Biology

2007 Senior Thesis Abstracts

(CLASS OF 2007: If your thesis abstract is not currently included on this page and you would like it to be, please follow this link.)

Making Sense of Unpredictable Environments: Variable Temperature Effects on Embryonic Growth in the Oriental Fire-Bellied Toad (Bombina orientalis)

Juliana Marie Arrighi

(Advisor: Kaplan)

Temperature has long been recognized as having a profound effect on plasticity in amphibian development. Before they hatch, frog embryos have very little control over their temperature environment, which is often highly variable. Looking beyond average temperatures and understanding the effects of thermal variation on amphibian development is crucial for predicting ontogenetic response to changing variable environments, especially in light of the role of climate change in amphibian declines. In previous laboratory studies, highly variable thermal regimes (24 ± 10?C), like those experienced in the field, decreased developmental and growth rates and resulted in smaller relative tail lengths in Bombina orientalis embryos. Low and moderate thermal variation (24 ± 3?C and 24 ± 6?C, respectively) did not induce a response significantly different from a constant 24?C thermal regime. It was unclear if these responses are the result of the large amount of thermal variation or high or low thermal tolerance thresholds being exceeded. In this study, Bombina orientalis embryos were raised in moderately variable (± 6?C) and constant thermal regimes at high and low average temperatures (27 and 19?C, respectively). These temperature treatments were used in two experiments in which morphological traits at hatching or embryonic developmental rate were measured. Variation in temperature, mean temperature and original ovum size each had effects on different aspects of embryonic development. Embryos in variable thermal regimes had lower developmental stage and smaller tail lengths, total lengths and tail musculature. Tail musculature and body size (snout-vent length) increased with egg size. Based on field research of hatching fitness and swimming performance, these observed effects of variable thermal regimes are expected to decrease hatchling performance.

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The Effect of Mutated Host Ribosomal Protein Genes on Barley yellow dwarf virus Stop Codon Readthrough in Saccharomyces cerevisiae

Amy Erin Campbell

(Advisor: Russell)

Programmed stop codon readthrough is an unconventional mechanism by which ribosomes are directed to bypass the translation termination signal and continue in-frame translation to create an extended protein product. This strategy is utilized by the plant pathogenic Barley yellow dwarf virus (BYDV) to produce a minor coat protein required for infectivity. While the viral sequences that program BYDV's stop codon readthrough have been characterized, the host factors involved are not known. Saccharomyces cerevisiae was used as a model host to characterize the effect of mutant ribosomal protein genes, rps2 (sup44) and rps9B (sup46), on BYDV stop codon readthrough. Mutation of these genes is known to result in a dominant omnipotent suppressor phenotype. Yeast strains with wild-type and mutant versions of each allele were compared. To quantify the gene's effect on readthrough, a b-galactosidase-firefly luciferase double reporter containing the BYDV readthrough sequences was utilized. To measure basal readthrough rates in these yeast, control double reporters were constructed. Basal readthrough levels were determined to be 2.5% and 4.3% for wild-type and mutant strains, respectively. These values are significantly different than the literature values for similar constructs. Mutated rps2 was shown to have a significant stimulatory effect on BYDV stop codon readthrough, increasing rates from 0.4% to 4.9%. The effect of rps9B was not characterized in this study due to inadequate protein activity during assays. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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The Effect of 17-ß Estradiol on FoxA1 Localization in MCF-7 Breast Cancer Cells

Leigh Ann Curran

(Advisor: McClellan)

Estrogen is linked to promoting breast cancer and interference with estrogen action is the primary effect of current breast cancer therapies. Unfortunately, drugs targeting estrogen synthesis and/or estrogen receptor (ER) function have devastating effects on quality of life. Recent genetic profiling studies point to the transcription factor, FoxA1, as a favorable prognostic factor and growth inhibitor in breast cancer cells. In this study the influence of estrogen on the subcellular distribution of the FoxA1 transcription factor in MCF7 breast cancer cells was examined using immunocytochemistry. Nuclear FoxA1 staining increased in MCF7 cells treated with estrogen for 30 minutes. The greater frequency of nuclear FoxA1 localization in estrogen treated samples was correlated with the presence of ER suggesting that ER mediated mechanisms trigger enhanced nuclear association of FoxA1. Both ER and FoxA1 interact to regulate some of the same genes involved in cell proliferation, survival and differentiation. Understanding these interactions is a key element in the development of a more targeted and effective breast cancer treatment.

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GABAergic Signaling Regulates Growth Cone Motility in Newborn Neurons of the Dentate Gyrus

Cooper A. French

(Advisor: McClellan)

Adult neurogenesis is limited to specific structures and circuits in the mammalian brain. The integration of newborn neurons into existing neural circuitry is guided by a complex synergy of growth factors, neurotransmitters, and synaptic activity. Recently a method for tracking newborn neurons expressing a fluorescent marker in the dentate gyrus was serendipitously discovered. Here I use this method to track the motility of dendritic growth cones in acute slices of adult mouse brain using a spinning disc confocal microscope. Stimulation of the perforant pathway increased growth cone motility adjacent to the cell body, and this effect was blocked by the γ-amino-butyric acid (GABA)A receptor antagonist, SR 95531. GABAA receptor antagonism shifted growth cone motility to distal regions of the primary dendrite indicating a role for the neurotransmitter GABA in the integration of newborn neurons in the dentate gyrus.

