Biology Department

1998 Senior Thesis Abstracts

(CLASS OF 1998: If your thesis abstract is not currently included on this page and you would like it to be, please follow this link.)

Central Nervous System Acetylcholinesterase Inhibition from Coexposure to Pyridostigmine and Blood-Brain Barrier-Weakening Stress

Andrew Bacelis

(Advisor: Arch)

ABSTRACT

Pyridostigmine bromide (PB), an acetylcholinesterase inhibitor, was administered prophylactically to over six hundred thousand persons deployed in the Persian Gulf War of 1990-1991 to increase survival rates after exposure to the lethal nerve agent soman. Pyridostigmine was believed incapable of crossing the blood-brain barrier; however, side effects indicative of central nervous system involvement were reported. In the present study, the procedure used in Friedmanet al. (1996) was modified to evaluate penetration of pyridostigmine into the brain under more realistic drug administration and stress conditions, and to explore the delayed effects of AChE inhibition. Adult male Swiss Webster mice were divided into four treatment groups (pyridostigmine versus no drug, crossed with stress versus no stress). These were further divided into two temporal delay conditions: the immediate group was euthanized immediately after their last drug and stress exposure, and the delayed group was euthanized five days after last drug and stress exposure. A dose approximating the human PB dose consumed during the Persian Gulf War was dissolved in the drinking water of animals in the drug conditions. Animals in the stress conditions were subjected to forced swim sessions. Brain acetylcholinesterase activity was assayed using a modified Ellman method. The present findings indicate that pyridostigmine can penetrate the blood-brain barrier under stress, as stress allowed enough pyridostigmine past the BBB to significantly inhibit brain AChE in the Drug+Stress group. A nonsignificant trend was observed suggesting pyridostigmine may exert delayed enzymatic effects on the central nervous system after AChE inhibition. The findings underscore the importance of investigating the neurotoxic potential of pyridostigmine under these conditions in cognitive studies.

The mechanism of axis specification in the marine wormUrechis caupo and phylogenetic implications

Meredith Calvert

(Advisor: Black)

ABSTRACT

In many spiralian organisms (such as annelids, molluscs and echiurans) the establishment of normal body symmetry during development is dependent upon the specification of cells within the D-quadrant. In some molluscs, this occurs by an inductive interaction between the macromeres of the D-quadrant and the micromeres of the animal pole. The mechanism of axis specification utilized in the marine echiuran wormUrechis caupo was investigated by treating developing embryos with an extracellular matrix perturbant, monensin. This suppresses macromere-micromere contact in molluscan embryos, and should prevent normal axis formation inU. caupo if macromere-micromere induction is important in this species. Treated embryos were observed using scanning electron microscopy and light microscopy. The results showed that a macromere-micromere induction is occurring inU. caupo, and that this mechanism is necessary for normal axis formation. The different inductive mechanisms utilized by spiralian organisms are examined within an evolutionary context.

Agrobacterium rhizogenes mediated transformation in soybean Glycine max

Sarah Davidson

(Advisor: Dalton)

ABSTRACT

Root nodules of nitrogen-fixing legumes, such as soybean (Glycine max ), are exposed to various active oxygen species produced by the strong reducing conditions needed to convert molecular nitrogen to usable ammonia. Nodules have several enzymes that scavenge active oxygen species, including ascorbate peroxidase (AP) which scavenges H2O2 in the nodule cytosol. In order to look at the role that ascorbate peroxidase plays in the nodule, it is desirable to study the effects of down regulation. One method of accomplishing such a goal is to introduce a sequence for ascorbate peroxidase in the antisense orientation, thus blocking translation of AP transcripts. The novel bacterium,Agrobacterium rhizogenes, is an excellent mediator of such transformation, as it transfers a segment of its T-DNA to the plant and induces the plant to produce transformed roots. In this study,Agrobacterium rhizogenes K599 carrying a binary vector with a cDNA for ascorbate peroxidase fused to a GUS reporter gene was introduced into young soybean hypocotyls and cotyledons. Fluorometric, histochemical, and opine detection assays were used to assay for GUS expression and cucumopine synthesis respectively, which are indicative of successful transformation of the plant byA. rhizogenes. As indicated by the GUS and opine assays no transformed soybean tissue was obtained. As soybean has historically been resistant to most methods of transformation available today, it is hoped that by applying the AP antisense construct to the commonly transformed legume,Lotus corniculatus, transformed roots will be obtained which will allow us to elucidate the role of ascorbate peroxidase in legume root nodules.

