Biology

2016 Senior Thesis Abstracts

(CLASS OF 2016: If your thesis abstract is not currently included on this page and you would like it to be, please follow this link.)

Sink or fim: Regulation of the fim operon by PerC in Enteropathogenic Escherichia coli
Lily Ben-Avi
Adviser: Jay Mellies

Enteropathogenic Escherichia coli is a diarrheal pathogen that represents a significant health threat for people in the developing world. It has been shown that a plasmid- encoded protein PerC is responsible for regulating a number of genes that control EPEC’s virulence strategy, and previous RNA sequencing data suggests strong down regulation of expression of genes in the fim operon, which encode type I fimbriae. This study attempts to verify this finding at various levels of expression using wild type (WT) EPEC, an isogenic ∆perC deletion strain, and a perC complemented construct. First, a PCR-based assay was used to examine the orientation of a genetic switch element that controls transcription of the fim operon; this showed that while the switch had both orientations in the strains lacking PerC, those that contained PerC were entirely off, suggesting that PerC is down regulating fim genes via this regulatory element. Then, a western blot for the structural FimA protein was performed, with inconclusive results. Because type I fimbriae are potent immune stimulators, an IL-2 ELISA was performed to measure differential immune response, and the results were also inconclusive. Finally, epifluorescence microscopy was performed to visualize type I fimbriae on samples of each strain, and it was observed that PerC-containing strains had no expression of type I fimbriae, while those without PerC did, which matched the hypothesis formulated based on the PCR-based assay. Taken together, these results provide strong support that PerC is down regulating the expression of type I fimbriae in EPEC via the inversion of a regulatory switch element.

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The Effects of Reduced Impact Logging On Biodiversity in Tropical Forests: A Meta-Analysis and Qualitative Review
Emily Beck Binford
Adviser: Keith Karoly

Reduced Impact Logging (RIL) is a form of logging in the tropics characterized by the use of a planned harvest of pre-indicated commercial tree species, a minimum cutting diameter, and set cutting cycle. RIL was developed as a response to forest degradation resulting from unplanned selective logging. Proponents of RIL aim to increase long-term timber yields by allowing the forest to regenerate by limiting cutting cycles and maintaining a population of young potential crop trees. Many environmentalists are supporters of RIL as well, arguing that decreased residual tree damage encourages forest ecosystem health. However, the overall effects of RIL on biodiversity levels have yet to be resolved in the scientific literature. My thesis aimed to quantify the overall impact of RIL on biodiversity through the completion of a meta-analysis. A meta-analysis is a way to quantitatively compare and combine data from separate studies of a common research area. Using Web of Science, I collected and processed 107 studies, finding a total of six studies that quantified the biodiversity effects of RIL. Three of these studies used species richness (total number of species) as their measure of diversity, allowing me to calculate a response ratio for each study to serve as an effect size. Each effect size was weighted by a modified variance in order to yield a mean effect size. These three studies addressed butterfly, bird, and bat diversity. The mean effect size was calculated to be -0.1, indicating a small, negative effect from these studies of RIL on biodiversity. A more coarse cross-study summary method called “vote-counting” was completed, which assessed each study’s reported effect of RIL on biodiversity as a binary minor/significant. Of the six original studies, five reported RIL to have a minor effect on biodiversity. Considering the small number of studies available and the heterogeneity of taxa examined in these three studies, these results cannot be considered conclusive with respect to the biodiversity effect of RIL. However, each of the six original studies revealed some difference in community structure in response to RIL, indicating that RIL significantly alters species assemblage, which will require more research to determine its implications for the overall forest ecology.

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The Role of Actin-Microtubule Cross-Linking in Cell Migration
Mari Shiratori Cobb
Adviser: Derek Applewhite

Migratory cells are responsible for dramatic cellular processes ranging from devel- opment to tumor initiation and metastasis. One of the factors that contributes to cancer metastasis is migration, which could be affected by disorganization in the cytoskeleton. In this thesis, we look into how the depletion of Shot, the main actin- microtubule crosslinker in Drosophila melanogaster cells, affects cell migration and adhesion. The techniques we used to examine these effects were RNAi depletion, scratch wound assays, western blots, and immunofluorescence microscopy. Following Shot depletion, cells were observed to migrate statistically significantly faster and have a statistically significantly higher concentration of F-Actin in sites of cell-cell adhesion. In-vivo studies of whether there was an increase of F-actin in the delami- nation area of Drosophila border cells proved inconclusive. We think that the results found using Drosophila melanogaster could also apply to other organisms.

