Undergraduate Research Fellows 2002-2003

Charisma Bautista Effect of Hydrastis canadensis isoquinoline alkaloids on the growth of Fusarium spp. MENTOR: Dr. George Bean, Department of CBMG

Hydrastis canadensis is a plant of significant medicinal value that has been included as an endangered species in Appendix II of CITIES. The plant has been used to treat mucous membrane inflammation. Moreover, H. canadensis contains isoquinoline alkaloids (IAs) that can be isolated and used as antimicrobial agents. The three major IAs are canadine, berberine and hydrastine. Several species of pathogenic fungi have been isolated from the roots of Hydrastis, including Fusarium oxysposum and F. proliferatum. This study will focus on the effects of these three major isoquinoline alkaloids present in Hydrastis, both individually and in combination, on fungal growth, in order to determine if Fusarium species isolated from H. canadensis are more adapted to this plant than other Fusarium species. For comparison, two Fusarium isolates from the barley plant will be used in the study as well.

Eric Buchner Second-site suppressor analysis of CheA -CheW interactions in Escherichia coli chemotaxis system. MENTOR: Dr. Richard Stewart, Department of CBMG

Movement of bacteria such as Escherichia coli towards and away from specific chemical gradients is called chemotaxis and is controlled by a signal transduction pathway. This pathway starts with the binding of a ligand to a transmembrane receptor protein, which in turn activates the protein kinase CheA. An additional protein, CheW, assists CheA in phosphorylating a third protein in this sequence, CheY. Activated CheY can then interact with the regulatory switch of the flagellar motor, changing the direction or pattern of bacterial movement. The proposed experiments would characterize the CheA-CheW interactions by creating and isolating mutated CheA proteins to suppress mutations in CheW known to inhibit interaction. Using mutagenesis polymerase chain reaction (PCR), it is possible to create a large variety of CheA mutants which can then be tested in a yeast two-hybrid selection scheme. In this selection method, only yeast with mutated CheA proteins that suppress known CheW mutations will grow, allowing for easy isolation. These proteins can then be sequenced and analyzed to find key interaction points with CheW.

A better knowledge of how the ear has adapted to a deep-sea environment will assist in understanding the evolution of the inner ear and the implications of ear morphology on its function. Prior studies have shown several trends in the anatomy of certain deep-sea species, such as elongated stereocilia bundles and enlarged saccules in sound-producing fish. This study will attempt to confirm these prior observations and look for general anatomical trends among the species studied. An effort will be made to identify the evolutionary forces driving these adaptations and explain the function these adaptations serve in the organism. The ears of five deep-sea fish species will be removed from the skull and their general morphology observed. The sensory regions of the ear will be dissected out, analyzed under high magnification using scanning electron microscopy and transmission electron microscopy, and variations in the orientation and structure of the sensory cells recorded. Observations will be analyzed for shared characteristics across the species studied, and the data compared with shallow-water species to determine what adaptations are unique to a deep-sea environment.

The objective of this project will be to study the involvement of the mitochondrial and the CD95 receptor pathways in Sulfur Mustard (HD)-induced programmed cell death, or apoptosis. We want to better understand the Sulfur Mustard(Hd) induced apoptosis pathways because sulfur mustard is used today as a chemical warfare agent that causes skin blisters. The first pathway is via the mitochondrial pathway which is dependent upon calcium and calmodulin. The second pathway is via the FAS ligand or CD95 receptor pathway named after the protein enzyme that binds at that receptor. Both of these initial events in both pathways leads to the caspase cascade that breaks down molecules important to the cell's viability, ultimately leading to the cell's death. Apoptosis inhibitors will be studied to try to protect against the HD-induced apoptosis. The specific inhibitors that we will study are: W7, a calmodulin (CaM) antagonist; M3/M38, CD95 antibodies used to block the CD95 receptor; ACDEYD-CHO, a caspase-3 specific inhibitor; and Z-VAD, the general caspase inhibitor. The effects of HD on cell viability will be measured by a Calcein-AM fluorescence assay and a LDH release assay. Apoptosis, or cell death, will be measured by a Caspase-3 assay.

The Jeffery laboratory studies the developmental differences between the cave-dwelling and surface-dwelling populations of Astyanox mexicanus. The cavefish populations have degenerate eyes and various degrees of pigmentation loss. Previous studies on A. mexicanus show that a complex pattern of gene expression leads to cavefish eye degeneration. A reduction in the expression of the Pax6 gene in the eye fields of the cavefish embryo contributes to eye degeneration. The over-expression of the gene sonic hedgehog (shh) leads to suppression of the Pax6 gene during eye development of cave-dwelling A. mexicanus (Strickler et al., 2001). Previous studies on mice show that the expression of the gsc gene regulates the expression of the shh gene (Filosa et al., 1997). I hypothesize that altered expression of gsc in cavefish may lead to the over-expression of shh and the subsequent events of eye degeneration. Understanding the role of the gsc gene in cavefish eye degeneration should provide a clearer picture of the complicated process of vertebrate embryonic development.

Thomas Charpentier The effects of TBP stirrups on the topology of the DNA-TBP complex. MENTOR: Dr. Jason Kahn, Department of Biochemistry

Dr. Kahn's research lab studies the topological and geometric effects induced by the TATA Box Binding Protein (TBP) upon binding DNA. TBP is an essential protein that identifies promoters on DNA, leading to transcription initiation. Our project seeks to determine whether the TBP-DNA complex can alter its shape to either induce writhe or not depending of DNA strain. Our research team is planning to test a mutated TBP that is predicted to cause less writhe in the DNA. We are testing to see if it is possible to show that binding of the mutant TBP may require unwinding of the DNA. The minicircle DNA we are using is a model system for DNA in chromatin. My part of the project initially is to mutate the gene for wild type TBP and purify the newly mutated protein. Future research will try to establish thermodynamic relationship between nucleosome and the TBP bonding, and quantify how much energy is needed for the initial twisting and writhing of the DNA strand.

Daniel Coady Lipophilic Cucurbiturils. MENTOR: Dr. Lyle Isaacs, Department of Biochemistry

The goal of the research is to create a lipophilic glycoluril that can be assembled into a lipophilic cucurbituril, which can be situated into a cell membrane. The glycoluril will be constructed first from dihydroxyfumaric acid, and other reagents in three separate reactions. Once pure glycoluril is obtained, the substituents can be added. These substituents will be long alkyl chains, or cholesterol. Long hydrocarbon chains were chosen because of their hydrophobic nature, and ease of assembly. Cholesterol will used because it is already abundant in membranes, and is readily accepted. Cell membranes are composed of phosphoslipids, in a bilayer. The phospholipid has a polar head group that will associate with the aqueous environment on either side of the membrane, and two fatty acid chains that will form the hydrophobic interior of the bilayer. It is this hydrophobic core the lipophilic cucurbituril is intended to fit. Once a fully constructed lipophilic cucurbituril is assembled it can be tested in a liposome. If successful then the cucurbituril will be tested for ion transport in a membrane.