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The Role of Dopamine Reuptake and Degradation on the Amplitude and Kinetics of Dopamine-Mediated Inhibitory Postsynaptic Currents

Stephanie Christine Gantz

(Advisor: Arch)

Dopamine cells in the substantia nigra (SN) and ventral tegmental area (VTA) are thought to contribute to learning, attention, movement, and pleasure, as well as the rewarding effects of drugs of abuse. These parts of the midbrain are unique, in that dopamine is released dendrodendritically. Dendrodendritic release of dopamine has been the subject of research since its proposal some thirty years ago. However, the mechanistic components of this release have yet to be determined. Recently, the identification of a D2 receptor-mediated inhibitory postsynaptic current (IPSC) evoked from dendrodendritically released dopamine has enabled the physiological combination of release and receptor activation to be investigated (Beckstead et al., 2004). As a crucial component in dendrodendritic dopamine transmission, identifying regulatory mechanisms of the IPSC is necessary to advance research. The IPSC has been found to be regulated tightly by DAT-mediated reuptake, yet no other regulatory mechanisms have been investigated. The aim of the present study was to evaluate the role of dopamine reuptake and investigate the role of intra- and extracellular dopamine degradation on the amplitude and kinetics of IPSCs elicited from dendrodendritic dopamine release. Whole-cell recordings were made from dopaminergic neurons in the VTA in slices from mouse brain. The blockade of reuptake with cocaine increased the amplitude, area under the curve (AUC), and time to peak, of the IPSC. Inhibition of monoamine oxidase (MAO) reduced the typical cocaine-induced increase in IPSC amplitude, while having no significant effect on AUC and time to peak. The inhibition of catechol-O-methyltransferase (COMT) had no significant effect on the typical cocaine-induced increase in amplitude, AUC, or time to peak, of the IPSC. In summation, dendrodendritic dopamine transmission is regulated primarily by DAT-mediated reuptake, with some contribution by intracellular dopamine degradation.

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PINX1: A Potential Telomerase Inhibitor in Xenopus laevis

Jayne Taylor Gaubatz

(Advisor: Shampay)

The ends of linear chromosomes, known as telomeres, are highly regulated DNA and protein complexes. Telomere maintenance is critical to genome stability and normal cell division, as well as to a number of disease processes and aging. In most cells, telomere length is maintained by the ribonucleoprotein telomerase, which is composed of a reverse transcriptase and an RNA template. While in humans telomerase activity is suppressed in most somatic tissues, all tissues in the frog Xenopus laevis contain active telomerase, making it a convenient organism in which to study telomerase regulation. The protein PINX1 has been identified as a potent telomerase inhibitor in humans and in yeast, and has been suggested as a potential tumor supressor. In this study, a potential PINX1 homolog in Xenopus was identified and cloned. xlPINX1 shares significant sequence similarity to human PINX1, particularly in the N-terminal half, and is ubiquitously expressed in Xenopus tissues. A comparison of PINX1 expression and telomerase activity in various Xenopus tissues indicated that a potential negative correlation may exist between the two; however, more research is necessary to determine whether xlPINX1 is also an inhibitor of telomerase, and to further characterize its function. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation and a grant to JS from the National Science Foundation.

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Tankyrase in Xenopus laevis

Angela Maria Gibbs

(Advisor: Shampay)

The ends of linear chromosomes are composed of repeated, noncoding DNA tracts called telomeres. Telomeres protect the cell from DNA degradation and damage by acting as a buffer to the end replication problem, whereby incomplete replication shortens the telomere after every population doubling. Telomerase is a novel reverse transcriptase that extends telomeres. The actions of telomerase are regulated by the complex shelterin, which prevents or allows telomerase access to the telomere. In humans, the shelterin component TRF1 remodels DNA and recruits other proteins to prevent telomerase activity, making it a negative regulator of telomere length. Tankyrase 1 modifies TRF1 by catalyzing the addition of poly(ADP-ribose) chains onto TRF1, resulting in the removal of TRF1 from the telomere. In this way, tankyrase is a positive regulator of telomere length. Tankyrase has been found in human, mouse, and chicken, but it has never been described in Xenopus laevis. Here, a putative tankyrase gene in Xenopus laevis was identified based on sequence similarities to human tankyrase 1. Xenopus tankyrase is 87% identical to full-length human tankyrase. If only the shared domains (ANK, SAM, and PARP; excludes HPS domain which xTNKS does not have) are compared, the two genes are 96% identical. A clone of the putative Xenopus tankyrase was acquired, its structure confirmed, and prepared for characterization. A coimmunoprecipitation assay will be able to determine if Xenopus tankyrase binds to Xenopus TRF1, which has been cloned and sequenced. A positive interaction would suggest that Xenopus tankyrase functions like human tankyrase, despite Xenopus TRF1 having a shortened acidic domain (where human tankyrase binds human TRF1). Further work will be needed to assess this interaction and determine if the putative Xenopus tankyrase gene has any functions or interacting partners in common with human tankyrase. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Manganese, Glutamate, and the Pursuit of Specificity: Origins of Metal Binding Selectivity in the Manganese Transport Regulator MntR

