Biology

2008 Senior Thesis Abstracts

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Associative Nitrogen Fixation Ability Does Not Predict Symbiotic Nodulation Ability in Burkholderia tropica

Christopher Kyle Black

(Advisor: Dalton)

Symbiotic nodulation of legumes by diazotrophic bacteria was long believed to be the exclusive realm of a few alphaproteobacteria closely related to Rhizobium. Recent work on tropical legumes has revealed nodulation ability in many strains of Burkholderia, a ubiquitously distributed genus of betaproteobacteria that was previously noted mostly for its opportunistic pathogens of plants and humans. Previous work at Reed College has showed that Burkholderia tropica is capable of endophytic nitrogen fixation in dune grasses (Ammophila arenaria and Elymus mollis) from Oregon. To determine whether Oregon Burkholderia might also have the ability to induce nodulation, a greenhouse inoculation trial was performed. Red Alder (Alnus rubra), Scotch Broom (Cytisus scoparius), Yellow Bush Lupine (Lupinus arboreus), Miniature Lupine (Lupinus bicolor), and Siratro (Macroptilium atropurpureum) were inoculated with B. tropica strain Aa1 (isolated from dune grasses) and grown under N-limited hypoxenic greenhouse conditions. Inoculated Macroptilium and Cytisus showed small increases in size, but no nodulation was observed. Diverse bacteria were recovered from surface-sterilized Cytisus roots, but none of the isolates fixed nitrogen in liquid culture and no strain clearly corresponding to Burkholderia was recovered. In a concurrent study, PCR was used to probe Burkholderia tropica for the presence of the core nodulation genes nodABCD. Products apparently corresponding to nodA and nodC were recovered, but nodB and nodD were not. Burkholderia tropica strain Aa1 seems to be incapable of nodulating plants and may lack a functional nodB. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Promoter-Regulated Effects of TIS11 Overexpression on the Rate of Barley Yellow Dwarf Virus Stop Codon Readthrough in Saccharomyces cerevisiae

Rachel Katherine Bond

(Advisor: Russell)

Stop codon readthrough is the process by which the stop signal separating two contiguous in-frame genes is misread as a sense codon, resulting in the synthesis of a fusion protein. In Barley yellow dwarf virus (BYDV), a plant pathogenic positive sense RNA virus, this very rare process is programmed to occur at a low rate. In BYDV, readthrough from the major coat protein gene to the adjacent readthrough domain sequence results in a minor coat protein required for virus transmission by aphids. Because the virus life cycle depends on the host, host factors are assumed to be involved in viral processes such as stop codon readthrough. Identification of these host factors can lead to a better understanding of the viral life cycle, and eventually to strategies for thwarting infection. BYDV sequence programmed stop codon readthrough has been shown to function in the yeast, Saccharomyces cerevisiae, a genetically tractable organism used in this study as a model system to study the effect of host genes on readthrough efficiency. A double reporter plasmid, utilizing the BYDV readthrough sequences, was used to quantify the readthrough rate in S. cerevisiae. This double reporter plasmid incorporates a stop codon between two reporter genes encoding the enzymes β-galactosidase and firefly luciferase, allowing the readthrough rate to be calculated by measuring the activity of each reporter enzyme. One method of identifying host factors involved in stop codon readthrough is through overexpression of host genes, which can cause the readthrough process to be perturbed and result in a change in readthrough efficiency. The host factor gene TIS11, a putative translation factor with a zinc-finger motif, was identified in a previous screen as increasing the readthrough rate when overexpressed. In this study, the effect of TIS11 on readthrough efficiency was investigated by overexpressing it with two promoters, the stronger glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and the weaker translation elongation factor 1α (TEF) promoter. TIS11 overexpression under the TEF promoter significantly increased the readthrough rate by 28%, while GPD-driven overexpression increased the readthrough rate non-significantly, by only 2%. To investigate this unexpected result, the levels of TIS11 mRNA produced under each promoter were examined using quantitative real-time PCR. Depending on the gene used for normalization of expression, TEF-driven levels of TIS11 mRNA were 26.3- or 1.6-fold greater than TIS11 produced by the natural promoter, while GPD-driven levels of TIS11 mRNA were 20.8- or 0.8-fold greater than baseline TIS11 mRNA levels. In the light of this data, the original hypothesis that greater TIS11 levels induce greater readthrough efficiency is supported. However, it is unclear why the promoter-regulated TIS11 mRNA levels do not correlate with promoter strength. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Assessing the Presence, Stability, and Linearity of Dominance Hierarchies Among Female Cichlids of the Species Astatotilapia burtoni