An Investigation of the Signal Transduction Pathways Responsible for Differential Exocytosis in the Bag Cells ofAplysia

Adam Douglass

(Advisor: Arch)

ABSTRACT

The molecular mechanisms governing the differential exocytosis of acidic peptide (AP) and egg-laying hormone (ELH) from the bag cells ofAplysia californica were examined by monitoring secretionin vitro. Following radiolabeling of all newly-synthesized proteins, secretion from isolated bag cell organs was stimulated by chemical depolarization. Peptide synthesis was found to decrease during winter months, in agreement with earlier studies. Studies of particular focus were conducted in the presence of GTPgammaS or aluminum tetrafluoride ion, both of which are known to activate heterotrimeric G-proteins. Peptides released into the secretion medium were desalted by gel filtration and then separated by isoelectric focusing. The amounts of each were quantified by liquid scintillation spectroscopy. Inability to obtain an adequate supply of animals prevented the completion of the intended experiments and replicable data were not obtained for either of the treatments. Potential complications resulting from the saponin treatment that was used to facilitate the transport of GTPgammaS into the cell interior are discussed in light of the apparent reduction in secretory response that was observed in the presence of the chaotropic reagent. Mechanisms by which signal transduction pathways might mediate differential peptide release are discussed and an attempt is made to reconcile them with the current model for regulated exocytosis.

Growth factor response in feline oviductal stromal cells

Lucy Goodwin

(Advisor: McClellan)

ABSTRACT

The cellular effects of estrogens and progestins on the uterine and oviductal lining are thought to be dependent on interactions between lumenal surface epithelial cells and the underlying stromal cells. A general model contends that female sex steroid hormones act through the stroma via autocrine and paracrine production of growth factors like EGF and IGF-1 that then affect the epithelium. Identifying cellular sites of peptide growth factor action in female reproductive tract tissues has been hampered by the use of mixed or semi-purified cultures of stromal and epithelial cells, and specific roles for epithelial and stromal cells in indirect models of estrogen and progestin action have not been confirmed. Here, ERK activation and changes in tyrosine phosphorylation patterns in cloned feline oviductal stromal cells were examined as early markers of EGF and IGF-1 action. Effects on signaling pathways were correlated with morphological changes and cell growth measured by tritiated thymidine and thiazolyl blue (MTT), a cell-number assay. The two growth factors elicited distinct patterns of ERK activation and tyrosine phosphorylation yet both peptides acted as short-term mitogens. IGF-1 promoted cell survival and shape changes associated with decreased adherence. The data herein support the notion that EGF and IGF-1, peptides whose concentrations are increased by E2 act directly on stromal cells in the oviduct to promote limited DNA synthesis and differentiation. This study was funded in part by a HHMI Undergraduate Research Program grant.