Kristen Grauer-Gray

The manganese transport regulator (MntR) is a metal-binding regulatory protein from Bacillus subtilis. MntR binds two manganese ions in a binuclear cluster, with the binding sites known as the A and C sites. The binding of metal to MntR is selective. In the presence of Mn2+ or Cd2+, MntR is activated to bind its cognate operators and repress manganese uptake. In the presence of a variety of other divalent cations, it is not activated to bind DNA. Previous studies attributed the specificity of MntR to the presence of a selectivity filter based on interactions between the two metal-binding sites. This hypothesis gives each of the binding sites an independent role. Occupancy of the C site is thought to be essential for activation of DNA-binding, while the A site is thought to mediate selectivity by arranging the C-site residues only in the presence of a cognate metal. Subsequent studies with mutants have revealed weaknesses in the selectivity filter hypothesis. While the selectivity filter posits that specificity originates in the A site alone, mutants have been crystallized in which the C site alone binds metal, and yet selectivity is retained. This thesis proposes a revised selectivity filter, in which the A site can arrange the C site for metal-binding, but the C site can also arrange itself in the absence of an A-site ion. In this hypothesis, selectivity depends on both the A and C sites, rather than on the A site alone. The revised selectivity filter hypothesis is tested in this thesis through studies of two mutants: H77A, which lacks one of the seven A-site ligands observed in wild-type MntR, and E99D, which shortens a ligand that bridges the two metal-binding sites. Solution assays on E99D were inconclusive, but hint that E99D has diminished selectivity against Co2+. Crystal structures of apo H77A, H77A•Mn2+, and E99D•Mn2+ provide structural information that complements the results from solution assays. The structure of H77A•Mn2+ supports the revised selectivity filter model, in that the C-site appears to bind metal without first having its residues arranged by the A site. The structure of E99D•Mn2+ reveals the elimination of one bridging interaction, but the interpretation of this structure depends on a more conclusive study of the specificity of E99D. Future studies of specificity in MntR should build on the work done in this thesis by examining the interactions of E99D and H77A with cobalt, and by elucidating the effect of mutations to the bridging residue Glu102.

 

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Failure of a Chimeric Egg Laying Hormone in an Endogenous System

Meridith Amanda Griebeler

(Advisor: Arch)

Most neuropeptides derive from an inactive precursor. Two such neuropeptides, found in the Aplysia californica bag cells, egg-laying hormone and the acidic peptide, derive from the same precursor. When secreted these two neuropeptides should appear in equal quantities. However, ELH and AP are released with different kinetics. To resolve the mechanism behind the differential secretion of ELH and AP two plasmids coding for fluorescent ELH or AP were created to visualize the processing of the precursor and secretion of its products. The ability of these chimeric proteins to be processed and secreted was the question of this study. The Aplysia bag cells were transfected with the plasmids and the tissue was then exposed to a high-potassium medium in order to induce regulated secretion. After the secretion challenge was completed, the tissue and precipitates were run through 12% SDS-PAGE. The proteins were transferred from the gel to NC paper and probed for the chimeric tag with polyclonal antibody. Immunreactive proteins were visualized using an a-IgG coupled to HRP and a chemiluminescence reaction catalyzed by HRP was performed. Conclusions about the regulated secretion of proELH constructs are unsure. It appears that the proELH-EGFP precursor can be secreted into medium if the bag cells are transfected within their connective tissue. The significance of this secretion is unclear because the precursor was never found at sizes smaller than the complete, unprocessed precursor. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Comparative Phylogenetic Analysis of 5.8S rRNA Hairpin Variation

Ginger Chin-Chi Jui

(Advisor: Karoly)

The comparative method in evolutionary biology seeks to discover and explicate general patterns of evolutionary change. Evidence of an association between environmental and phenotypic variables across a range of taxa may illuminate common processes of natural selection and adaptation in shaping biological characters. Recent studies of genetic variation in Pacific Northwest larkspur have focused on the interplay between a neutral process of genetic drift and a selective regime resulting in local environmental adaptation. DNA sequencing of the internal transcribed spacer (ITS) region in Delphinium nutallii and D. leucophaeum has revealed a single nucleotide polymorphism (SNP) in the 5.8S ribosomal RNA gene localized within a highly conserved GC-rich hairpin structure. The two hairpin alleles have different predicted RNA secondary structure and estimated thermostability. Additionally,the two alleles show a nonrandom geographic distribution, with the more stableform of the hairpin associated with higher maximum temperature and precipitation across sites.

In my thesis, several approaches were undertaken to extrapolate a general pattern of 5.8S rRNA hairpin evolution based upon the association between free energy phenotypes and local climate variables observed in Delphinium. A literature search of all molecular phylogenies published in the American Journal of Botany and Molecular Ecology over a 2-year period was conducted to determine the availability of 5.8S rRNA sequences and GPS coordinates for sampled taxa. Candidate phylogenies for comparative analysis were identified using criteria of containing 5.8S rRNA variation, distribution within the continuous United States, detailed location information and complete sampling at the genus level or below. Fifty ITS phylogenies were recovered out of a total of 116 molecular phylogenies, but no phylogeny met selection criteria. A phylogeny of 17 western North American Erythronium previously known to contain hairpin variation and collection locales was selected for comparative analyses of hairpin stability and climate variables using phylogenetically independent contrasts.

The independent contrasts method improves upon non-phylogenetic comparative methods, which, in the simplest model, takes correlations between trait variables across taxa without accounting for the non-independence between taxa due to Phylogenetic relatedness. Independent contrasts, however, take into account both the structure of relatedness between species and an explicit model of trait evolution. The unit of comparison in PIC is the difference in trait value between two operational taxonomic units (OTUs) at tip or ancestral nodes.