Evan Freer Bremer

(Advisor: Renn)

The African cichlid fish Astatotilapia burtoni has often been used in studies of social behavior; the dominance systems of the males are particularly well understood. Studies focusing on the females of the species have been of their affiliative preference with respect to their reproductive status. Since they school in the wild and in laboratory settings, the potential for the development of a social structure is significant. I chose to study female A. burtoni with respect to their dominance structures and establish the groundwork for future studies of female dominance. To do this, I observed two groups of female A. burtoni for a total of 8 weeks, recording their agonistic interactions. I used two recognized methods of calculating dominance hierarchies to calculate ranks for the fish each week, and assessed these rankings for linearity and stability. My findings suggest that female A. burtoni form dominance hierarchies among themselves that are largely linear, and last about a day.

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Sex-Role Reversal and Hormonal Regulation of Social Behavior
in the African Cichlid fish Julidochromis marlieri

Seyram Ahiadeke Butame

(Advisor: Renn)

The fish family Cichlidae is characterized as monogamous and biparental. Typically, the males are larger and more aggressive and hence are able to dominate their mates. The genus Julidochromis or "Julies" as they are affectionately called, is part of this family of fish localized to Lake Tanganyika in Eastern Africa. Julidochromis marlieri, is one of five species belonging to the genus. They are unique, in that they differ from other species by having the females larger than their male counterparts and hence, females develop the potential to dominate the males. Size differences between males and females may lead to the development of a polyandrous mating system, though they are typically monogamous. Such differences have led researchers and cichlid enthusiasts to suggest that J. marlieri are a sex-role reversed species. Some of the work in this area has centered on the size difference between the males and the females and the species.
The aim of this investigation was to examine possible sex-role reversal from the perspective of social interactions between males and females. I set up different treatments with different size combinations of males and females and recorded the levels of aggression of each individual. Hormone samples were collected from test subjects using a new, non-invasive method to detect correlations between aggression and the concentrations of key sex hormones. The results corroborated the idea that in situations when the female is larger, the female tends to be more aggressive and dominant; and in situations where the male is the larger of the two sexes, the male becomes more aggressive and hence dominates its mate. Hormone data, suggested a correlation between the levels of aggression and the concentration of testosterone released by the individuals sampled. There was lack of correlation between 17b-estradiol, progesterone, 11-ketotestosterone concentrations and aggression levels. Lastly, each treatment had one individual that was more aggressive, compared to others, suggesting that in a social setting there may be a social hierarchy of some kind, which is typically headed by the largest individual. This study supports the idea that J. marlieri is a sex-role reversed species, given that in typical social settings, the females are bigger, more aggressive and hence dominate their male counterparts. This study was funded in part by a James F. and Marion L. Miller Foundation grant.