Anne Colgrove Cloning of the TCV coat protein gene for over expression. MENTOR: Dr. Dorothy Beckett, Department of Biochemistry

Turnip Crinkle Virus, a plant RNA virus, is a member of the Carmovirus genus that causes crinkling of leaves in turnip plants. It is associated with a nonessential satellite RNA, called sat-C RNA, which intensifies the symptoms observed in the plant upon infection with TCV. The coat protein, a structure that is important to the assembly of the virus, also alters the outward expression of symptoms in an infected plant. Recently, it has been observed that a reduction in the amount of CP can block the normal intensification of symptoms observed when a TCV infection of Arabidopsis thaliana is accompanied by sat-C. Also, when sat-C RNA was present in the infection, the virions had less RNA than normally expected. We hypothesize that this is because a competition exists for CP between the sat-C and the genomic RNAs. In order to better understand the symptom modulation at a biochemical level, we will study the interaction between CP and sat-C RNA. The immediate focus of this research is to produce large amounts of TCV coat protein by cloning the gene and over expressing the protein in E. coli. The cloned gene will be sequenced in order establish that the correct sequence has been retained. Once this process is complete, the coat protein will be purified in large quantity.

Nesseria gonorrhoeae is the causative agent of gonorrhea, one of the most prevalent STDs in the country. These gram-negative diplococci primarily infect the mucosal linings of the urogenital tract in men and women. Disseminated infections can lead to secondary conditions like infertility, particularly in women. Attempts to study the pathology and the immune response to the infection have been hindered by a lack of a suitable animal model for the disease, as N. gonorrhoeae is a strict human pathogen. A mouse model for gonococcal infection has recently been developed. The major goal of this study is to use this mouse system to characterize the local mucosal immune response to gonococcal infection. I will analyze the pathology and the local mucosal immune response of the genital tract of gonococcal infected mice. I will identify the regions gonococci preferentially colonize and the cells gonococci principally reside in, as well as determining the levels of IgA, leukocyte-homing receptors, cytokines, and the numbers and types of immune cells in the infected genital tract using enzyme-linked immunosorbent assay and immunohistochemistry. This study will greatly increase our understanding of gonorrhea disease process and may provide new strategies for the prevention and treatment.

Kathleen Daly Role of estrogen and estrogen receptors in early stages of colon cancer in female rats. MENTOR: Dr. Bernadene Magnuson, Department of Nutrition and Food Sciences

Recently, it has been suggested that estrogen has a protective effect on colon cancer. Males are statistically more likely to develop colon cancer, and numerous studies have suggested that hormone replacement therapy (HRT) reduces the risk of colon cancer in post-menopausal women. The proposed study will examine the association between estrogen levels and estrogen receptor expression and early stages of colon cancer in differing ages of female rats. Recent studies suggest that higher levels of a specific estrogen receptor (ERb), located in the colon, may reduce the susceptibility and development of colon cancer. This study will determine if changes in levels of ERb in the colon occur with age, and if these changes have an effect on development of early stages of colon cancer. The information that maybe gained from this study will further our understanding of the role of estrogen in colon cancer development in varying ages of females. Increased knowledge on this subject will show further implications of HRT on women. Furthermore, this study may provide information for investigating preventative effects of some dietary compounds that have estrogen-like activities such as soy phytochemicals.

Danny Gold Effects of tendon-vibration on disordered kinesthesia in Parkinson's Disease during voluntary movements. MENTOR: Dr. Jose Contreras-Vidal, Department of Kinesiology

Parkinson's disease (PD) is a neurological disorder traditionally characterized by a variety oi motor deficits such as tremor, hypometria and bradykinesia. However, recent behavioral studies suggest the notion of PD being a purely motor disorder is not correct, and that the motor disabilities may result from problems that exist on the sensory rather than the motor level. Thus, a preliminary study I conducted (Gold & Contreras-Vidal, in preparation) showed that PD patients, compared to controls, undershoot targeted movements under conditions that require proprioceptive guidance of limb movement (e.g., blind reaches). This suggested that the "internal model" for hand motion is disrupted in PD. This innovative vision of the disease requires quantification of the extent to which sensory disturbances contribute to the debilitating motor deficits that PD patients display. Now, I propose to use the tendon-vibration paradigm to assess the effect of proprioceptive distortion in PD's control of hand movement. By applvqng vibration to the distal tendon of the biceps and anterior deltoid of the dominant limb during a drawing task with vision occluded, proprioception can be disturbed. We expect the PD patients to undershoot movement less than controls (in contrast to studies without tendon-vibration where patients undershoot more than controls). The results of this study will provide an assessment of the extent to which sensory disturbances affect the movements of patients, while also serving to validate the view that PD must be viewed as a sensory-motor disorder.

Upon infection, organisms activate transcription factors which bind to target sites that regulate genes important to the immune response. Discovering the sequence and location of these sites helps us understand the specificity of transcription factors, and consequently, the mechanics of the response. Microarray studies provide whole genome expression data that enable scientists to map out transcriptional networks on a genomic scale. One approach to outlining these gene regulation networks is to mine the data from microarrays to develop generalizations of the target sites, and then to search through the genome for sequences which fit these prototypes. Most popular computational methods used to generate prototypes and predict binding sites make assumptions that may reduce the efficacy of the search. Neural networks are an alternative computational approach and they can be constructed such that they are free of most prior biases upon the similarity of its training set. The following proposal demonstrates how a novel neural network adaptation can assemble a sophisticated prototype by learning the motif of sequences upstream and downstream from the genes identified through microarray studies. When this prototype is compared against the genome, predictions are confirmed by wet-lab assays and a transcriptional network will be mapped.

Aydin Haririnia Assignment of amino acid residues of UBA domain of HHR23A utilizing NMR spectroscopy. MENTOR: Dr. David Fushman, Department of Chemistry-Biochemistry

The proposed research involves the use of nuclear magnetic resonance (NMR) spectra to analyze and assign the amino acid residues of UBA (Ubiquitin Associated) domains of hHR23A (Human homologue of Rad23A). UBA-ubiquitin interactions are linked to many human diseases, including cancer and neurodegenerative disorders. The UBA domains are present in various factors that function in DNA repair, proteolysis and signal transduction. It is now the subject of one of the most important fields in medical research. The role of ubiquitin and UBA in proteasome degradation, which controls the level of proteins in the cell by removing misfolded and unnecessary proteins, is an issue that needs to be addressed to understand the complex process of ubiquitin-mediated proteolysis. UBA is involved in modulating ubiquitin related degradation. Thus, understanding the interaction between UBA and ubiquitin could provide valuable insights into proteasome degradation. Characterization of the interaction between these proteins requires a map of the interface between their domains. In order to produce this map of the interface, assignment of NMR signals is necessary. The proposed research aims to assign the peaks of UBA(1) and UBA(2) in the context of hRAD23 and in its complex with polyubiquitin chains using NMR spectroscopy.

Mika J. Hunter Effects of organic insecticides on biological control agents in organic agriculture. MENTOR: Dr. Galen Dively, Department of Entomology

Among the many vegetables grown by organic growers, green bean is one of the major cash crops. However, a major constraint to the success of this crop is the Mexican bean beetle. Originally a major pest of soybeans in the United States, the Mexican bean beetle (MBB) is the primary insect pest of green beans. Unfortunately, the MBB is difficult to suppress. This factor becomes even more difficult for organic growers to deal with due to the availability of OMRI-approved (Organic Materials Review Institute) products. One alternative is the use of biocontrol agents. In the case of green beans, a parasitic wasp, P. foveolatus, can be released to control the MBB population. However, after P. foveolatus releases, insecticide applications may be necessary to suppress MBB if they are reaching economically significant levels. Currently, few studies have been conducted exploring the effects of these products on P. foveolatus and whether there are ways to use insecticides to minimize disruption of P. foveolatus. This experiment will determine these effects by looking at different OMRI-approved products and conducting a surface-treated exposure test and a foliage-treated feeding study. Adult mortality, time to adult parasitoid emergence, number of progeny per host, sex ratio, and body weight will be the data endpoints collected.