Using maximum parsimony, maximum likelihood and punctuational models of trait evolution, no significant correlations were found between hairpin phenotypes and climate for western North American Erythronium. However, a consistent and significant relationship was observed between precipitation and maximum temperature, providing an internal test to the applicability of an independent contrasts to climatic data. As an alternative to the adaptation hypothesis, rates and direction of nucleotide substitution were compared across 11 plant phylogenies to test for a broad pattern of GC-biased gene conversion in hairpin evolution. However, there was not a significant bias in rates of substitution that increased or decreased GC content (t= -0.58, P-value = 0.5771). Finally, further in vitro gel migration assays of Delphinium hairpin sequences were conducted to confirm predicted differences in hairpin secondary structure. However, anomalous results in control experiments precluded the replication of previous hairpin folding experiments.This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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The CfaR Positive Regulator of the Colonization Factor Antigen I (CFA/I) Pili is Necessary for Enterotoxigenic Escherichia coli Infection of the Nematode Caenorhabditis elegans

Rebecca Jean Kreston

(Advisor: Mellies)

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen implicated in the deaths of millions of children living in impoverished countries. ETEC relies on pili to colonize and affix to host-specific receptors on intestinal epithelia, thus mediating bacterial adherence and colonization, the first steps common to all infections. Colonization Factor Antigen I (CFA/I) pili are the most commonly encountered pilus type found on the cell surface of ETEC. Encoded by a four-gene operon, CFA/I attaches to sialoglycopeptide receptors in the epithelia of the small intestine in a number of mammals. CfaR, a positive gene regulator of CFA/I transcription and expression, governs the ability of ETEC to colonize the small intestine via these pili. This research uses the nematode Caenorhabditis elegans, a simple invertebrate that has been widely implemented in several diverse bacterial pathogenesis studies, to examine CFA/I and its regulator CfaR in the process of infection. Using two types of growth media, nematode growth medium (NGM) and colonization factor antigen (CFA), ETEC strains devoid of the CFA/I pili, its regulator or both were used to construct Kaplan-Meier survival plots. Log-rank analysis of these survival curves and paired t-tests of the LT50 s, the time in which 50% of the population reached death, were also analyzed. An ETEC strain containing only the CfaR regulator showed equivalent survival rates and LT50 s to that of the prototypical virulent ETEC strain on NGM agar, demonstrating that the CfaR regulator is the most important virulence factor for the advancement a fatal ETEC infection on NGM. On CFA media, the wild-type H10407 strain was the most virulent ETEC strain. Using brightfield and darkfield microscopy, images showed nematodes with distended guts and gut blebs, suggesting that ETEC uses a mechanism of enteric colonization and infection in the nematode. This research suggests that the pilus regulator is important for an ETEC-mediated enteric infection in the nematode Caenorhabditis elegans.This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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The Ontogeny of Phenotype in a Thermally Fluctuating Environment: Effects of Temperature Variation on Hatchling Morphology and Performance in the Frog Bombina orientalis

Ezra Samuel Lencer

(Advisor: Kaplan)

Many studies have implicated a role for the thermal environment during development in inducing phenotypic variation in poikilothermic organisms. Laboratory studies often only investigate the effects of constant temperature environments, and field studies may only quantify the thermal environment as an average temperature. The thermal environment during development for many organisms includes often dynamic daily temperature fluctuations, and assumptions of average or constant thermal environments may be unrealistic. Increasingly, attention is being turned to the effects of temperature variation on phenotypic development.

This thesis explores the effect of maternal investment and diurnal temperature variation during early development on hatchling morphology and performance traits for the frog Bombina orientalis. Embryos were exposed to different magnitudes of diel temperature variation throughout the course of early development using a programmable incubator. Only the greatest amount of temperature variation had an effect on tadpole phenotype. The consequences of the environment during development for sprint speed were shown to interact with the water temperature which individuals were performing in. Swimming speed was measured at three temperatures, and temperature variation was shown to have a detrimental affect on tadpole performance at 23ºC and 29ºC, but not at 15ºC. Partially, the effects of temperature variation were shown to be mediated through affects on body allometry. Temperature variation decreased tail length which was shown to affect swimming speed. Egg size increased both snout vent length and tail length and was shown to interact with the thermal environment. This work demonstrates that the amount of temperature variance organisms are exposed to during development needs to be considered for a more complete understanding of the role of the thermal environment for generating phenotypic variation.

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Ontogeny of Loxosceles ambo and pisco Phylogenetic Context

Judith Emily Patudith Levine

(Advisor: Black)

The embryonic development of the recluse spiders Loxosceles ambo and L. pisco was studied with high-resolution timelapse and classical histology techniques. The embryos of both species follow the general outline of spider development, showing a primitive plate, cumulus, 'runners,' and germ band. In addition, before internalization of the primitive plate and cumulus, Loxosceles embryos form multiple cell islands, which are isolated cells that internalize at many points on the blastoderm. Loxosceles embryos also show a heretofore undescribed mass migration of cells late in gastrulation, which I term the 'second runners.'