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Phytostabilization of Abandoned Diatomite Mine Sites in Terrebonne, Oregon, Utilizing Purshia tridentata and Soil Amendments

Samuel Dyer Chamberlain

(Advisor: Dalton)

Fossilized diatomite deposits of the Pleistocene era exist within Terrebonne region of central Oregon. The relative purity and commercial value of the deposits led to sustained excavation for many years until operations were no longer profitable. As a consequence of operations and insufficient environmental regulations the mined land was abandoned and continues to be in a state of disrepair to this day. Strip mining techniques have left large areas of exposed diatomite capable of entering local airways. Inhalation of diatomite poses the threat of respiratory damage leading to lung cancer and silicosis. For this reason diatomite must be stabilized in order to reduce quantities available to wind erosion. While conventional physical and chemical mine reclamation strategies can be costly and labor intensive, phytostabilization presents itself as a low-cost environmentally friendly alternative. Phytostabilization is defined by the use of plants to immobilize pollutants within a soil environment. This technique not only stabilizes pollutants but also begins the process of returning an area to a functional biological community. For the Terrebonne mine Purshia tridentata was used as a phytostabilizer. P. tridentata was grown in diatomite amended with vermiculite, steer manure, and transported topsoil from regions supporting healthy P. tridentata populations. Plants were grown in these conditions from seed and also transplanted from topsoil to these conditions. Additionally, population sizes of Frankia capable of nodulating P. tridentata seedlings were determined within the transported topsoil amendment and diatomite. The effect of hydrogel amendment on P. tridentata growth under drought conditions was also assessed. No soil amendment had an overall positive effect on plant growth or establishment, and the topsoil amendment contained infective Frankia populations too low to be accurately determined. Hydrogel amendment did encourage plant growth in drought conditions. None of the amendments are recommended to aid Purshia tridentata growth on-site with the exception of hydrogel amendment to overcome drought stress conditions. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Formation of Filaments by H-NS and Ler

Garon Coriz

(Advisor: Mellies)

Both the enterohemorrhagic (EHEC) and enteropathogenic (EPEC) pathotypes of E. coli carry a pathogenicity island known as the locus of enterocyte effacement (LEE). This locus is responsible for the ability of these pathogens to attach and efface lesions on epithelial cells. The heat-stable, nucleoid-structuring (H-NS) protein is a known silencer that operates at the LEE. An anti-silencer called Ler (LEE-encoded regulator) is a major agent in a regulatory pathway influencing the expression of LEE, which counteracts the activity of H-NS. Here we investigated why Ler and H-NS proteins have completely different effects as gene regulators though they are composed of similar amino acid sequences. It was predicted that H-NS would form collar-like structures while Ler would straighten DNA at the LEE5 operon. Surprisingly, with the aid of electron microscopy, Ler and H-NS were found to form large filaments up to nearly 3 µm in length when combined with linearized plasmids containing the regulatory regions of the LEE5 and proU operons. A follow-up study using a ~1.2 kb DNA fragment containing the rrnB P1 promoter was conducted to determine if RNAP was necessary to form collar-like structures. Yet, filamentation with the combinations of H-NS+RNAP+DNA and H-NS+DNA showed RNAP not to be necessary. In addition, RNAP was demonstrated to be sufficient for filamentation by the presence of filaments in the RNAP-only control. The RNAP filaments also proved that filamentation was not DNA-dependent, but reaffirmed that this is a protein-dependent phenomenon. Unfortunately, inconsistencies with filamentation in the experimental and control samples limited the drawing of further general conclusions.

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Ontogeny and Phylogeny of Two Spider Species, Cheiracanthium mildei and Latrodectus mactans

Allison Elizabeth Edgar

(Advisor: Black)

This thesis describes the development from oviposition to hatching in two species of spider, Cheiracanthium mildei and Latrodectus mactans, with a particular focus on gastrulation. C. mildei's development resembles in many ways the canonical model of spider gastrulation, while L. mactans's development more closely resembles that of the
non-canonical species Zygiella x-notata. The data collected from these species are used in a comparative analysis with nine other species, each in a different family, scored from the literature on spider development. Several prominent heterochronies are detected, and these, along with other morphogenetic data, are used with an outgroup (Opilionidae) to create an evolutionary hypothesis of the Araneae. The monophyly of the orb weavers, previously upheld largely by behavioral data alone and disputed by genetic data, is upheld by developmental data which suggest several key synapomorphies, including timing of developmental events and novel sites of cell internalization. This study was funded in part by a grant from the James F. and Marion L. Miller Foundation.