Rebecca Iskow Cytoskeletal function in the determination of cell fate in the water fern, Marsilea vestita. MENTOR: Dr. Stephen Wolnick, Department of CBMG

Programmed cell death is important for the proper development of all higher organisms. Although tremendous progress has been made in understanding how cell death is regulated, the identity of many proteins has yet to be determined. I propose to study this pathway during steroid-regulated destruction of Drosophila melanogaster salivary glands by profiling the proteins present before and during cell death. Samples will be collected using standard protein extraction techniques and then analyzed by two-dimensional SDS polyacrilamide gel electrophoresis (SDS-PAGE) and finally matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

Marnie Layton Genetics of C. briggsae Sex Determination. MENTOR: Dr. Eric Haag, Department of Biology

Sex determination genes are very important because they are some of the fastest evolving genes in the genome. The pathways of sex determination are also changing very rapidly, and many distantly related species can use entirely different sets of genes to deterrnine sex. A main focus of the Haag lab is an attempt to determine whether C. elegans and its sister species, C.briggsue, are using the same genes to control hermaphrodite development. My job is to help reconstruct the sex determination pathway in C. briggsue, the less studied species, so it can be later examined for the individual steps that lead to its sex determination. To do this I will perform F2 screens for sex determination mutants of C. briggsae mutated by EMS. After identifying the Tra mutants that disrupt specific genes in the sex determination pathway, I hope to map and balance the mutants. The mutants will then be used in screens for suppressors of the Tra phenotype. The other lab members will then use this information to perform molecular cloning techniques on the affected mutants.

Hansel E. Lee The Pathway of Carotenoid Synthesis in the Dinoflagellate Gonyaulax polyedra. MENTOR: Dr. Elisabeth Grantt, Department of CBMG

Carotenoids are found in all photosynthetic organisms where they aid in light absorption for photosynthesis, and serve as antioxidants to protect from free radicals produced by light. These pigments are also the major source of vitamin A in the human diet. Although the biochemical synthesis pathway of carotenoids in terrestrial photosynthetic organisms is fairly well understood, very little is known about the pathway in aquatic organisms including algae. This is significant because nearly all photosynthetic activity occurs in aquatic systems, and not on land. It seems likely that the algal pathway will be similar to that of land plants, however major carotenoids in algae such as peridinin and fucoxanthin are not found in land plants. The goal of this project is to identify the genes and study the enzymes of the carotenoid pathway leading to peridinin in the dinoflagellate Gonyoulax polyedra. In addition to new information on the carotenoid synthesis pathway of algae, the results may also add to our understanding of the origin of carotenoid pathway genes during dinoflagellate evolution.

Victoria Lilling Establishment of a Synthetic RNA Double Stranded Virus in the Yeast, Saccharomyces cerevisiae. MENTOR: Dr. Jonathan D. Dinman, Department of CBMG

The L-A double-stranded RNA (dsRNA) virus of yeast acts as a helper virus to support the M₁ and X dsRNA satellite viruses. The genome of M₁ encodes a secreted toxin, and L-A and M₁ infected cells secrete a toxin that kills uninfected yeast cells. X is a deletion mutant of L-A that acts as a defective interfering particle, excluding M₁. The lack of a reverse genetic system for dsRNA viruses precludes any study the function of the genes contained the genomes of this class of viruses. In my project, I will create a reverse genetic system for L-A. I will start by making a synthetic RNA satellite virus containing the 5'- and 3'-ends of X and a selectable marker to monitor transfection in the yeast cells. I will also use the loss of the M₁ satellite virus as an indicator that the synthetic dsRNA satellite virus has been transfected into the yeast cells. Upon successful establishment of the synthetic X-virus construct, I will make a synthetic L-A virus from a cloned cDNA and transfect both the synthetic L-A and synthetic satellite virus into a yeast cell that lacks the L-A virus. Selection for the X virus based selective marker will enable selection for self-propagating synthetic L-A.

Milton Liu Rational Control of Molecular Adsorption by Chemical Design. MENTOR: Dr. Robert Walker, Department of Chemistry-Biochemistry

A clearer understanding of the factors that affect how a molecule binds to a surface, or adsorbs, would be useful in not only chemistry, but biology and engineering as well. Hydrogen bonding, electrostatics, temperature, and molecular structure all have some effect on the kinetics of molecular adsorption and the equilibrium that exists between solutes in solution and solutes adsorbed to a solid surface. Quartz slides of different polarities will be exposed to bulk solutions of varying concentrations and allowed to come to equilibrium. The number of molecules bonded to a surface will be detected using excitation and emission spectroscopy. Preliminary proof of principle results for Rhodamine 6G are included. Future experiments with Coumarin dyes will vary structure, temperature, and substrate composition in order to determine how these factors affect the kinetics and thermodynamics of molecular adsorption.

Long T. Nguyendo Identification and Characterization of a Gene in the Biosynthesis Pathway of Neisseria sicca. MENTOR: Dr. Daniel Stein, Department of CBMG

Neisseria gonorrhoeae is the pathogen that responsible for the sexually transmitted disease, gonorrhea. Its pathogenicity is largely dependent on the type of surface structures that it produces. Typically, Neisseria strains produce lipooligosaccharide (LOS); however, certain non-pathogenic strains display lipopolysaccharides (LPS) on their outer membrane. This suggests that non-pathogens may be altered due to differences in the outer membrane structures. From our previous work, we have been able to isolate LPS mutants by screening for antibiotic resistance. This study is based on the hypothesis that these LPS mutants have defective genes involved in LPS biosynthesis. We will randomly insert a known sequence of DNA into the bacterial genome to create LPS mutants with the same LPS structures that previously conferred antibiotic resistance. Using the inserted sequence, we will clone and sequence the target gene, and then, attempt to identify its function by searching for other previously characterized genes with sequence homology to the target gene in the national gene database.

Sweet corn containing genes from Bacillus thuriengensis (Bt) express Cry1Ab protein which is specific to caterpillars. Although the protein expressed in pollen has no known toxicity to honeybees, studies have not addressed sublethal effects on bee behavior. This study will determine the effects of Bt sweet corn on the foraging ability of honey bees to find sources of food and communicate locations of these sources to other workers. The experimental design will involve two plots of sweet corn (Bt versus nonBt) planted 500 meters apart. One week prior to pollination, two hives will be placed in each plot and allowed to produce new brood by utilizing pollen from corn plants and from pollen cakes made from pollen collected in each plot. At 2 and 5 weeks after the exposure, bees from each hive will be marked with a unique color, and a honey station will be established midway between plots. For two days thereafter, records of the number of marked bees finding the honey station and those returning to their respective hives will be used to test whether bees from hives in the Bt and NonBt plots are exhibiting the same foraging and orientation behaviors.

Fungi are pathogens of both animals and plants. A significant factor in both is the ability of the fungus to tolerate antifungal compounds, such as antibiotics used to treat animals or natural defense compounds in plants. Studying the regulation of resistance genes in plant pathogenic fungi provides an understanding of the ways that fungi can evolve resistance. The pea pathogen Fusarium solani (also a human pathogen) is able to detoxify an antifungal isoflavonoid compound, pisatin, produced by the host plant. Expression of a cytochrome P450 pisatin detoxification gene (PDA1) is triggered by the exposure of mycelium to pisatin. A 40 bp sequence in PDA1 is responsible for pisatin induction. A protein (PRF) that binds this site has also been identified along with its gene sequence. The aim of my project is to test the hypothesis that this PRF protein acts as a nuclear receptor, binding both pisatin and the 40 bp DNA site. To test this, I will express the PRF protein in a yeast strain in which the 40 bp pisatin-responsive site sits behind a lacZ reporter gene. I will see if the engineered yeast strain will modulate the lacZ reporter activity in response to pisatin treatment. Function of this protein as a nuclear receptor would be significant in identifying the signaling pathway for the control of this resistance gene.