Runners in other species are cells that internalize via a central blastopore and migrate en masse in a centrifugal direction. The cells are thought to be the progenitors of the mesendoderm of the germ band. In Loxosceles, there are two rounds of internalization of runners. The first occurs as the cumulus detaches from the primitive plate and the second occurs when the cumulus has migrated to the equator. Timelapse analysis shows that the second runners come to populate the segment that will form the fourth walking legs. Finally, Loxosceles embryos locate yolk in the opsithosoma via the ancestral mechanism of yolk translocation rather than inversion. The observed difference from the general spider developmental program before (cell islands, second runners) and after (yolk translocation) the germ band stage is consistent with the hourglass model of development.

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Proliferation of Primordial Germ Cells and Sex Differentiation in Threespine Stickleback (Gasterosteus aculeatus)

Zachary Robert Lewis

(Advisor: McClellan)

The differentiation of organisms into males or females is a crucial developmental event and a necessary precondition for sexual reproduction. Interestingly, no universal mechanism for triggering male versus female development has been discovered despite the anatomical conservation of ovaries and testes across animal taxa. One possible factor in sex determination is the ratio and distribution of somatic cells to germ cells within the gonads. Female sex differentiation may occur in gonads with a higher number of germ cells and/or a lack of surrounding somatic cells. This hypothesis is addressed in the threespine stickleback fish (/Gasterosteus aculeatus/) using molecular markers for definitive sex assignment. The early morphological development of the gonads in two stickleback populations was histologically examined by light microscopy. The first indication of sex differentiation was an increase in the number of primordial germ cells attributed to rapid mitotic expansion prior to meiosis. Sexually dimorphic patterns of somatic cell distribution and apoptosis were correlated to this event. This data shows that primordial germ cell proliferation is a sign of female sex differentiation and potentially plays a role in stickleback sex determination.

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Steroid-Induced Transcriptional Regulation of IL-10 in Macrophages: Understanding the Effects of Sex Hormones in Mitigating Th1-Mediated Autoimmune Disease

Alison Emilia Mahan

(Advisor: Ruben)

Many autoimmune diseases are more prevalent in females than in males, and understanding the causes of this dichotomy are important for the development of new treatments for both women and men. One possibly relevant factor in understanding this difference is the observation that secretion of the anti-inflammatory protein interleukin 10 (IL-10) is significantly higher in men than in women. Additionally, studies performed in mice have shown that mitigation of disease symptoms after treatment with certain sex hormones corresponds to an increase in IL-10 production. Previous work with this protein has focused on a subset of T-cells known to mitigate specific autoimmune diseases. This project sought to investigate whether this effect exists in an additional class of immune cells, macrophages, that are known to produce IL-10. The results of this project establish that these cells do not exhibit the same sensitivity observed in the T-cells studied previously. Interesting and unexpected results, however, have created additional questions to be investigated in future research projects. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Isolation of Insulin in Aplysia californica Provides Insight as to Its Role as a Hormone and Neuropeptide

Gretchen Elise Metzenberg

(Advisor: Arch)

Endogenous insulin was isolated from various tissues of Aplysia californica using a novel immunomagnetic isolation technique. This technique allowed for protein isolation from tissue homogenate and hemolymph samples. Insulin's presence was confirmed using SDS-PAGE and western blotting methods. It is thought that insulin acts primarily as a neuropeptide in this organism, making it an ideal model to study its neuromodulatory effects as they relate to cognitive signaling in the vertebrate brain. Insulin was identified in all central ganglia as well as hemolymph and connecting nerve fibers between the pleural and pedal ganglia. The identification of insulin in the circulating hemolymph indicates insulin may act as a hormone in addition to its actions as a neuropeptide; in this way, it is similar to its actions in vertebrate organisms. Isolation of endogenous insulin could be used for future physiological or biochemical studies of insulin's function in Aplysia.This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Clinal variation in ribosomal RNA in the Northwest Larkspur Delphinium nuttallii: Selection or Drift?

Brook Taylor Moyers

(Advisor: Karoly)

The Northwestern Larkspur species Delphinium nuttallii has a single nucleotide polymorphism in the ribosomal RNA, resulting in two forms of the same molecule. This polymorphism is non-randomly geographically distributed among D. nuttallii populations in Oregon and Washington. One phenotype is found in populations in the warmer and wetter Willamette Valley and at the west end of the Columbia Gorge, while the other is found in two colder and drier areas: the eastern Columbia Gorge and in southwest Washington. rRNA folding models predict that the rRNA variant found in populations in warmer locations is more stable at higher temperatures, potentially indicating that selection is shaping the geographic distribution of the polymorphism.

This polymorphism is a single base pair substitution located in a GC-rich hairpin of the highly conserved 5.8S ribosomal RNA. Helpfully, the base pair substitution disrupts a restriction enzyme cut site, which makes the use of PCR-RFLP (polymerase chain reaction restriction fragment length) analysis possible to genotype plants.

In this study I investigated which evolutionary force is primarily responsible for the geographic distribution of the polymorphism in the 5.8 S rRNA of D. nuttallii. I took a locus comparison approach by developing variable, neutral markers that were then used to infer the pattern of genetic variation within and among populations in the absence of natural selection. I chose to focus on one of the two clines for the 5.8 S polymorphism: the East-West cline along the Columbia River Gorge. A subset of twelve populations along the cline was chosen and DNA extracted from collections made from those populations. Fifteen plants from each population were genotyped.