The Effects of Maternal Eating Disorders on Fetal Development: The Little Fetus that Couldn't

Deanna Grant

(Advisor: Black)

ABSTRACT

Women with poor nutritional status before and during pregnancy may give birth to babies who fail to grow to their genetic potential. The consequences of intrauterine growth retardation can be severe, including increased perinatal mortality, delayed physical and neurological development, and a higher incidence of adult diseases such as non-insulin dependent diabetes and ischemic heart disease. Some researchers contend that increased maernal weight gain has no beneficial effects. My analysis, however, supports a positive relationship between maternal weight gain and birthweightin both normal and underweight women. Thus, an obstetrician can increase the chance that a malnourished woman gives birth to a healthy infant by encouraging her to gain adequate weight during pregnancy. This is particularly important in the case of eating-disordered women. When a woman's percentage of body fat falls below a certain threshold, the body suspends reproductive function. This prevents unsuccessful pregnancies. Nevertheless, by using fertility drugs, eating-disordered women can reverse this defense mechanism. This practice ignores the data that show that underweight and amenorrheic women have complicated pregnancies and give birth to an abnormally high proportion of growth-retarded babies.

The Effects of Maternal Eating Disorders on Fetal Development: The Little Fetus that Couldn't

Mark William Jarvis

(Advisor: Ruben)

ABSTRACT

Apoptosis is a kind of genetically programmed cell death. It is used by organisms to remove useless or, more importantly, deleterious cells. A cell which has lost its ability to control proliferation is particularly detrimental, and if not eliminated, could grow into a tumor. Apoptosis is one possible mechanism by which the organism can eliminate a potential tumor. The occurrence of tumors in Amphibia is rare and is reflected in an increased resistance to chemically-induced tumor formation. A potential basis for this anomaly could be that amphibians regulate apoptosis differently from mammals or other vertebrates. Cells which lose their ability to die would presumably be more susceptible to transformation into cancer. Since several members of the TNF family of cytokines and their associated receptors have been implicated as one inductive mechanism of apoptosis, determining if apoptosis ofXenopus laevis lymphocytes is similarly regulated by TNF-a, is of interest. Functional experiments revealed that apoptosis ofXenopus laevis splenocytes could be enhanced by treatment with TNF-a, provided the cells were first incubated in IFN-g. While these results were consistent within experimental groups, they were not statistically significant when the entire population is taken into account. Further lines of experimental evidence suggest that apoptosis in these cells may be regulated by TNF-a. Here, I provide evidence for the enhanced expression of the TNF receptor p55 in splenocytes treated with IFN-g. Concurrent with the mode of action proposed in the mammalian literature, IFN-g treatment increased the binding of the anti-TNFR55 antibody in a splenocyte extract. Western blot analysis of the splenocyte cell extract to identify the TNF receptor p55 was inconclusive due to global binding to many of the proteins in the extract. Despite inconsistencies, pre-treatment with IFN-g, followed by TNF-a treatment never produced a statistically significant decrease in apoptosis, only an increase. The TNF-a mechanism of killing appears to be similar inXenopus to that in mammals and other vertebrates. Thus, the increased resistence ofXenopus to tumor formation is not likely to be due to the absence or a reversal in function of TNF-a regulated apoptosis.

A Metapopulation Model of a Multi-Species Marine Fisheries Reserve

Pema Kitaeff

(Advisor: Childress)

ABSTRACT

Marine Fisheries Reserves have been advocated in the recent literature as an efficient method of sustaining an exploited population of marine organisms. Dispersal from a reserve which protects a portion of the population may enhance the abundance of individuals outside the reserve. To assess this effect for four commercial Northwest species, a mathematical model representing a real-life MFR was constructed based on Levin's metapopulation model (1969). Life-history data for the Japanese Abalone, Red Sea Urchin, Dungeness Crab, and Kelp Bass were used as input for the model to determine what proportion and type of patches would be necessary to support viable populations for all four fished species. Results showed that a fairly large reserve (70%) would be necessary to support sustainably viable populations of all four species simultaneously. Results also showed that animals with similar habitat requirements may be protected by the same reserve.