Tam Quach Role of the Receptor Phosphorylation in Regulating the Intracellular Trafficing of the B Cell Antigen Receptor. MENTOR: Dr. Wenxia Song, Department of CBMG

The binding of B cell antigen receptor (BCR) to exogenous antigens initiates signaling cascades through Iga/Igb heterodimer of the receptor and induces the internalization of BCR/antigen complex. Previous studies indicate that the signal transducing through the BCR regulates BCR internalization processing. However, the mechanism for such regulation remains to be elucidated. The goal of this proposal is to examine the role of the receptor phosphorylation in the intracellular trafficking of the BCR by mutating phosphorylation sites in the immunoreceptor tyrosine-based activation motifs (ITAM) and other regions in the Iga cytoplasmic tail.

Jonathan Reichel Does IGF-1 Genotype Predict Muscle Mass Response to Strength Training in Women and Men at Risk for Sarcopenia? MENTOR: Dr. Ben Hurley, Department of Kinesiology

The loss of strength and muscle mass with age, known as sarcopenia, is associated with an increased susceptibility to a variety of diseases and disabilities. Because strength training (ST) can reverse this process, it is often considered the intervention of choice for the prevention and treatment of sarcopenia. However, there is a large inter-individual variability in muscles' response to ST and twin studies indicate that heredity may explain this variability. In this regard, insulin-like growth factor one (IGF-1) is a leading candidate gene for stimulating increases in muscle size (hypertrophy). Therefore, I propose to test the hypothesis that IGF-1 gene profile (genotype) will influence muscle hypertrophic response to ST in middle-aged and older adults. To test this hypothesis, 50 to 80 year old women and men will undergo genotyping for the IGF-1 gene at baseline (before kaining) and computed tomography (CT) scans before and after training to assess muscle hypertrophy as a result of a 10 week ST program. This data will be used to determine the association of muscle hypertrophy with IGF-1 genotype. The significance of these results is that it will add new information to the current literature on the contribution of a specific candidate gene in explaining variability in muscle hypertrophic response to ST for those who are at risk for sarcopenia. This information in combination with other follow-up investigations could lead to individualized exercise prescriptions for the prevention and treatment of sarcopenia in the elderly.

Many microorganisms of the domain Archaea, one of the three domains of life aIong with the Bacteria and the Eukarya, live in extreme environments such as hydrothermal areas. These microorganisms are called hyperthermophiles. One example is Pyrococcus furiosus that grows optimally at 100°C. In this environment its DNA undergo extensive damage, including mutation. When accumulated, these mutations will kill the cell. All DNA-based microorganisms, bacteria and eukaryotes, have a similarly low genomic spontaneous mutation rate resulting from the action of specific DNA repair mechanisms. No such repair mechanism has been found in any of the ten-archaeal genomes sequenced so far. I propose to find out if the genomic spontaneous mutation rate in P. furiosus is similar to that of other DNA-based microbes, suggesting that P. furiosus has a novel system to repair spontaneous mutations. Alternatively, a high mutation rate would suggest that P. furiosus does not have a system to repair spontaneous mutations. I will also obtain mutants with abnormally high mutation rates with the long-term goal to study DNA repair pathways in P. furious, and ultimately understand how hyperthermophiles can live in such extreme environments.

Ceramides, naturally occurring lipids known for their involvement in cellular apoptosis, or programmed cell death, are theorized to form channels in the outer membranes of mitochondria. Cytochrome c, a protein needed in the cytoplasm to trigger the process of apoptosis, is apparently released from the mitochondrial intermembrane space into the cytoplasm through ceramide channels in the outer membrane. Evidence that ceramide channels are specific to the outer membranes of mitochondria leads us to investigate the reason for this membrane specificity. By detecting channel formation in liposomes made up of a variety of different lipid components, a lipid dependence can be determined. Also, experimentally testing the current model for ceramide channels will be informative in determining the characteristics of the ceramide molecule that are important to channel formation. Such results could explain reasons for any lipid dependencies that exist. Overall, the research will be geared toward discovering the propensity for channel formation in various different settings, allowing us to determine why, in fact, cerarnide channels are membrane specific. Such knowledge will lead to an increased comprehension of the apoptotic process, and thus will have many biomedical implications.

Robert Stuntz The Role of Hairpin 5 in the Relication of TCV's SatC. MENTOR: Dr. Anne E. Simon, Department of CBMG

Turnip crinkle virus (TCV), a 4054 base long (+)-sense RNA plant virus belonging to the genus Carmoviridae, is associated with several satellite RNAs, including the 356 base long satellite RNA C (satC). SatC shares a portion of its sequence with TCV, including several elements found to be important in replication. Among these replication elements shared by TCV and satC is Hairpin 5, a hairpin whose internal loop can be found in nearly all Carmoviruses. Mutations made in this loop in Hairpin 5 of TCV have been shown to cause a loss of replicase fidelity and, in some cases, a complete loss of TCV replication. Is satC's Hairpin 5 of equal importance to satC replication? The goal of my research is to elucidate the importance of Hairpin 5 in the replication of TCV's satC.

Adrianna Szczpaniec The Effects of Systemic Insecticide, Imidacloprid, on Fecundity and Natural Enemies of Pest Species in Mites. MENTOR: Dr. Michael J. Raupp, Department of Entomology

My independent research project deals with the effects of imidacloprid, a systemic insecticide developed to target specific pests, on the communities of beneficial insects that play a crucial role in pest control. The disruption of the populations of natural enemies of insects is thought to be the primary reason behind the pest outbreaks. The system I study involves ornamental plants, boxwoods and hemlocks, in urban settings as well as in a greenhouse. The pests used in the experiments are boxwood mites, spruce spider mites and hemlock rust mites. My research involves monitoring the communities of beneficial insects on imidacloprid treated shrubs and trees at two study sites in Washington, DC, as well as performing bio-assays in the laboratory. Sticky traps placed on trees and shrubs are used to monitor the populations of natural enemies at the field sites. The laboratory studies involve exposing pest mites to imidacloprid, offering them to natural enemies and measuring the mortaiity of beneficial insects. Another aspect of my studies involves examining the phenomenon of increased egg production by pest species of mites when exposed to imidacloprid. The food offered to mites varies in the levels of insecticide, and the egg production is compared to the fecundity of mites not exposed to imidacloprid. Biochemical techniques will be used to evaluate the level of imidacloprid in plants and, if possible, insects.

Salmonella typhimurium DT104 is an emerging pathogenic strain of enteric bacteria. It is a principle cause of gastroenteritis in the United States and exhibits widespread antibiotic resistance. S. typhimurium possesses numerous survival mechanisms which enhance its ability to thrive under adverse conditions. One such mechanism is the formation of rugose colonies at 25°C, a morphological change. The presence of rugose colonies is correlated with the presence of an extracellular substance encapsulating the cells and the ability to aggregate. Rugose colonies exhibit an increased resistanee to acid, salt, chlorine and oxidative stress. These are conditions that are potentially experienced in the transition between host and external environment. Initial studies have demonstrated that the formation of rugose colonies is density dependent. Gram negative bacteria, such as Salmonella spp. engage in cell-to-cell communication, called quorum sensing. Autoinducer signaling molecules control gene transcription as a function of cell density. This project will attempt to determine if autoinducer production affects rugosity. The second goal is to determine where, and at what point, the autoinducer affects the genetic pathway regulating rugose formation.