The next step of my research was to develop neutral and informative markers for D. nuttallii. I screened for inter-simple sequence repeat (ISSR) markers that amplified a large number of polymorphic loci and genotyped each individual for those loci. For two ISSR primers, sixty-four polymorphic marker loci were scored. The third step of my research was to confirm the clinal distribution of the 5.8 S polymorphism by genotyping a larger number of individuals per population at the 5.8 S locus. The genotype data generated for the rDNA and ISSR loci were then used to generate estimates of population genetic variation that could be compared.

Discordance of the neutral marker pattern from the strong clinal variation in the 5.8 S rRNA polymorphism allowed me to discard genetic drift as the primary driving force for the cline and consequently to support selection as that driving force.This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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The Nile Perch Fishery in Lake Victoria: Management Proposals for Fishery Sustainability and Biodiversity Conservation

Leah Nevada Page

(Advisor: Kaplan)

The sustainability of the Nile perch population in Lake Victoria and the survival of their endemic haplochromine cichlid prey both rely on effective management of the lake's ecology and fishery. The effect of fishery gear type and fishery extraction pressure on the Lake Victoria Nile perch population was modeled using RAMAS® Metapop software. Fishery gear type management proposals that (a) prevent gill net harvests of Nile perch below 50 cm in total length, or (b) effectively enforce an existing ban on beach seine fisheries, were both found to improve the sustainability of the Nile perch population. Instituting a gill net minimum increased the estimated average abundance after 50 years from 227 million to 628 million and decreased the probability of a 50% terminal population decline within 15 years from 53% to 34%. Preventing illegal beach seine harvests increased the estimated average abundance after 50 years to 856 million and decreased the probability of a 50% terminal population decline to 31%. Reducing fishery extraction pressure also yielded similarly positive results. The estimated average abundance after 50 years increased from 227 million to 862 million in the reduced fishery extraction pressure scenario and the probability of a 50% terminal decline decreased from 53% to 26%. A significant finding of this study is that, even if one of the proposed fishery gear type changes is enforced, the projected increase in fishery extraction pressure, if the fishery remains open-access, will likely lead to fishery collapse. There is a 70% chance of a 50% terminal population decline after 15 years if only the gill net minimum is enforced and a 54% chance of 50% terminal population decline if only the beach seine ban is enforced. If no fishery gear type restrictions are enforced, and if fishery extraction pressure continues to increase, the Nile perch fishery will likely collapse (78% chance of a 50% terminal population decline within 15 years). In response to previous Nile perch simulation studies, the potential for fishery gear type management to result in a population structure that favors resurgence of the endemic haplochromine cichlid stocks was also explored. This study found that the proportion of Nile perch in piscivorous age classes was not responsive to changes in fishery gear type management and therefore management efforts other than fishery gear type restrictions are needed to aid the recovery of haplochromine cichlid populations.

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Die Embryonalentwicklung des Arsch Endes der Spinnen Zygiella x-notata

David Alan Rasmussen

(Advisor: Black)

During early embryogenesis in the spider Zygiella x-notata a novel structure we call the caudal bud appears to play important roles in both gastrulation and morphogenesis of the posterior body. In spiders and all other arthropods, gastrulation organizes the main body axis of the embryo into a germ band, which is the primordium of the entire body. The germ band then divides to form the metameric segmented body-plan characteristic of arthropods. Segmentation in "short-germ mode" arthropods such as spiders originally generates only the anterior segments of the body while the posterior segments form from a posterior growth zone after the germ band elongates. In Z. x-notata, the caudal bud begins to form as a raised ring of cells at the center of the embryo as gastrulation is coming to a close. High resolution time-lapse videography revealed that cells internalize through the caudal bud. Later morphogenetic movements of the caudal bud suggest an important role for this cell internalization event. During germ band elongation, the caudal bud migrates to the posterior pole of the embryo where it appears to form the caudal lobe, which is believed to be the posterior growth zone that generates the opisthosomal (abdominal) segments. Cell labeling experiments confirm that the caudal bud contributes to the formation of the caudal lobe. Moreover, some labeled caudal bud cells localize to leg-bearing segments, suggesting the caudal bud has expanded growth zone properties and contributes to the formation of prosomal (cephalothoracic) segments as well as the opisthosoma. The caudal bud therefore appears to represent evolutionary modifications to gastrulation and the development of the posterior body in spider embryogenesis. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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An Attempt to Characterize the Function of a Telomere Protection Protein in Xenopus Laevis

Jacob Aaron Schwartzman

(Advisor: Shampay)

