Lucinda Carnell - US grants

Animals have the ability to sense, process and respond to signals important for navigating their environment. These behavioral responses to external signals rely on the nervous system?s ability to detect and integrate these signals. A continued exposure to a stimulus over time results in the animal diminishing its behavioral response, a phenomenon referred to as adaptation. Adaptation is critical for an organism to detect and respond to a new, possibly threatening, stimulus while in the presence of a continuous, existing stimulus. Modification of neural connections called synapses is thought to form the basis for adaptation, and other simple forms of learning. A central question regarding this process is how the nervous system modifies its connections to produce this diminished response. The goal of this project is to identify the gene products (proteins) mediating adaptation. The project will utilize the nematode, Caenorhabditis elegans (C. elegans), a well-characterized model organism for identifying genes involved in behavior. A method for measuring adaptation of the egg-laying behavior to long-term exposure to the neurotransmitter, serotonin, has been developed and will be used to examine the synaptic connections required for adaptation as well as to generate mutant worms defective in adaptation of this egg-laying response. Isolation of mutant worms defective in adaptation will provide a means to identified genes involved in this behavior. Because serotonin is an important neural signal that mediates behavioral responses such as feeding, mating behavior and aggression, this research may provide insight into how other serotonin-dependent behaviors are altered by continual stimulation. The project will have a broader impact on education through the applicant?s participation in the NSF-sponsored STEP program, which serves to increase participation and retention of students, including under-represented groups in the sciences. In addition, the applicant utilizes research in C. elegans in teaching molecular biology, genetics and neurobiology.

DESCRIPTION (provided by applicant): Type 2 diabetes is a serious metabolic disorder that has reached epidemic proportions worldwide. Decreased insulin secretion associated with the pancreatic 2 cells is a risk factor for the development of the disease. One model for the development of this risk factor is increased mitochondrial dysfunction and reactive oxygen species (ROS) generation. Support of this model comes from identification of a genetic link between defective nicotinamide nucleotide transhydrogenase (NNT) gene function and decreased insulin secretion (Freeman 2006b). NNT is a mitochondrial membrane protein that generates NADPH, which is required for the reduction of glutathione, an important antioxidant in ROS scavenger pathways. Another factor linking type 2 diabetes to mitochondrial dysfunction is intracellular lipid accumulation. Consumption of high- fat diets is an environmental factor that is associated with disease onset and with mitochondrial damage through the generation of ROS. Exactly how high-fat diets or intracellular lipid accumulation lead to mitochondrial dysfunction or how mitochondrial proteins contribute to this process is unclear. Thus, the effects of saturated and unsaturated fat diets and the role of the mitochondrial protein, NNT, on mitochondrial function will be investigated using the nematode, C. elegans. C. elegans is an established model for examining mitochondrial function and its physiological consequences. Previous studies have demonstrated that nnt-1 mutants have increased sensitivity to oxidative stress and recent preliminary data suggests that animals fed a high saturated fat diet have increased ROS production and movement deficits. Whole animal studies measuring mitochondrial function can be performed in conjunction with physiological function over the life span of the animal thus allowing a direct comparison between mitochondrial function and overall health of the organism. The hypothesis that NNT mutations and high-fat diet in combination will exacerbate mitochondrial and physiological dysfunction via increased ROS production and lipid peroxidation will be tested. The prediction that treating animals with mitochondrial-targeted antioxidants will diminish adverse effects will also be tested. These studies will clarify the origin of ROS and indicate whether or not further studies on mitochondrial-targeted therapeutic treatments of patients with type 2 diabetes are warranted. PUBLIC HEALTH RELEVANCE: Type 2 diabetes is a serious disease that has reached epidemic proportions worldwide. Recent evidence suggests that mitochondria may play a critical role in the development of the disease, however, mitochondrial dysfunction has not been clearly linked to physiological consequences. The proposed work will address the potential effects of dietary fat and genetic pre-disposition on mitochondrial and physiological dysfunction. Results will clarify the role of mitochondria and indicate whether or not further studies on mitochondrial-targeted therapeutic treatments of patients with type 2 diabetes are warranted.