Programmed Cell Death (PCD) is a fundamental process that eliminates obsolete cells and allows organisms to develop properly. Defects in PCD are known to cause tumors, birth defects, and neurodegenerative diseases. In Drosophila, the steroid hormone 20-hydroxyecdysone (ecdysone) triggers metamorphosis. During metamorphosis, ecdysone activates larval tissue destruction by PCD and adult imaginal tissue differentiation. During this process, the steroids are bound by a receptor complex that induces chromosome puffs, sites of active gene transcription. Previous research has shown that the ecdysone regulated E93 gene, that was isolated from the 93F puff, is an important gene in the PCD pathway. I propose to analyze other chromosome puffs with similar characteristics as 93F to identify another gene involved in PCD.

Rodolfo E. Zamora Manipulation of TCV Hairpin 5. MENTOR: Dr. Anne E. Simon, Department of CBMG

Turnip crinkle virus (TCV), a 4054 base long (+)-sense RNA plant virus belonging to the Carmovirus genus, is associated with several satellite RNAs, including the 356 base long satellite C (satC). SatC shares similar stretches of sequence with TCV and these sequences have been shown to be important in viral replication. One important common motif between the two is Hairpin 5, a hairpin whose internal loop can be found in nearly all Carmoviruses. Mutations introduced in this loop in Hairpin 5 have been shown to cause a loss of TCV replicase fidelity and greatly reduced accumulation of TCV in plants and protoplasts. These effects of Hairpin 5 in TCV may not only be the result of its sequence, but also of the size and orientation of the element. What effect does inverting Hairpin 5 or moving it to other locations in TCV have on the replication of TCV? I plan to make a construct of TCV with an inverted and moved Hairpin 5 and test its replication activity relative to other constructs.

Past research has shown that sex determination genes often evolve rapidly in comparison to other genes. Often sex determination genes are species-specific, and even closely related species can have very difFerent sequences for the same gene. What remains unclear is the method by which these genes evolve. This is one question asked in the Haag lab. Specifically, we are trying to determine if the differences in the fem-3 sex determination genes in C. elegans, C. briggsae, and C. remanei are a result of an ongoing process of continuous rapid mutations or if they are a product of a sudden sweeping change at the time of speciation. My job is to determine if the variation of sex genes seen at the species level is also present within the population and to determine if the variations are due to natural selection of if they are neutral. To do this I will amplify fem-3 via PCR from multiple strains of a species, sequence the gene, compare the sequence to a reference strain, and then analyze the data to determine what variants are present in the population. These data can then be used to determine how the sex determination genes are changing with time.

This experiment will create fluid flow that moves particles as desired. Thus far, simulations show that pressure differences on tiny intersecting flow channels can move particles as desired. The next stage in this research is to test the results from these simulations in a lab. To carry out the experiment, micro-channels will be fabricated by cutting through a sheet of plastic and packing the patterned sheet between two glass layers. Tiny liquid pumps will be placed at the openings of the channels. Four different experimental setups will test various pressures applied at each channel, in order to sort particles, separate clustered groups of particles, collide particles, and to sort the same particles into the same channels.

Many microorganisms can navigate towards sources of nutrients (chemo-attractants), and away from harmful substances (chemo-repellents). This capacity, called chemotaxis, is the result of a coordinated signaling system which converts information about the surrounding environment into a behavioral response. In E.col, the fundamental mechanism underlying chemotaxis involves a set of cytoplasmic signaling proteins called Che proteins that regulate the swimming pattem of the bacterium. Of these, the protein kinase CheA plays a central role, forming a complex with CheW (an adaptor protein) and receptor proteins that directly bind attractants and repellents. Following CheA autophosphorylation, a phosphate is transferred from CheA to CheY. Phosphorylated CheY can then bind to a flagellar switch protein, FliM, causing a change in the rotation of the flagella. I am currently in the process of characterizing mutant versions of CheW, specifically dominant-negative mutant alleles, in the hopes of elucidating the roles of CheW. Characterization has involved sequencing the mutant alleles as well as investigating how the mutants affect the biochemical activities of purified CheW protein. The swimming patterns of cells expressing the cheW alleles will also be observed in liquid culture and other assays to study altered flagellar rotation. The bacterial chemotaxis system serves as an excellent model for studying protein-protein interactions and sensory signal transduction processes that are necessary in all cells.

The Cossidae is a family of moths (order Lepidoptera) that occurs worldwide. It is composed of approximately 700 species, all of whose larvae are woodborers. The Cossulinae, one of the six subfamilies of the Cossidae, is restricted to the Americas in its distribution. The proposed research involves identifying the diversity of the Cossulinae of Costa Rica. The Costa Rican fauna of Cossulinae is estimated to comprise 17 extant species. ln order to systematically identify specimens from Costa Rica, morphological analyses will be conducted, dissections and nomenclatorial changes will be made accordingly, and species descriptions will be generated.

The movement of substances within the cytoplasm is important for a variety of cellular functions. Transport within a cell relies on proteins that interact in special ways. Some of these proteins form filamentous cytoskeletal networks, while motor proteins move along the filaments, carrying substances to distinct destinations. Several motors have been identified, and their functions have been documented in multiple kinds of cells. The working paradigm for motor function is that each motor moves in only one direction along a particular kind of filament. During the period of support, I will concentrate on a specific group of motors, known as the kinesins. I will study kinesin involvement in movements that lead to the differentiation of motile gametes (spermatozoids) in the water fern Marsilea vestita. This organism exhibits extraordinary spatial and temporal precision during development, which relies on the translation of stored mRNAs. RNA interference (RNAi) experiments eliminate specific mRNAs in the gametophyte, and the null phenocopy provides the means to determine specific functions for particular kinesins in development and differentiation. I expect each motor to affect development in different ways and at different times. By observing development in the absence of each of these motors I can assess their functions.

Hemophilia is an inherited bleeding disorder that affects people from all races and ethnic groups. The most common form of this disease is hemophilia A, which is typically caused by genetic mutations in the X- chromosome linked gene Factor 8 (F8C). Many mutations have been previously discovered in this gene in affected individuals; however, there is very little known about the pattern of variation in this gene for "normal" samples of individuals from global populations. I intend to screen different regions of the F8C gene for mutations in many human groups including samples from both Sub-Saharan Africa and regions outside of Africa for a total of ~300 individuals. DNA sequence analysis and family relatedness analyses of these different groups will finally tell us something about the F8C gene and the mode of genetic inheritance in both infected and uninfected individuals. In addition, by examining the association and pattern of mutations across the F8C gene in different ethnic groups, we can learn about the age, origin, and impact of these mutations and their association with hemophilia A.

Mutant forms ofthe gene BRCA1 have been associated with a significantly increased risk for breast, ovarian, and prostate cancers. The gene product of BRCA1 is known to have a large variety of functions within the human body, most notable are its roles in cell growth regulation and tumor suppression. This project aims to determine the structure of the BRCA1 protein in the domain where it interacts with another tumor suppressor, the retinoblastoma protein, or RB. Such a structural determination is important, as an understanding of the three-dimensional structure of the RB-interactive domain of BRCA1 is important in understanding the nature ofthe interaction of these two proteins, as well as the means by which BRCA1 functions. Understanding ofthe structure of BRCA1 would allow researchers to target specific functions of BRCA1 to treat or prevent cancer in patients who carry mutant versions of the gene.