A Yeast Virus Replication: Initiation of the Life Cycle using a Yeast Cytoplasmic Transcription System that is not Dependent on Homologous Recombination

Andre Limnander de Nieuwenhove

(Advisor: Russell)

ABSTRACT

In order to study the cis acting sequences that are necessary for replication of the M1 double stranded RNA virus of the yeastSaccharomyces cerevisiae, it is necessary to establish an efficient system for cytoplasmic transcription of viral RNAs from an M1 cDNA clone. Establishing this system would provide a launching platform for M1 infections. The system used thus far is based on the pGKL plasmids of the yeastKluyveromyces lactis, which have been introduced into S. cerevisiae. This system, however, is dependent on in vivo homologous recombination, which decreases its efficiency with respect to systems that can be manipulated in vitro. The purpose of this study was to create a small linear plasmid vector that can be manipulated in vitro , and that can replicate upon transformation into cells containing pGKL2. The first step to create this linear plasmid is to clone the terminal inverted repeats (TIR) of pGKL2 into the ends of the MCS of a plasmid cloning vector. The design allows cutting of this plasmid with a blunt cutting restriction enzyme, thereby releasing a linear plasmid (pLIN) with termini exactly like those of pGKL2 and the MCS in the middle. Because these termini are responsible for binding pGKL2-encoded replication proteins, we expect pLIN to replicate efficiently. In the present thesis I report successful cloning of the first TIR into the plasmid cloning vector. The cloning of the second TIR has been unsuccessful thus far. Perspectives and future experimental approaches are discussed, as well as the implications that the development of pLIN would have on the long-term objectives of the laboratory.

Shortcomings of Current Crop Pollination Strategies: Lessons From Natural Pollination Systems

Margaret M. Mayfield

(Advisor: Karoly)

ABSTRACT

Over half of all crops used by humans require animal pollination for reproductive success. Most crops in the US and Europe are pollinated exclusively by the European honey bee (Apis mellifera). In recent years declining numbers of European honey bees, caused by diseases and mite infections, have increased public and scientific interest in crop pollination and this honey bee pollination strategy. Although honey bee pollination is considered to be the best strategy for ensuring maximized crop yields, little is truly known about how efficient this pollination strategy is, or whether alternative strategies, such as the management of wild bees, could provide equivalent or better pollination to crops. In this study, I examine reproductive success, pollen limitation, pollinator visitation variability, and pollinator covariation in natural and crop systems. I also look at the frequency of generalist and specialist pollination strategies in nature. From these data I suggest that the honey bee pollination strategy is not providing crops with above normal pollinator services or improving yields above natural levels. The data from this study suggest that alternative pollination strategies, such as the management of wild bee populations, should provide more effective alternatives to the honey bee pollination strategy. My results also suggest that honey bees are needed to compensate for the severely reduced number of wild pollinators in agricultural areas, at least until effective land and pollinator management programs are enacted in this country and around the world.

Behavioral Responses of Callianassa californiensis and Upogebia pugettensis (Decapoda: Crustacea) to anoxic stress

Rigel D. Pearce

(Advisor: Childress)

ABSTRACT

Studies have shown that  Upogebia pugettensis  and  Callianassa californiensis  have physiological differences which lead to differing tolerances to low oxygen conditions. This study illustrates the differences in the behavioral strategies to such conditions between these two species.  Upogebia pugettensis  and  Callianassa californiensis  were put in specialized narrow tanks which were then sealed for 36 hours. The behavioral responses to anoxic stress were observed and compared between species. As predicted based on physiological differences,  Upogebia  exhibited stress-related behaviors sooner than  Callianassa. By 36 hours,  Upogebia  performed very few behaviors due to oxygen deprivation, whereas  Callianassa  was still fairly active.

Synthesis of 5-iodonorvaline from N-carboxybenzyl-glutamic acid-1-benzyl ester: A Novel Compound for Phasing in the X-ray Crystallographic Analysis of Proteins

Michael Quon

(Advisor: Glasfeld)

ABSTRACT

5-iodonorvaline was synthesized in this experiment as a possible vehicle for the incorporation of the heavy atom iodine into proteins. It is believed that this 'un-natural' amino acid will aid in the production of heavy atom derivatized protein isomorphs for phase determination in x-ray crystallography.