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Characterizing Serpentine Soil Tolerance in Mimulus guttatus (Phrymaceae)

Margaret Frisbie Hendrick

(Advisor: Karoly)

The physiological mechanisms of serpentine soil tolerance in plants are not well understood. It has been proposed that the tolerance mechanisms are highly complex and multi-faceted. However, much evidence points to the importance of tolerance of low Ca:Mg ratios that are characteristic of all serpentine environments. This study investigated the mechanisms of serpentine tolerance in Mimulus guttatus, a bodenvag species that occurs both on and off serpentine soils. Six populations of M. guttatus, three serpentine and three non-serpentine, were used in this study in order to examine both divergence between populations from different soil types as well as divergence among populations within the same soil type. Fifteen maternal lines were chosen for each population, with one individual representing each maternal line. Morphological and phenological traits were measured in a common garden greenhouse setting. These traits included percent flowering, corolla length, petal lobe width, plant height, specific leaf weight, leaf water percentage, leaf area, and leaf perimeter. Above and below-ground Ca, Mg, and biomass dynamics were measured in response to hydroponic treatment. Two treatment extremes were chosen for the hydroponics portion of research to discern divergence between serpentine and non-serpentine populations, the first treatment having a Ca:Mg ratio of four and the second having a Ca:Mg ratio of 0.01. Two cuttings were made per plant and one cutting was placed in each treatment solution. These treatments were administered hydroponically in order to assay selectively for tolerance of a low Ca:Mg ratio environment and to create a serpentine-like environment that was not so extreme as to kill non-adapted individuals outright. Tissue concentrations of Ca and Mg were determined using Neutron Activation Analysis (NAA) at the Reed College nuclear reactor. This research found evidence that populations of M. guttatus are diverged according to soil type for traits that fit a pattern of local adaptation. Serpentine adapted M. guttatus flowered earlier and had smaller flowers and smaller leaves than non-serpentine M. guttatus in a common garden setting. Serpentine endemic and non-serpentine populations responded differently to hydroponic treatment. Serpentine endemic M. guttatus were found to have significantly higher foliar and root Ca:Mg ratios as compared to non-serpentine individuals in both environments. Comparison of above and below ground responses between soil types suggests a possible mechanism for serpentine soil adaptation. This study was funded in part by James F. and Marion L. Miller Foundation grant.

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The Change in Localization of the Ubiquitin Ligase Nedd4 in the Presence of 17-β Estradiol in MCF-7 Breast Cancer Cells

Emily Li Pin Kam

(Advisor: McClellan)

Estrogen is a powerful hormone known to have many functions in the human body. However, it has also been found to promote breast cancer and current therapies aim to interfere with estrogen action. Although efficient, these treatments compromise quality of life. Nedd4 is an important ubiquitin ligase shown to regulate tumor suppressors and transcription initiation. Recently, a Nedd4 tethering protein, N4BP3, was identified as an estrogen target gene. In this work, the influence of estrogen on the subcellular distribution of Nedd4 in MCF-7 breast tumor was studied using immunofluorescence. Interestingly, confocal images of Nedd4 immunofluorescence revealed a novel cytoplasmic distribution of this protein in linear, punctate aggregates that appeared to associate with cytoplasmic filaments. Further investigations could include double staining experiments to verify this filamental association.