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A Gene By Any Other Name: Reevaluating Gene Annotation in Astatotilapia burtoni
Ameliah Raine Cooper
Adviser: Suzy Renn

The National Center for Biotechnology Information (NCBI) has been an invaluable resource in cataloguing genome sequences and annotating genes. As of the completion of this thesis, at least six cichlid genomes have been sequenced and uploaded to this database. As the number of viable genomes increase, the necessity for new gene annotation algorithms within their pipelines increases. Pipelines help to automatically categorize different sections of the genomes into predicted coding regions, noncoding regions, transcripts, and proteins. However, the algorithm that labels them based on their relationship to known genes is poorly managed, thus potentially misnaming many of these predicted regions. By manually curating some of these predicted genes through alignment against similar genes within their families, particularly those that code for receptors that regulate feeding behavior in Astatotilapia burtoni , this study seeks to rectify or confirm the labels of certain predicted proteins for the Renn Laboratory database. In performing this study, ways in which the pipeline algorithms can be revised to be more accurate in the future are examined, as well as what types of results we might expect within a family with immensely diverse speciation.
The results show several mislabeled predicted neuropeptide Y receptors in A. burtoni, among other cichlids, but also several accurate labels within the same tree, which implies that the algorithm is not entirely nonfunctional, but requires some fine-tuning. The melanocortin receptor tree supports this by correctly placing the predicted receptors in the relevant subtype clusters on the tree except for one predicted receptor. Similarly, the leptin receptor tree also seems to show mostly correct labels.

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From feces to families: Genotyping beavers in the Reed Canyon
Hanna Dornhofer
Adviser: Todd Schlenke

The North American Beaver, Castor canadensis , is considered a keystone species because of its central role in creating and maintaining North American freshwater ecosystems. A population of C. canadensis inhabits Reed College Canyon in southeast Portland, part of the Johnson Creek Watershed. The Reed Canyon provides an abundance of resources to the surrounding community, ranging from storm drainage to educational and recreational opportunities. Beavers have been incorporated into the Reed Canyon management plan for reestablishing a healthy wetland since 1999. But beavers may raise lake levels in some areas with their damming habits. To date, no extensive research has been conducted on the Reed Canyon beaver population. To enable continued coexistence of humans and beavers in the area, basic information on the C. canadensis population is needed. Night-vision video cameras installed at beaver dam and den sites recorded beaver activity at the east end of Reed Canyon in January and April. Beaver DNA was extracted from fecal matter and amplified at 10 microsatellite loci previously used in beaver kinship studies. Genotypes at each locus were determined for 14 samples and showed that a minimum of five beavers inhabit Reed Canyon. Genotypes of four fecal samples collected from populations living either upstream of Reed Canyon or in a neighboring watershed were less similar to the Reed samples. This study improves the current understanding of the Reed Canyon beaver population and offers a new technique for the noninvasive study of beavers.

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Cross-Species Inhibition of Telomerase by PinX1
Ajit Elhance
Adviser: Janis Shampay

A cell experiences progressive shortening of the chromosome ends as it undergoes each division. To counteract this shortening, the enzyme telomerase adds tandem repeats of a short non-coding sequence to the ends of the chromosomes to create telomeres, protecting the coding DNA from degradation. Telomerase activity in humans is regulated in part by the protein PinX1. PinX1 is capable of suppressing telomerase activity through a telomerase inhibitory domain near the C-terminal of the protein. While somatic human cells do not contain high levels of active telomerase, the African clawed frog, Xenopus laevis , expresses active telomerase in all tissues. A Xenopus ortholog of PinX1 was previously discovered and found to have a similar inhibitory effect on Xenopus telomerase activity. However, despite the similar inhibition patterns, it is currently unknown whether the mechanism of telomerase inhibition is conserved between the two species. In this experiment, PinX1 orthologs from mouse, human, and Xenopus laevis were cloned into a GFP-fusion vector and transfected separately into mouse NIH/3T3 cells to determine if the proteins were capable of inhibiting native telomerase activity.  The cells were analyzed by fluorescence microscopy, western blot, and a Telomeric Repeat Amplification Protocol (TRAP) assay. Despite the presence of the plasmid DNA in the transfected cell cultures, no detectable GFP fluorescence was observed, nor was any protein found by western blot, which suggests that a suppression of gene expression was occurring in the cells. The TRAP assay reflected this, with no clear pattern of telomerase activity inhibition by any of the PinX1 treatments. Surprisingly, PinX1 transfectants appeared to exhibit greater telomerase activity compared to cells transfected with an empty pEGFP-C1 vector. However, these results were not conclusive and further research will be required to determine if a cross-species inhibition effect exists.

