecular studies on arsenic complications, one of the aims in our project is to directly test placental integrity in arsenic exposed mice. Groups (n=8) of pregnant mice will be exposed to water (control) and to increasing levels of arsenic. Placental integrity will be tested using the VEVO 770 High Resolution Ultrasound Imager. Microbubbles are injected (see Figure 1 attached) intravenously into the maternal circulation. If the placenta is functioning properly and maintaining a barrier, the microbubbles will stay in the maternal circulation i.e. they will not cross into the embryonic circulation. We hypothesize that arsenic will degrade placental integtrity and the microbubbles will be found in the embryonic circulation of the arsenic-exposed mice. In addition, we will monitor blood pressures (using the VEVO) to assess whether arsenic exposure causes maternal hypertension. The latter is a sign for preeclampsia, a common problem of unknown etiology in humans. Maternal hypertension combined with placental insufficiency would suggest that environmental toxicity could be a cause of human preeclampsia.
This study will use high resolution ultrasound to monitor placental ingegrity and cardiovascular physiology and performance. The student will learn animal handling techniques and will explore the many capabilities of the high resolution ultrasound technology. In addition, the student will assist in other studies to include PCR, ELISA, immunoprecipitation/immunoblot and fluorescence microscopy.
"Histological Comparison of Animal Models of Silicosis"
Dr. Celine Beamer and Dr. Christopher Migliaccio
Chronic exposure to crystalline silica results in pulmonary inflammation and fibrosis. Unfortunately, diagnosis of the disease occurs too late in the process to render treatment effective. The use of animal models allows us to research the potential mechanisms involved early in the disease process in the hope of finding a therapeutic target prior to fibrosis. While many studies have implicated the role of Th2-immunity in the development of a fibrotic environment in some mouse models, recent experiments in our laboratory suggest distinct pathways based on the mouse strain being utilized. Therefore, in this study we will analyze lung tissue from multiple strains of mice, including null and/or cellular depleted strains, in an effort to delineate key elements of fibrosis.
In this project, the student will learn a variety of histological methods including sectioning and mounting of tissue, immunohistochemical staining procedures, and confocal microscopy.
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