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Is Dam Removal A Panacea? Salmon, the Sandy River, and the Marmot Dam

Matthew Joseph King

(Advisor: Kaplan)

During the summer of 2007, the Marmot Dam on the Sandy River was removed. The following fall, the river was allowed to flow free for the first time in almost a century. The reservoir behind the dam had amassed roughly 750,000 cubic meters of sediment during that time and the river now had access to all of it at once. The bulk of this sediment was predicted to move through the river system of the course of one winter flood season, and the effects of the sediment load were not fully assessed before the dam removal took place. This thesis aims to assess the potential negative effects of the sediment load on the downstream reaches, particularly on salmon. The flood season runs concurrently with salmon spawning seasons and it is possible that salmon redds could have been inundated during a sediment laden flood. I evaluated sediment aggradation at Fall Chinook salmon spawning sites in Oxbow park, monitored the sediment as it moved from the reservoir toward the lower reaches, and tested a technique to monitor within redd conditions over the course of a removal. I found that despite predictions made prior to the removal the bulk of the sediment did not reach the main spawning grounds for Fall Chinook. Also, the technique I tested for measuring within redd conditions proved to be quite effective and could be applied during future dam removals.

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Green Roofs and Urban Biodiversity: Arthropod Conservation in Portland, Oregon

Georgia Helena Kirkpatrick

(Advisor: Kaplan)

Like many western cities, Portland is experiencing rapid population growth and increased densification, resulting in the loss of open space and habitat. As urban biodiversity declines, the potential of green roofs - roof systems that support vegetation - to provide foraging and nesting habitat for arthropods, birds, and other urban fauna is being seriously considered. This thesis explores the role that green roofs have played and can play in urban biodiversity conservation strategies, with a particular focus on insects and spiders. Green roofs provide several important ecosystem services; they retain and evapotranspirate stormwater, insulate buildings, and reduce urban heat island effects. Prompted by research in Switzerland, Germany, and the United Kingdom, where green roofs are relatively common, increased attention is being commanded by the biodiversity benefits of green roofs in Portland. In addition to reviewing existing green roof biodiversity studies and consulting with local green roof experts, original research was conducted on two of Portland's 99 green roofs. This study has three aims. First, to collect preliminary data in what will hopefully be a longer survey of arthropod presence on Portland green roofs. Second, to determine how microclimates affect arthropod presence. And third, to assess experimental design frameworks for comparing arthropod biodiversity on urban green roofs. Green roofs can best contribute to urban biodiversity as part of a comprehensive strategy that involves parks, street trees, green facades, and considers connectivity as well as providing a diversity of habitats. While green roofs have some limitations as habitat they also have some significant benefits, such as protection from ground-dwelling predators. The thesis concludes by offering design recommendations to green roof developers to enhance the biodiversity benefits offered by green roofs.

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Lactoferrin as a Potential Anti-Proliferative Agent: Lactoferrin Exhibits Inhibitory Transcriptional Control over Skp1

Claire Elizabeth Matturro

(Advisor: McClellan)

Lactoferrin, an iron-binding protein present in most exocrine secretions, is reported to have anti-proliferative effects in a variety of cancer cells. Lactoferrin's antitumor action may be independent of its iron binding function and may involve direct transcriptional control of important cell cycle genes. In breast cancer, estrogen also regulates cell cycle genes to effect increased proliferation, and strategies to combat the disease are aimed at blocking all hormonal effects. In this work I investigate whether lactoferrin treatment alters transcription of two opposing cell cycle regulatory genes, S-phase associated kinase protein 1 (Skp1) and cyclin G2 (CCNG2) in MCF7 breast cancer cells. Using quantitative real time PCR I show that lactoferrin acutely inhibits SKp1 transcription, a protein necessary for the transition to the proliferative state. However, lactoferrin was unable to override estrogenic effects on the negative cell cycle regulator, CCNG2 and estrogen blocked the lactoferrin effect on Skp1. The results produced by this study, when taken in the context of previously published findings, suggest that lactoferrin may represent a promising therapy in non-estrogen responsive cancers. This study was funded in part by a James F. and Marion L. Miller Foundation grant.