Phagocytosis is a process that must be regulated by different genes, whether to recognize pathogens, to ingest them or other processes to keep the organism from infection. Previous research suggests that in Drosophila, there are several genes of interest that may be involved in one or more of these processes. Using flies known to have mutations in these genes of interest, I have been performing an assay to test the phagocytosis of fluorescently labeled Escherichia coli, Staphylococcus aureus and Saccharomyces cerevisiae particles into Drosophila cells. The flies are injected with these particles then time is allowed for phagocytosis to occur. After sufficient time, the flies are injected with trypan blue dye to mask the fluorescence of any particles not engulfed by a fly cell. The flies are then examined under a fluorescence microscope with specific light filters to distinguish between different fluorescent particles. Drosophila phagocytes lie on the dorsal vessel in the fly abdomen so fluorescence in that region is considered positive for phagocytosis. If no fluorescence is observed, the fly is considered negative, possibly due to the mutation in question. After identifying which mutations are defective in phagocytosis of one or more particle types, the genes in question will be mapped and formally characterized to determine their role in Drosophila immunity.

Though very similar in appearance to bacteria, the domain Archaea was not discovered until the late 20th century. Ranging from hydrothermal vents to hypersaline water bodies, or acidic hot springs, these microorganisms thrive in the extremes, thus stretching the possibility of survival to the utmost. Hyperthermophiles such as Pyrococcus and Thermococcus, belong to one such group of anaerobic archaea that flourish in temperature ranges varying between 80°C-100°C. Recent studies show that these organisms are capable of incorporating external DNA fragments from other organisms into their genome by a process known as lateral (horizontal) gene transfer (LGT), and that a significant amount of DNA exchange has taken place between the two genera Pyrococcus and Thermococcus. Research also indicates that the gene exchange process between hyperthermophiles may be occurring as a result of 'jumping genes' or transposons. My study will put this hypothesis to test by firstly isolating and characterizing a large number of hyperthermophilic strains obtained from hot springs on the island Vulcano in Italy. The pattern of transposon distribution in their genome will then be determined, and a correlation, if any, to chromosomal rearrangements in the isolated strains will be recorded. This research in the long run will not only provide a better understanding of the cellular mechanisms involved in microbial evolution, but would also open avenues for further research in the field of archaeal genomics.

Meiotic drive is a genetic phenomenon manifested in insect populations whose males carry the Sex-Ratio X chromosome (SR^pse). These males produce a disproportionate number of female progeny, thus leading to populations dominated by the female sex. The sperm of SR^pse males bearing the Y chromosome are rendered inviable, resulting in solely X-bearing sperm. Paracentric inversions (reversed segments of DNA) commonly characterize the SR chromosome. Considering that inversions often act to conserve genomic sequences and that these sequences have been identified across species subject to meiotic drive, it is probable that these inversions are critical to the expression of the SR^pse phenotype. After visually identifying the exact locations of these inversions, genetic markers developed by Dr. Gerald Wilkinson would then be hybridized to the SR^pse chromosome. Through fluorescence in situ bybridization (FISH), the location of these markers and their complementary gene sequences could be distinguished. In doing so, those sequences which have been preserved by the chromosomal inversions could be identified, and the conclusion could be made that the corresponding genes are critical to the mechanism which renders Y-bearing sperm of the SR^pse male unviable. These concluslons would elucidate the mechanism(s) of meiotic dnve.

This study will clarify the mechanism of ATP hydrolysis by the chaperonin GroEL, a molecular machine that assists in protein folding. I will examine the roles of the carboxylates D398 and D52 as catalytic bases, particularly with respect to which is responsible for ATP hydrolysis in the T- and R-states of GroEL. A stock of D398A and D52A mutants created through mutagenesis will be purified using nickel-chelate chromatography. ATPase activity will be measured in a series of experiments exploiting the variables of D398A and D52A mutations along with GroEL rings locked into the T- or R-state. I anticipate these results will show that D52 and D398 have a much greater impact on GroEL ATPase activity than has been shown in previous experiments where contamination from chromosomally expressed, wild type GroEL was a likely factor. These results may also reveal that different carboxylates are responsible for the hydrolysis of ATP in both the T- and R-states, which would be an interesting and unprecedented observation. The examination of the roles of D398 and D52 in ATP hydrolysis, particularly with respect to the T- and R-states, will contribute to a more in-depth understanding of the mechanism of ATP hydrolysis by GroEL.

In 2001, the sales of vitamin and herbal supplements in the United States exceeded $17 billion. The majority of these botanicals are bought and consumed by women who have become the industry's largest consumer. There has been a dramatic rise in the popularity of women's supplements geared towards improving women's health coupled with the recent controversy surrounding synthetic hormone replacement therapy, causing them to seek out natural alternative therapy. The vast majority of botanical supplements are imported into the U.S, often from third world countries where quality control is often lacking. At best, only 50% of these botanicals are treated to prevent insect pests and microbial contaminants. There are increasing concerns to consumers that purchase these herbal products that include the possibility of product adulteration with toxic ingredients such as pesticides, or of an even greater concern, the presence of toxic fungi or fungi that can produce mycotoxins, which are potentially detrimental to human. Aflatoxins are the most frequently occurring mycotoxin and are produced by the fungus Aspergillus flavus, which is often found on stored plant products giving rise to the concern that aflatoxins may be present in botanical supplements. Aflatoxins are important in that they are potent carcinogens to humans and their intake should be limited. I hypothesize that these botanical supplements will contain aflatoxins and this will be determined surveying popular woman's botanicals for the presence of toxigenic fungi and their toxic metabolites, such as aflatoxin. The aim of this proposal is to develop and validate a sensitive analytical method to accurately quantify the presence of aflatoxins in botanical supplements.

The prevalence of asthma is increasing and poses a great public health concern. This highlights the need for improved therapeutic strategies. It is evident from current data that the expression of the disease involves the type 2 T cell (Th2)-associated allergic inflammation. Dendritic cells (DCs) which regulate T-Cell are important antigens present cells. DCs are presented in many tissue and they can capture antigens by endocytosis. After capturing antigen, DCs will migrate to lymphoid organs and secrete interleukin (IL)-12 and 23 to initiate immune responses. IL-12 and IL-23 induce the production of interferon-c (IFN-c) and increase the type I T helper cell (Th1)-mediated responses. It is believed that the balance between Th2 and Th1 response determines the expression of allergic asthma, wherein a reciprocal regulation of Th1 and Th2 responses has been demonstrated and a deficiency in Th1 response in allergic asthrna has been proposed. The goal of this project is to design nanoparticles targeting the dentratic cells for activating dendritic cells and delivering desired biomolecules such as DNA and proteins into dendritic cells thereby modulating the Th2 type immune response. The accomplishment of this project will help us not only to understand more about DC biology but also to provide experimental basis for developing new therapy for asthma.

Supramolecular chemistry focuses on the study of noncovalent interactions. One prominent area in supramolecular chemistry is self-assembly. Our group studies the self-assembly of lipophilic nucleosides. This work is initiated by fact that guanosine self-assembles through H-bonds to form a tetrameric structure, the G-quartet. A deeper understanding of this bio-mimetic assembly could provide us inside information to design better model systems. The resulted highly functional artificial self-assembly can be used as a new approach to solve biological and chemical problems. My research focuses on determining whether a derivative of a self-associating compound will still aggregate in solid state. Once the compound is synthesized and purified, ESI-MS, 1H NMR, X-ray crystallography, and analytical ultracentrifugation will be applied to analyze this new guanosine derivative.