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Holocene Extinction; The next "Mayan Calendar" Apocalypse?
Robert Treat Haas
Adviser: Sarah Schaack

The Holocene Mass Extinction has been postulated by some scientists to be the greatest challenge facing modern Earth, but research is equivocal on the subject. Throughout natural history, other mass extinctions may offer clues, but comparisons between modern extinctions and historical ones often fall short, as data about modern extinctions are compared to scant fossil evidence. Anthropogenic factors, such as climate change, deforestation, and pollution, are some causes of our current biodiversity crisis, but scientific endeavors to spur positive social change in society have limitations. To combat what could be an impending mass extinction, some scientists are trying to preserve lost genomes by storing them frozen in repositories, or even by expressing these ancient genes in vivo. Using a variety of techniques, these researchers have even caused an extinct species to live again. The ultimate goal of de-extinction efforts is to reintroduce these species into the wild, but existing conservation efforts already are helping the environment in other ways. Species-based conservation, and environmental engineering offer hopes for the future, so it may not be too late for concerted efforts to help better mitigate the on-going biodiversity crisis.

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Correlating Hormones and Behavior in the Cycle of the Mouthbrooding Cichlid, Astatotilapia burtoni
Mara Elizabeth Kaiser
Adviser: Suzy Renn

Extensive research has been done to correlate aggression and male behavior. Female behavior has been sidelined, often because the females of a given species are assumed to represent subordinate behavior while males are thought to be dominant. Female fish behavior may be limited by the group housing conditions that disallow the fish to exhibit the full scope of maternal care behaviors. The first goal of this thesis was to determine if a reliable estimation of hormone rate of release over the days of the brooding cycle could be constructed; secondarily, to examine the relationship between androgenic and estrogenic hormones and maternal care behavior. Female Astatotilapia burtoni underwent one-hour water collection to quantify the amount of steroid hormones the fish passively released. Water collection was determined to be an accurate method to measure hormone concentrations relative to circulating plasma concentrations. Reproductive stage was noted on the day of hormone collection. Behavior was measured using an ethogram of maternal, aggressive, and stress behaviors. The amount of individual variation in hormone release over a reproductive cycle was too great to create a reliable time course of androgenic and estrogenic hormones. Significant correlations could not be found between the rate of hormone release and brooding stage, or between specific behaviors and hormone release. There was a significant correlation between specific behaviors and brooding stage.

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Estimating the rate and spectrum of mitochondrial mutation in Daphnia using next-generation sequencing
Fenner Benjamin Macrae
Adviser: Sarah Schaack

Mutation is the ultimate source of genetic variation, and thus is a driving force of  evolution. Despite this fact, rates of spontaneous mutation have only been  directly estimated in a few organisms, and little is known about their patterns of  variation across taxa. Estimating rates of mutation in the mitochondrial genome  is of particular importance, due to the widespread usage of mitochondrial genes as phylogenetic markers. In the present study, we detected mutations in next-generation  sequencing data from a set of Daphnia pulex mutation accumulation lines. The  majority of mutations were low-frequency heteroplasmic insertions and deletion  (indels) at homopolymeric tracts, consistent with previous examinations  of mitochondrial mutations, but several base substitutions were also detected. We  estimated the base-substitution rate to be 22.7 x 10−8 , and the rate of insertions and  deletions (indels) to be 8.0 x 10−8, resulting in an overall mutation rate of 32.3 x 10−8. This differs significantly from a previous estimate in D. pulex, which found an  increased rate of indels, but a decreased rate of base-substitutions. We suspect that  differences in the error profiles of next-generation sequencing and Sanger sequencing  may partially explain this discrepancy. This suggests that caution should be taken in  comparing the results of mutation-accumulation experiments which utilize differing  sequencing methodologies.