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Differential Regulation of the proU and LEE5 Operons of Enteropathogenic Escherichia coli by the Repressor H-NS and Anti-Silencer Ler Requires more than Competition for a Shared Binding Site

William Clement McNitt

(Advisor: Mellies)

Enteropathogenic and enterohemorrhagic Escherichia coli infect the human intestinal epithelium, causing extensive damage to the brush border of epithelial cells by forming attaching and effacing lesions. The bacteria induce cytoskeletal changes in the host cells by injecting effector proteins into the host via a type III secretion system. All of the genes necessary for the type III secretion system are found on the locus of enterocyte effacement (LEE) pathogenicity island. Many of the operons contained within the LEE are silenced by the ubiquitous gram-negative bacterial repressor H-NS, and this silencing is relieved by the LEE-encoded regulator (Ler). Another E. coli operon, proU, is also silenced by H-NS, but is not affected by Ler. The present study employed electrophoretic mobility shift assays on polyacrylamide gels to determine whether or not a small difference in the binding kinetics of Ler and H-NS at the regulatory sites of the LEE5 and proU operons explains the difference in regulation by Ler. The data demonstrate that the difference in binding kinetics of Ler and H-NS at the two operons cannot explain the differential regulation of the operons, suggesting that other protein regulators or structural differences in the DNA contribute to the regulation of the operons. This study was funded in part by a James F. and Marion L. Miller Foundation grant.

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Intraspecies Recognition in Asatilatopia burtoni Cichlids

Catherine Ariel Mingoya

(Advisor: Renn)

All species of cichlid fish are extremely dedicated to the growth and development of their offspring into sexually mature adults (Keenleyside, 1991). From fertilization through offspring independence, parental care can last up to 9 weeks and requires a tremendous investment of time and energy funneled into egg and school maintenance and protection (Alemadi & Wisenden, 2002; Barlow, 2000; Gunther, 1972). Since such detailed care and high expenditures of energy are required to raise the young to maturity, evolutionary theory stipulates that in the case of the cichlid, in order to maximize efficiency in benefiting it's own reproductive success, it would be important for it to expend the least amount of energy in deriving the greatest benefit in caring for its biological offspring (Hamilton, 1964). This raises the question of if and how cichlids may identify their family members from the family members of others in order to appropriately direct nepotistic preference.
Young fry possess the ability to respond to chemosensory and visual cues related to their species (Alemadi & Wisenden, 2002; Nobel, 1939) and mothers have been shown to discriminate between their young and the young of other species (Barlow, 2000). Here, wild caught Asatilatopia burtoni mothers and their lab-born broods are engaged in three experiments to 1) identify the presence or absence of a preference of females to orally accept their young over the young of others of the same species when threatened 2) identify the presence or absence of a preference for fry to congregate with their mother over another mother when threatened and 3) to identify the presence or absence of a chemosensory cue in kin-recognition. The results show that there is a preference for mothers to accept their young into their buccal cavities in times of distress. In addition the data suggests that fry play a role in the congregation process and are not passive recipients of parental care.

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The Potential Use of Two White Rot Fungi, Pleurotus ostreatus and Trametes versicolor, in the Bioremediation of Used Motor Oil

Daniel Luther Sullivan

(Advisor: Dalton)

White rot basidiomycetes are the only organisms capable of fully decomposing the complex plant polymer lignin. Unlike most metabolic pathways, lignin decomposition is achieved through a non-specific, oxidative mechanism involving oxidases and peroxidases. A fortuitous effect of this capability allows these abditive fungi to also decompose recalcitrant organopollutants such as polychlorinated biphenols and polycyclic aromatic hydrocarbons. In the present research, selected strains of two white rot fungi, Pleurotus ostreatus and Trametes versicolor, were grown in chopped straw amended with used motor oil. Both fungi were able to colonize the substrate, though T. versicolor with more success. The activities of two ligninolytic enzymes, manganese peroxidase and fungal laccase, were monitored at 7, 21, and 42 days post amendment. The enzyme activities of both controls and amended cultures were low compared with expected activities. However, P. ostreatus and T. versicolor cultures were able to reduce the level of used motor oil by 8% and 22% in 56 days, respectively, compared with uninoculated controls. During degradation, T. versicolor cultures accumulated an aqueous layer of metabolites containing partially degraded hydrocarbons. This study was funded in part by a James F. and Marion L. Miller Foundation grant.