Central Washington University (CWU) is a Baccalaureate and Masters Granting Institution located in a rural agricultural region about 110 miles east of Seattle on the other side of the Cascade Mountains. CWU has a student population of about 9,000 students with about 20% minority enrollment. The five investigators from the departments of Biology and Chemistry are requesting acquisition of instrumentation to enhance their existing microscopy facility. This acquisition will greatly enhance the teaching, training and research infrastructure at CWU.

The specific instrument is a new Leica DM5500 compound microscope system with fluorescence and differential interference contrast (DIC) capabilities. The proposal includes a vibration isolation table and a computational workstation that are necessary for utilizing the full capabilities of the microscope. To facilitate fluorescence visualization in multiple colors the microscope will be equipped with fluorescence-compatible objective lenses; a broad-spectrum (ultraviolet through infrared), high intensity light source; and multiple filters that allow specific wavelengths to be used for different experiments. Fluorescence microscopy will be used by nearly all the investigators to analyze fluorescently labeled cell organelles such as nuclei, mitochondria and axons. It will also be used to characterize multiple genes and proteins that are present in the cells of organisms ranging from extremophile bacteria to microscopic worms to developing chick and adult mouse brains. The DIC capability will allow investigators to visualize (and ultimately measure sizes of) structures within a tissue or within cells that have not been treated with a fluorescent or histological stain. This optical capability is necessary for investigators who study the growth of diverse fungi and those who need to visualize unstained cells and tissue in the worm and chick brain. Similar to the fluorescence capabilities, these DIC optics require highly specialized objective lenses, filters and prisms in the light path of the microscope.
In order to document results of experiments, digital cameras and a computer are necessary. Because some investigators will photograph with brightfield (normal white) light and others will photograph fluorescent images, two different cameras are required. Each is specialized for high image quality and accurate, quantitative representation of the brightfield or fluorescent specimen. Additionally, intense computational power is required for many of the analyses that will be performed on the images obtained. The requested computer has storage space for the many large images that will be collected, and it will be equipped with state-of-the-art software for performing the following functions: Deconvolution is a technique to analyze multiple focal planes of a specimen to digitally subtract unfocussed material in order to highly clarify the final image. Quantitation of fluorescence intensity will allow automated counting and measurement of sizes of labeled structures such as bacteria, axons, and mitochondria; it will also allow comparison of biochemical and DNA differences among experimental groups. Computerized control of the microscope stage allows stage movements to be calibrated and quantitated in order to measure the size of a three-dimensional specimen that cannot be visualized completely in one image.

The investigators involved in this proposal are studying areas of Biology that are as diverse as cell and developmental neuroscience, systematics and biology of fungi and fungus-like protists, microbial ecology of extreme environments and mitochondrial mechanisms of protection from oxidative stress. Each of these areas requires the scientists to be able to correlate molecular and biochemical changes with changes in cell structure and behavior. The most advanced microscopy instrumentation is required to analyze and document cellular and subcellular processes, such as changes in neuronal connections during development and behavioral adaptation, morphological differences among known and newly characterized fungi and protists, viral interactions with extreme microbes, and alterations to mitochondria in response to stress. Acquisition of this instrument will enable the investigators to establish and extend their research in these areas and to continue to train undergraduate and graduate students in their laboratories. Indeed, the Biology and Chemistry departments at CWU are able to incorporate many undergraduate students in the ongoing research. Undergraduates find it relatively easy to get involved with at least one research professor and typically disseminate this research through presentations and publication. Furthermore, the faculty are committed to incorporating inquiry-based teaching and technical training of students in laboratory classes. The acquisition of this instrumentation will allow even more undergraduates and masters-level graduate students to have access to state-of-the-art research microscopes as they train to become future scientists. CWU has established and provided support for several programs such as the Science Honors Program for undergraduate research and competitive funds for research and travel. CWU also has externally funded programs such as the NSF-funded Science Talent Expansion Program and Department of Education's McNair Scholars Program, both of which include efforts to increase recruitment and retention of underrepresented groups in science. CWU is implementing interdisciplinary watershed research into local middle and high schools through the NSF-supported GK12 Grant, Yakima WATERS, which places graduate fellows into schools to incorporate inquiry-based education into the curriculum. The investigators on this proposal are actively involved in these programs.