Seasonal and Spatial Variation in Oxidative Stress in an Old Growth Pseudotsuga menziesii Canopy

Ellen Francis Roberts

(Advisor: Dalton)

ABSTRACT

Oxidative damage to various cell components (proteins, lipids and DNA) is one of the most universal effects of environmental stress in plants. Environmental stresses, such as drought stress or extremes of temperature and light, result in the increased endogenous production of activated oxygen species, such as superoxide (O₂-^.)), hydrogen peroxide (H₂O₂), hydroxy radicals (OH^.) and singlet oxygen (¹)O₂). Plants are equipped with an elaborate array of antioxidants and antioxidant enzymes which protect the cell by scavenging these activated oxygen species. Thus oxidative damage is a measure of the extent to which the protection system is unable to keep up with the rate at which activated oxygen is being produced. This study investigated the effect of canopy position and season on oxidative stress as measured by bioindicators for lipid peroxidation -thiobarbituric acid-reactive species (TBARS)- and protein oxidation -protein carbonyl content- in the foliage of old growth Douglas-fir ( Pseudotsuga menziesii ). Oxidative stress as estimated by TBARS was greatest at the lowest position in the canopy and decreased at all positions during the winter. Protein carbonyl content followed the same seasonal pattern, but showed no significant effect of position.

Microsatellite-Based Estimates of Individual Inbreeding and Relatedness for a Population of the Wildflower, Mimulus guttatus

Andrea Sweigart

(Advisor: Russell)

ABSTRACT

To better understand the evolutionary transition from self-fertilization to outcrossing, individual inbreeding coefficients were estimated and the relationship between geographic distance and genetic relatedness was examined for a population of Mimulus guttatus that has an intermediate selfing rate. Extremely polymorphic microsatellite loci were amplified using PCR in M. guttatus individuals sampled in pairs along two transects. Expected heterozygosities for five loci ranged from 0.79 to 0.93. Using observed microsatellite allele frequencies, the population inbreeding coefficient ( F ) was calculated to be 0.19 (SE = 0.023). A method-of-moments estimator developed by Ritland (1996) was used to estimate the distribution of inbreeding among and relatedness between individuals of a natural population. The mean individual inbreeding coefficient ( F = 0.16) did not differ significantly from the population-level estimate. Individuals sampled from one transect showed significantly more inbreeding than individuals sampled along the other (p = 0.005). There was no apparent relationship between interplant distance (range: 0 to 14 meters) and mean genetic relatedness between individuals. However, there was a trend indicating that variance in relatedness declines with increasing interplant distances. These results represent the first application of polymorphic microsatellites to estimate fine-scale genetic population structure and will provide the opportunity to explore novel realms of population evolutionary dynamics.

Tailoring New DNA-Binding Specificities in the Escherichia coli Purine Repressor Through A Mutation at Histidine 20

Danielle K. Vincent

(Advisor: Glasfeld)

ABSTRACT

The Escherichia coli purine repressor functions as the master regulatory protein in de novo purine nucleotide biosynthesis through binding of the pur F operator upon binding the corepressor, hypoxanthine. The thermodynamic importance of histidine 20 in the binding of PurR to the pur F operator was assessed through the mutation of residue 20 to alanine, arginine, and glutamine. The dissociation constants of the alanine and arginine mutants, determined with fluorescence anisotropy, are 1000 nM and 20 nM, respectively. The glutamine mutant demonstrates only nonspecific binding to the pur F operator. The dissociation constants of these three mutants suggest histidine 20 may contact the DNA via a charged, water-mediated hydrogen bond. Site-directed mutagenesis of a double mutant, in which histidine 20 and arginine 26 were mutated to an arginine and a histidine, respectively, was successful. PurR belongs to the GalR/LacI protein family and is structurally similar to LacI. Binding studies performed with the double mutant and the pur F and lac O operator sequences should determine whether PurR can be engineered to mimic LacI specificity.