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Ecophysiology and Pleiotropy of Foliar and Floral Anthocyanins in Delphinium nuttallii and Delphinium leucophaeum

Rebecca Anne Susko

(Advisor: Karoly)

Anthocyanin-based flower color polymorphisms have historically be studied only from the perspective of the floral tissue as it relates to plant/pollinator interactions. Anthocyanins are known to also be present in foliar tissue, though no definitive link between anthocyanin biosynthesis in floral and foliar tissue has yet been confirmed. Because anthocyanins and their flavonol precursors have been shown to confer a diverse array of stress tolerances, their study in all tissue types is warranted. Specifically, anthocyanins have been linked to amelioration of stress due to variation in light intensity. This research examined anthocyanin and flavonol concentrations in the foliar tissue of the blue-flowered Delphinium nuttallii and the white-flowered Delphinium leucophaeum grown in shade and light environments to determine if foliar pigment levels are pleiotropically linked to floral pigment levels, and to assess the potential ecophysiological consequences of such a linkage. Chlorophyll fluorescence was used to measure the physiological stress response of 30 D. nuttalii and 13 D. leucophaeum under shade and light conditions in a common garden experiment. Spectrophotometry, high performance liquid chromatography (HPLC), and reflectance spectroscopy were used to determine foliar anthocyanin and flavonol concentrations in each light environment. Chlorophyll fluorescence measurements indicated that D. leucophaeum suffered a greater degree of stress than D. nuttallii in the light environment. Interestingly, anthocyanin concentrations measured spectrophotometrically in D. leucophaeum increased relative to D. nuttallii in the light environment, though overall no species difference was observed. Pigment concentrations determined using HPLC were orders of magnitude less than concentrations determined spectrophotometrically, though the methodologies showed a high degree of correlation. Reflectance spectroscopy was not correlated to pigment concentrations determined spectrophotometrically. Because there was no observed species difference in pigment concentrations, there is no support for the hypothesis that foliar and floral pigment concentrations are pleiotropically linked. The general trend of increased stress paralleled with induction of anthocyanin biosynthesis that was observed in D. leucophaeum but not in D. nuttallii suggests that the light environment may be an indirect selective force acting to maintain this pigment polymorphism in natural populations.

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The Evolution of Phenotypic Plasticity Under Alternative Population Dynamics

Jonathan R. Sweeney

(Advisor: Karoly)

Two strategies exist for organisms in an heterogeneous environment. They may evolve adaptive phenotypic plasticity and adjust their phenotype to the local environment to maximize their fitness, or they may evolve local specialization and produce a single phenotype with maximum fitness in a single environment. In this study I asked what conditions favor each strategy, using a modeling approach. I explored the evolutionary consequence of migration dynamics, environmental heterogeneity, and plastic response accuracy on the evolution of adaptive phenotypic plasticity and local specialization in four population models. For each model I constructed the population of one plastic genotype (P), two specialist genotypes (M1 and M2), and two environments. The plastic genotype could respond to the environment with an accuracy given by (a), while each of the two specialists had a fixed phenotype that was well adapted to only one of the two environments. I implemented two previously developed models in R, then extended the second model to include a calculation of the global fitness landscape. I then considered three subpopulations. In the single population model adaptive phenotypic plasticity was favored when environmental heterogeneity was high and when plastic response accuracy was high. In the two subpopulation model high levels of migration between sites favored adaptive phenotypic plasticity over a wide range of parameter space. The highest point of fitness occurred when specialists dominated each subpopulation or when the plastic response accuracy was 1. In the third model under island migration, low migration between the three subpopulations sustained a population consisting of all three genotypes. As migration increased, adaptive phenotypic plasticity was favored. In the fourth model under stepping-stone migration, the area that supported all three genotypes and the area that supported only specialist 1 and 2 was smaller than under island migration.