Apoptosis, programmed cell death, is a process that is important in the body in terms of eliminating unwanted cells and keeping the body in homeostasis. However, undesirable apoptosis can lead to loss of important tissue in heart disease, stroke, and neurodegenerative disease and suppression of apoptosis contributes to the invasive growth of cancer cells. It is hypothesized that closure of the mitochondrial channel, VDAC, located in the outer mitochondrial membrane in all eukaryotes, is required for induction of apoptosis in many cell types. To test this hypothesis, mutated versions of the VDAC protein will be designed in our laboratory, which will stay either open or closed. When such a protein is achieved, this protein will be expressed in mice in place of the wild type to look for effects on apoptosis. The portion of the research that I will be involved in will be to determine the electrophysiological properties of the mutated VDAC channels to determine if they remain in an open or closed state. Depending on my findings, new mutations will be engineered into the protein to achieve the desired behavior. The long-term purpose is to develop treatments for patients since apoptosis is fundamental to the leading causes of death in western countries.

Many researchers have attempted create artificial ion channels. Artificial ion channels will become important in the future as they may play a role in drug delivery and cellular recognition. The focus of this research will be the testing of whether a self-associating compound called calix[4]arene-guanosine (CG) will be able to function as an ionophore and bind salt ions. Once this compound is synthesized using a previously optimized process, a reaction work-up will be performed that allows it to self-associate, and perhaps bind salt ions. Upon completion of this reaction work-up, the complex formed by CG and salt ions will be analyzed using ¹H NMR, mass spectrometry, elemental analysis, and if possible, x-ray crystallography. After the complex is characterized with these types of spectroscopy, the exact structure of a CG-salt complex will be elucidated, as well as the ion-binding preferences of CG.

Activated macrophages are principal mediators of inflammation in the body. When stimulated by bacterial products, such as lipopolysaccharide (LPS), these cells become activated to produce potent proinflammatory cytokines. Previously, the Mosser lab has elucidated an alternate macrophage response to stimulation with LPS. The classically activated response to stimulation with LPS is inflammatory, characterized by the release of the cytokine interleukin 12 (IL-12). However, when the macrophage Fc-c receptors are ligated prior to LPS stimulation, the macrophage shifts its cytokine profile. IL-12 production is turned off and the anti-inflammatory cytokine, IL-10, is produced in high quantities (Capiello, et al., 2001; Gerber and Mosser, 2001). These macrophages have been termed Type II activated macrophages. Three intracellular pathogens, including Leishmania sp., the parasite studied in the Mosser lab down regulate IL-12 production to delay the immune response (Sutterwala and Mosser, l999). Furthermore, an excessive inflammatory response can lead to acute endotoxemia. The Mosser lab has previously shown that the Type II activated macrophage response can rescue mice from acute endotoxemia (Gerber and Mosser, 2001). Understanding this pathway may lead to clinical treatments for inflammatory diseases and may clarify Leishmania pathogenesis. This research will examine the genes specifically expressed by Type II activated macrophages.

REVERSION TO ETHYLENE SENSITIVITY (RTE) is a gene first identified in Arabidopsis thaliana as a component in ethylene hormone signaling. RTE has homologs in all animals, including one in Caenorhabditis elegans with 60.7% amino acid similarity, yet the function of the animal RTE genes are unknown. Preliminary data suggests Aralbidopsis RTE serves as a copper chaperone, which is substantial to the field of metallochaperones. In addition, mutant copper chaperone genes are connected to several human diseases like Menkes disease. Studies to understand RTE function are currently being performed in Dr. Chang's lab in the plant model system Arabidopsis thaliana. In order to understand the function of RTE in animals, I will be researching the function of RTE in the animal model system, C. elegans. My research will be focused on answering two questions: 1. Where does the RTE protein localize? and 2. What is the mutant phenotype of RTE? I will determine RTE's spatial and temporal expression pattern using a green fluorescent tag to be transformed into C. elegans. A mutant phenotype of RTE will be accomplished by using the powerful tool of RNA interference (RNAi). In C. elegans, introduction of a double-stranded RTE RNA (dsRNA) by injection or feeding of transformed E. coli silences the post-transcriptional RTE producing the desired mutant. Mutant phenotypes can be determined by comparing transformed to non-transformed C. elegans. Further research will be guided by the copper chaperone hypothesis including adjustment of copper levels in C. elegans growth media and its effects upon development, behavior, and viability. The findings of this project will give valuable insight into the recently discovered RTE gene family.

The urea cycle is an essential metabolic pathway that converts ammonia into urea. Hyperammonemia is a disease in which, due to a failure of the urea cycle, ammonia builds up in the body and causes serious neurological problems. N-acetylglutamate synthase (NAGS) catalyzes production of N-acetylglutamate (NAG), which is an essential activator of carbamylphosphate synthetase I, the first enzyme of the urea cycle. NAGS has the potential to regulate the urea cycle by supplying variable amounts of NAG for activation of carbamylphosphate synthetase I. The mammalian NAGS gene has been recently cloned. This allows initiation of studies into this gene and its product. Three avenues of investigation will be pursued in this project: 1) The DNA sequence of the NAGS gene will be analyzed using bioinformatics tools. This will provide insights into the regulation of NAGS gene activity. 2) An R509Q mutation, found in a patient with mild hyperammonemia, will be introduced into the NAGS gene. Mutant NAGS protein will then be expressed and purified from bacterial cells. 3) Biochemical characterization of wild type and mutant NAGS proteins will yield information about the function of the NAGS protein and its role in regulation of the urea cycle.

Werner Syndrome (WS) is an autosomal recessive genetic disorder that is caused by a dysfunction in the WRN protein. Mutations in the WRN gene are believed to predispose patients to accelerated aging and the WRN protein plays a critical role in preventing heart disease, cancer, and premature aging. The WRN protein is involved in DNA repair and helps repair damaged DNA during replication and recombination, and this project is designed to study a WRN nucleaselDNA complex. Since WRN nuclease is an enzyme, an enzymatically inactive mutant must first be produced for the structural studies of the complex by Nuclear Magnetic Resonance Spectroscopy (NMR) methods. The structural information about the complex can be used for proposing the function of the WRN protein and its role in WS disease. Once the structure is determined researchers can begin to find targeted gene therapy to prevent premature aging and heart disease.

The presence of one or two cellulose binding domains (CBDs) in bacterial cellulases confers these genes the ability to hydrolyze crystalline cellulose. It has always been assumed that plant cellulases are endo b-1,4 glucanases that are similar to bacterial cellulases family 9 in their catalytic domain but lack the CBD(s) and hence, do not hydrolyze crystalline cellulose. This assumption has been challenged with the inspection of the Arabidopsis genome where three genes with a unique C-terminal extension that resembles CBD has been found. In this proposal, we begin the study of a cellulase with this unique structural extension at the C-terminal referred to as F27J15.28 (Atlg48930). To determine the extent of this gene involvement in Arabidopsis plant development we are pursuing two approaches. First, we will prepare a promoter-reporter gene construct which will be introduced in Arabidopsis plants by Agrobacteria transformation. Thus, analysis of the reporter gene will reveal the location of Atle48930 pene expression. Second, we will attempt to down regulate the expression of the Atlg48930 gene through the use of RNAi. To produce RNAi the 3'UTR portion of the Atlg48930 gene, will be placed in both sense and antisense orientations behind a strong constitutive promoter. Qnce this construct is introduced into plants, a double-stranded RNA will be generated which will ultimately suppress Atlg48930 gene expression. We will also attempt to over-express the full length cDNA of Atlg48930 gene under the control of a strong constitutive promoter. Transgenic plants with RNAi and plants that over-express Atlg48930 message and protein will be scored in terms of phenotypic and genotypic changes compared to wild type. Particular attention will be paid to the analysis of development of plant tissues where the reporter gene staining has revealed Atlg48930 expression. Tissue sections will be stained with a cellulose specific stain and compared qualitative and fluorometrically.