Vertebrate chromosomes terminate in heterochromatic regions composed of repeated TTAGGG sequences. These regions, called telomeres, solve the "end replication problem'' of the eukaryotic DNA replication mechanism, which cannot fully replicate the chromosomal termini. Shortening over many replications could threaten the coding regions. Telomerase averts this situation by reverse-transcribing its internal RNA template to generate the telomeric repeats. Telomerase expression in the germ and stem cells of most animals maintains telomere length. In human somatic tissues, telomerase is generally repressed, and telomere shortening eventually leads to cellular senescence in cultured cells. Telomerase expression has been found to be up-regulated in almost all cancers. A group of telomere-associated proteins act to regulate telomere length and prevent non-homologous chromosomal fusions between telomeres. The two Telomeric Repeat binding Factors TRF1 and TRF2 were some of the first proteins found to influence telomere structure. TRF1 is involved in length regulation, but TRF2 is primarily responsible for preventing the telomere from detection by DNA-damage response systems, which leads to fusions. The African clawed frog, Xenopus laevis has been used as an experimental organism in developmental studies. Telomerase is constitutively expressed in Xenopus (Bousman, 2003), leading to questions about the nature of its telomere length regulation systems. I attempted to characterize the function of TRF2 in Xenopus in comparison with its human analog. If TRF2 has a conserved function in telomere protection, perturbing its function in the frog embryo should produce a significant and deleterious phenotypic effect. If the embryo survives long enough for cells to replicate, telomere shortening and developmental abnormalities should be detectable. I used two parallel methods in an attempt to knock down TRF2 in vivo: expression of a dominant-negative construct and an RNA interference approach. The fluorescently-tagged TRF2 constructs were visualized in cultured human and Xenopus cells, and displayed a punctate nuclear localization pattern suggesting association with telomeres. Fluorescent TRF2 was expressed in Xenopus embryos but could not be confirmed to have telomeric localization. Silencing RNA was constructed and a quantitative reverse-transcription PCR method was developed to assay TRF2 knockdown. Due to difficulties with Xenopus embryos, I was unable to confirm the knockdown of TRF2 or deleterious phenotypes.This study was funded in part by a grant from the James F. and Marion L. Miller Foundation and a grant to JS from the National Science Foundation.

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Red Fish, Blue Fish. One Fish, Two Fish: Testing Speciation by Sexual Selection in Astatotilapia burtoni

Jessica Thompson

(Advisor: Renn)

Sexual selection has played an important role in the extraordinary species radiations of haplochromine cichlid fish in the Great Lakes of Africa. In these fish species barriers are maintained by sexual selection of mate color. Many cichlid species appear to be differentiated by little more than color. These observations have led to many studies of whether sexual selection might cause accelerated speciation. Sexual selection causes directional fixation of alleles. Two geographically isolated populations could easily diverge in sexually selected characteristics. The differential female preferences responsible for this divergence would also create a gene flow barrier, which would further isolate the populations, accelerating divergence and leading to speciation. This process has been proven theoretically in modeling experiments, but never documented in a living organism.

It is difficult to test for speciation directly, but evidence for population specific mate choice would support a hypothesis of speciation by sexual selection. This study tested whether by population-specific mate choice occurred among three geographically isolated populations of the haplochromine cichlid fish Astatotilapia burtoni. There was no preferential mate choice among the populations, which show that speciation by sexual selection is not ongoing in this species. Genetic data agree with this finding and suggest that the populations were not genetically isolated despite large geographical separation.

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Social Regulation of Neuropeptides in Astatotilapia burtoni

Cadence Aleece True

(Advisor: Renn)

A variety of hormones and neuropeptides have been shown to regulate reproductive behaviors but the mechanism for this regulation is still an important question requiring further research. In the African cichlid fish Astatotilapia burtoni males can be of a territorial or non-territorial phenotype. Territorial (T) males defend a territory and spawn with females while non-territorial (NT) males are reproductively repressed in behavior and physiology. This example of alternative male reproductive tactics has been well studied on the hormonal and molecular level and represents an ideal model for investigating the mechanisms that control reproduction, specifically courtship behaviors as well as physiological capacity. Another reason this teleost species represents a good opportunity to study neural control of behavior is because the new technology of microarrays has been applied to this species to screen for gene expression differences between T and NT males. Among some of these genes are many neurotransmitter proteins, including GABA and kainate receptors as well as the neuropeptides gonadotropin-releasing hormone (GnRH) and arginine vasotocin (AVT). Microarray results only indicate whole brain increases in GnRH and AVT mRNA within T males and the localization of these peptide differences is important for understanding the significance and regulatory effects of these molecules. Here immunocytochemistry was used to localize both GnRH and AVT neurons in A. burtoni. Territorial males showed larger numbers of AVT cells than NT males in the preoptic area. Importantly, it was discovered that a subpopulation of AVT neurons also contained GnRH. This is the first example of colocalization of these two neuropeptides and indicates a possible interaction for these molecules in regulating reproductive behavior and physiology. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Constitutive Expression of the Tumor Suppressor Protein p53 in Xenopus laevis

Daniel Morris Walker

(Advisor: Ruben)

Adult Xenopus laevis have a very low incidence of spontaneous cancer, as well as a high resistance to induced cancer formation. This species provides a valuable model to look at protective components of the immune system to prevent neoplastic transformation. The tumor suppressor protein p53 has been identified as one of these mechanisms in mammals and over 50% of human cancers display mutated forms of this protein. This protein is a cell cycle regulator that is activated in response to cell stress and DNA damage and can act as a transcription factor to trigger apoptosis or cell cycle arrest. p53 has been identified and characterized in X. laevis oocytes and embryos (Xp53), and previous thesis work has identified p53 in adult X. laevis using Western blots. This thesis seeks to confirm the earlier results using intracellular detection of Xp53 with flow cytometry and cytostaining. The results of both the flow cytometry and cytostain experiments agree with the earlier findings and suggest a high constitutive level of Xp53 in unstimulated X. laevis splenocytes. These cells appear to express p53 in both the cytoplasm and the nucleus, whereas mouse lymphnode cells only show cytoplasmic expression. UV-B exposure significantly decreases detection of Xp53 (p<.0001, DF=14), and may be due to phosphorylation of specific amino acid residues within the anti-p53 antibody binding site, thus inhibiting the binding affinity of the antibody. The functional role of Xp53 is investigated by inhibition of p53-dependent apoptosis using pifithrin-a. The high constitutive level and unique pattern of nuclear staining in unstressed X. laevis splenocytes suggests that Xp53 may play a role in the observed cancer resistance of X. laevis. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Repertoire Usage of the Southern Resident Community of Killer Whales (Orcinus orca)