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Organ Localization and Function of Immunosuppressive Enzyme Indoleamine 2,3-Dioxygenase and its Novel Isozyme, IDO2, in Xenopus laevis

Catherine Samuels Uram

(Advisor: Ruben)

Indoleamine 2,3-dioxygenase (IDO) is a highly conserved enzyme that catalyzes the first and rate-limiting step of catabolism of essential amino acid tryptophan. Enzymatic activity yields microenvironments of tryptophan depletion and accumulation of toxic metabolites, called kynurenines, which have immunosuppressive effects on thymus-derived lymphocytes (T cells). In normal activity, IDO affords necessary tolerance to foreign cells, such as non-pathogenic microbes in the digestive tract and fetal cells during pregnancy. However, ectopic expression of the enzyme can generate unwanted tolerance to tumor and viral antigens in the central nervous system and periphery. Therefore, IDO inhibitors are currently being developed for immunotherapies. Recently, lower activity isozyme IDO2 has been identified, but its functions have not yet been defined. Xenopus laevis, the South African clawed toad, is a useful model organism for immunology research. During metamorphosis, adult and larval cells are in the same biological space without eliciting an immune response, thus providing a model for maternal tolerance towards the genetically disparate fetus in mammalian pregnancy. Further, Xenopus are resistant to tumors, so the organism is used as a cancer research model. This study is the first to explore whether both IDO and IDO2 exist in adult Xenopus and experimentally test their potential functional homology to IDO proteins in other species. Organ distribution of the isozymes is also studied through Western blots and absorbance-based assays. This study lays the foundation for a variety of future investigations with regard to IDO and the immune system of Xenopus. This study was funded in part by a James F. and Marion L. Miller Foundation grant.

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Maternal Care and Aggression in the African Cichlid Astatotilapia burtonii

Alexandra Holbrooke Winters

(Advisor: Renn)

Maternal care and aggression were studied in two stocks of the African cichlid fish, Astatotilapia burtoni, during and after brooding. While the behavior of A. burtoni males has been previously characterized, the females had not been known to exhibit any maternal behavior until recent observations. Brooding females were observed from spawning until approximately two weeks after release of the fry. Wild caught fish were observed first to provide a basis for later observations. Then the F1s, the offspring of the wild caught fish, and Labstocks, fish that have been raised in laboratory conditions for the past thirty years, were observed and used for hormone sampling. Hormone sampling was done at time points before and after release of the fry. It was found that Fls show more maternal care behaviors than Labstocks, such as taking their fry back into their mouths after release. Hormonal differences between the stocks suggest mechanisms for the behavioral differences seen between the two stocks. This study was funded in part by a James F. and Marion L. Miller Foundation grant.

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Expression and Characteriation of Xenopus laevis POT1

Gillian Reed Woodruff

(Advisor: Shampay)

Protection of telomeres 1 (POT1) is a key component of the shelterin complex, which helps protect the ends of linear chromosomes (telomeres) and regulate their length. All POT1 proteins characterized to date bind single-stranded DNA. X. laevis is a useful model organism for telomere biologists because its somatic tissues contain constitutively active telomerase, an enzyme that lengthens telomeres. A putative Xenopus orthologue of POT1, xPOT1, has been identified. If it is indeed an orthologue of hPOT1 it should bind single-stranded telomeric DNA. Characterization of xPOT1 activity in vitro would help place X. laevis into a broader context of other model organisms' telomere protection and regulation mechanisms. The sequence containing the OB fold of xPOT1 had been cloned previously into an E. coli expression vector (Procopio, 2007). Although expression of the recombinant xPOT1 in this system produces large insoluble protein aggregates in all induction conditions studied so far, room temperature and 15 degree C induction conditions produce enough xPOT1 to be isolated and studied in vitro. The efficient isolation and purification of soluble xPOT1 described here will facilitate further characterization of this protein. This study was funded in part by a grant to JS from the National Science Foundation.

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