Speciation, the process by which new species form from an existing population, can begin with something as small as a change in a single gene. The process itself, taking anything from hundreds to millions of generations, is not thoroughly understood. In fact, even the definitions of what makes a species distinct sometimes conflict. The pea aphids of New York are one pair of incipient species in this "gray area" which have been studied in great depth. In these aphids, the precise areas of the chromosome containing the genes leading to specialization and reciprocal speciation have been isolated. Because alleles at these loci are disadvantageous in the opposing environment, we expect gene flow to be less at these loci than at those away from genetic regions not influencing specialization. From these expectations we have composed a sort of "genetic mosaic" model in which certain loci on chromosomes show more gene flow than others. Additionally, we can create a "geographic mosaic" model in which there would be variation in gene flow across the entire genome. By comparing data collected from Maryland populations with data from New York populations we can test these models, along with other hypotheses such as if new species are formed primarily through the migration and spread of particular specialized alleles or through independent mutation in different populations. By comparing New York aphids to the less specialized populations in Maryland yet another layer of understanding will be added to our knowledge of speciation and the groundwork will be laid for even further study.

The study of speciation, that is how new species emerged, is one of the most important areas of evolutionary biology. Countless studies have been conducted on this topic and yet there is still much which is not known about the subject. This project will attempt to discover how Laupala cerasina emerged on the big island of Hawaii. Currently, the populations of L. cerasina in both the north and south regions of the big island of Hawaii are classified as one species. However, recent studies using nuclear DNA and amplified fragment length polymorphisms (AFLP) have shown this classification to be inaccurate. This project's goal is to use AFLPs to confirm these recent studies as well as to create a phylogeny of the populations of L. cerasina. This project will also try to discover how L. cerasina came to live on the big island of Hawaii. Either the populations emerged from one common Maui ancestry in a single invasion pathway or the populations of L. cerasina emerged from two distinct Maui ancestries in a double invasion pathway. The discoveries which come about from studying L. cerasina can eventually be applied to all forms of life.

Nitrenium ions are short-lived reactive intermediates which contain a positively charged nitrogen atom. It is becoming increasingly clear that nitrenium ions are responsible for the carcinogenic nature of certain compounds. In living tissues these compounds are oxidized by various enzymes, ultimately creating the extremely reactive nitrenium ion intermediate. The ion then attacks DNA, interfering with DNA replication and creating persistent mutations. Dr. Falvey's group was the first to directly detect an aryl nitrenium ion using a technique called laser flash photolysis. These results and subsequent work in other labs showed that many nitrenium ions are very short-lived. In most cases they survive for only a few microseconds. Being highly electrophilic (electron-seeking) species, they are rapidly attacked by water and other electron donors. A significant portion of the proposed work is to develop an understanding of how nitrenium ions decay. In the end this should help us unravel the chemical origins of certain cancers.

In the water fern llarsifea vestita, the presence of water activates the formation of gametophytes from the dry meiotic products. Sperm development in the hydrated spore takes about eleven hours and results in the formation of 32 spermatids and 7 sterile jacket cells in specific locations within the spore walls at defined time intervals. When microspores are placed in water, they develop synchronously making it ideal for conducting experiments such as RNAi treatment, in situ immunolabeling, inhibitor and radiolabel uptake assays and biochemical isolations. My main hypothesis through the period of research is that Cytoplasmic movements cause the localization of ribosomes, proteins and other translational components into cytoplasmic domains that will differentiate into spermatogenous initials. The main experimental procedures that will be perfonned include disruption of actin interactions with myosin and disruption of actin arrays in gametophytes. In addition I will either disrupt or stabilize microtubule arrays in developing gametophytes and also perform a variety of localization assays using sets of antibodies and nucleic acid probes that have been collecteu and shown to become localized in spermatogenous cells. I anticipate that these experiments will provide further insight on the role of particular cytoskeletal elements and that of cytoplasmic movement in the determination of the fate of cells.

Dampwood termites (Zootermopsis nevadensis, Isoptera: Termopsidae) found colonies and live their entire life cycles in a single stump or log. They do not forage beyond their colony's host wood. Multiple colonies can infest the same tree. As colonies grow and expand their galleries in the wood, it is inevitable that they will meet neighboring colonies. Such interactions result in one colony eliminating the other colony's reproductives, the king and queen. The remaining workers are assimilated into a colony that is initially headed by the surviving king and queen from the "victorious" colony (Heath l927; Shellman-Reeve 1994). All individuals (except soldiers) retain the ability to differentiate into functional reproductives in the newly merged colony; within two months supplementary or replacement reproductives mature (Castle 1934, Light & Weesner l9SI, Noirot 1985). What is not known is whether the new reproductives differentiate from only one or from both of the original colonies. Determining the parentage of new reproductives found in merged colonies will elucidate the fitness dynamics for individuals involved in intraspecific interactions.

We propose to develop a classification system for low complexity proteins that can deliver important chemical and sequence information in a database search. Low complexity sequences are sequences whose patterns break down into simple repeating processes. While tools for identifying low complexity sequences have been developed, no real system of classification has been developed and such sequences are still often filtered out of database queries. Classification of low complexity domain lags behind that of more random proteins, even though studies suggest many important roles for such proteins. This type of classification system would be important to all proteomics work.

Since Galambos and Griffin (1942) determined how microchiropteran bats orient in the dark using what Griffin called "echolocation" (1944), there has been a great deal of research done to further understand echolocation in bats. Studies on prey capture in echolocating bats have been conducted in the laboratory and in the field. In field recordings, signals emitted when the insectivorous species of bats hunt prey have been recorded and analyzed (Neuweiler 1984, Surlykke and Moss 2000, Schnitzler and Kalko 2001). There are also recordings made in labs of bats capturing insects (Schnitzler and Kalko 1998, Valentine and Moss l998, Surlykke and Moss 2000). However in these lab recordings the insects are tethered. Some researches have run experiments where the tethered insects move in a circular path (Moss and Surlykke 2001). There has been little research done in the confines of the lab involving free-flying insects. The proposed goal of this proJect is to analyze how an insectivorous species of bat, most likely the big brown bat, Eptesicus fuscus, responds to and captures free-flying insects in a laboratory setting and to compare the response and capture to data gathered from field recordings and lab recording involving tethered insects.

Apolipoprotein E (APO-E) is a protein found in human blood that is involved in the transport and metabolism of fats and lipids. Genetic variation present in the human population gives rise to three different forms of APO-E. The form of APO-E a person possesses (APO-E genotype) has been shown to affect how much blood cholesterol levels improve following a regular, weekly exercise program. The goal of this study is to test for a similar relationship between APO-E genotype, exercise training, and postprandial lipemia levels. Postprandial lipemia is the increase in plasma lipid levels that occurs after a person eats a meal that is high in fat. How quickly a person's metabolism can reduce postprandial lipemia has been shown to have an affect on his or her risk for cardiovascular disease, and is influenced by APO-E genotype. Subjects in the study will be tested for postprandial lipemia before and after participating in a six-month exercisetraining program. Changes in postprandial lipemia following this exercise program can then be related to APO-E genotype to show which genotypes exhibited the most postprandial lipemia reduction due to exercise training.