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Action of Cytoskeletal Crosslinking Proteins in Nuclear Positing and Movement
Emily Beth Merfeld
Adviser: Derek Applewhite

The well-established role of the cytoskeleton in cellular movement and intracellular organization is coordinated in part by cytoskeletal crosslinking proteins, which bind both actin and microtubules. This coordination involves precise positioning of the nucleus in intracellular space, and in some cases, use of the nucleus to achieve proper migration and positioning of the cell as a whole. The role of two cytoskeletal crosslinking proteins, Short stop (Shot) and Pickled eggs (Pigs), in cellular movement and nuclear positioning were examined in a Drosophila melanogaster system. Shot and Pigs were enriched at the nuclear membrane in neurons and cancer-like cells but not glia. Nuclear localization of Shot and Pigs in cancer-like cells was disrupted by genetic and pharmacological perturbations of cytoskeletal components. Migrational analyses in these cancer-like cells demonstrated that knockdown of pigs caused reduced migration rate and altered alignment of the nucleolus, nucleus, and whole cell relative to one another. Taken together, these results implicate a role for Pigs in cellular movement and intracellular alignment, and beg further elucidation of the functional differences between Shot and Pigs.

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Regulation of anaerobic respiration by PerC in enteropathogenic Escherichia coli
Amy Diana Platenkamp
Adviser: Jay Mellies

Enteropathogenic Eschericha coli (EPEC) is a diarrheal disease-causing bacterium that most adversely affects children under the age of five in developing countries. Typical EPEC (tEPEC) contains the E. coli attachment factor plasmid (pEAF) that includes the perABC operon. The perABC operon encodes plasmid-encoded regulator C (PerC), a known regulator of virulence genes, specifically the LEE1 operon of the locus of enterocyte effacement (LEE) pathogenicity island (PAI). While the number and type of genes regulated by PerC is still largely unknown, recent RNA-sequencing data has elucidated possible regulated genes. RNA-sequencing data indicates that anaerobic genes such as nitrate reductases are increased in transcription in the wild-type EPEC strain compared to a Δ perC deletion strain. This study examined the growth of a perC- containing wild-type strain, a Δ perC deletion strain, a perC- complement strain, and an empty-vector strain in the presence and absence of sodium nitrate. The perC -containing complement strain had greater growth than the empty vector strain after four hours of incubation in anaerobic conditions with excess sodium nitrate; similarly, the wild-type strain trended towards greater growth than the deletion strain in these conditions. A competition assay between the wild-type and deletion strains provided a similar trend of greater wild-type strain growth in the presence of sodium nitrate after four hours. These results indicate that PerC positively regulates anaerobic respiration, which confers a growth advantage to tEPEC that aids in niche establishment. The characterization of gene regulation by PerC provides greater context to the role of perC -homologues ( pch ) and other plasmid-encoded virulence proteins in niche establishment among related bacterial pathogens.

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T cell responses to a fibroblast-tropic human cytomegalovirus
Eren Mustafa Veziroglu
Adviser: Jay Mellies

HIV is a highly aggressive virus which rapidly depletes the immune system and leads to AIDS, a fatal condition. The Picker lab has developed a Rhesus CMV engineered to stimulate a highly effective immune response against SIV in Rhesus macaque (Macaca mulatta), and discovered that CMV-elicited CD8+ T cells were breaking a long- established immunological paradigm by recognizing antigens through the MHC-II molecule. The Picker lab associated this phenomenon, and therefore the efficacy of their vaccine in treating SIV, with the genes encoding fibroblast-tropism in CMV. In this study, fibroblast-tropic strains of human CMV were generated and administered in small doses to human male volunteers. Peripheral blood was obtained from volunteers in order to ask whether a fibroblast-tropic human CMV can elicit CD8+ T cells recognizing MHC-II. T cells from a single volunteer, subject 36, were tested for reactivity against CMV using intracellular cytokine staining and flow cytometry in a manner consistent with the Picker lab’s report. The T cell response to a fibroblast-tropic human CMV in subject 36 was compared with the T cell response to naturally acquired CMV infection in a different volunteer. The T cell response to fibroblast-tropic human CMV in subject 36 was consistent with a wild-type infection. This consistency with the wild-type strain indicates that there are other factors contributing to nontraditional CD8+ T cell action beyond infection by a fibroblast-tropic CMV.

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