Monika Wieland

(Advisor: Renn)

The Southern Resident Community of killer whales (Orcinus orca) frequent the waters of Washington State and British Columbia. The three pods that make up the community (J-, K-, L-Pods) have been well studied since the 1970s. These long-lived marine mammals live in stable social groups and they are known for their complex communication system that involves a shared set of 26 discrete calls. Each pod has its own repertoire, or group-specific subset of the calls that are socially learned. While intensive acoustic studies occurred in the 1980s, much about discrete call function and repertoire structure remains unknown. Using recordings made on the west side of San Juan Island in the summers of 2005-2006, this study considers current repertoire usage of all three pods. Call type usage, frequency of call usage, two-call sequences, and mean call duration are analyzed for each pod and compared to 1978-1983 data to assess changes that have occurred in repertoire usage. Information theory and Zipf's statistic are also used to quantitatively compare the killer whale communication system to other species. Call types have been added and dropped from every pod's repertoire, demonstrating an unprecedented rate of change for killer whale communication systems. Other calls have changed in frequency of occurrence and many calls have increased in mean duration, perhaps as a result of increased vessel noise. Many two-call associations are also found, indicating sequential structure in the repertoire. Analysis using Zipf's statistic also demonstrates a non-random repertoire structure. These observed changes all have implications for call function and overall repertoire complexity. These results demonstrate cultural evolution, complex repertoire structure, and potential impacts of anthropogenic noise. The increased understanding of killer whale discrete call system presented here will set the stage for future studies of this endangered population of killer whales.

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Creation of a proU-LEE5 Hybrid Gene to Elucidate the Regulatory Mechanics of Ler in Enteropathogenic E. coli

Trevor Matthew Young

(Advisor: Mellies)

The ability of enteropathogenic Escherichia coli (EPEC) to damage the intestinal cells it colonizes requires the virulence genes encoded on the locus of enterocyte effacement (LEE), which contains the five operons LEE1 through LEE5. These operons are negatively regulated by the nucleoid-associated protein H-NS (histone-like nucleoid structuring protein) with the LEE2 through LEE5 operons also positively regulated by Ler, a regulator encoded on the LEE1 operon. Multiple H-NS proteins negatively regulate the LEE operons by binding in dimer form to the regions upstream and downstream of the promoter. To allow expression at the LEE2 through LEE5 operons, multiple Ler proteins bind along the upstream H-NS binding site, and in the process of binding they dislodge the H-NS proteins already bound there. However, all evidence suggests that Ler antagonizes H-NS repression only at operons involved in virulence and not at the many other genes H-NS regulates. This raises the question of how Ler only antagonizes H-NS repression at certain operons. Evidence has shown that at some non-LEE operons, like the proU operon, the mechanism of H-NS repression is for multiple H-NS dimers to bind upstream and downstream of the promoter with H-NS dimers at one binding site tetramerizing with dimers at the other, forming a loop structure that RNA polymerase is unable to proceed past. Research has indicated that this phenomenon may also occur at LEE5. One theory of why Ler is able to antagonize the H-NS loop structure at LEE5, and not at other operons, is that this loop structure is less stable due to the downstream and upstream H-NS binding sites being closer together. To test this theory I tried to create a hybrid gene containing the upstream H-NS binding site of proU and the downstream HNS binding site of LEE5 to see if Ler could antagonize H-NS repression of this hybrid and allow for transcription. However, due to cloning problems I was unable to generate the proU::LEE5 hybrid gene necessary to conduct this research. I also present evidence that the leucine-responsive regulatory protein (LRP) may be involved in LEE5 regulation, especially since there is a putative LRP binding site near where an unknown LEE5 regulator is thought to bind. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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A Comparative Study of Sex Roles in African Cichlid Fishes of the Genus Julidochromis: Implications for Sexual Selection and Sexual Conflict.

Victoria Halley Zero

(Advisor: Renn)

Sex roles are those behaviors that may be performed predominantly by one sex or the other, and are generally thought of as fixed for a species. However, these roles may be determined by a variety of social and environmental factors. In most animals, males compete for access to females, while females are faced with a choice of potential mates. This is generally explained by the ability of males to produce a larger number of selection pressure on males in the form of sexual selection. The mechanisms that determine sex roles and the strength of sexual selection are still much debated. The five species of the African cichlid genus Julidochromis are an ideal model for studies of these issues because some are reported to be sex role conventional and others reversed. Furthermore, these roles may be plastic in response to body size, mating system, and environmental conditions. In order to validate claims of sex role reversal, it is necessary to conduct a rigorous behavioral comparison. This study is the first genus-wide analysis of behavior for Julidochromis to be conducted using standardized measures. I examined the reproductive stage on the amount of aggression and parental care in all five species of Julidochromis. This study provides evidence to support the importance of sex and size for aggression and parental roles, particularly for J. marlieri. In female-largest pairs, females were more dominant than males. This study also shows that the intensity of sex roles responds to changes associated with the development of Julidochromis ornatus were observed to anticipate egg laying by increasing territoriality, and spent more time in the nest once eggs were laid. Hence these species exhibit temporal plasticity in sex roles and flexibility in their responses to the social environment.

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