2005 Senior Thesis Abstracts

(CLASS OF 2005: If your thesis abstract is not currently included on this page and you would like it to be, please follow this link.)

Cloning Xenopus laevis Telomere Binding Protein, POT1

Clea Maruxa Aguera-Arcas

(Advisor: Shampay)

Telomeres, the repetitive sequences at the chromosome termini, serve to protect the chromosome against sundry assaults specific to the DNA's end as well as to regulate cell cycle progression. As such, they serve to prevent chromosomal ends from being falsely recognized by the cell as double-stranded DNA breaks, are responsible for protection against chromosome degradation associated with the end-replication problem, and act as a buffer for the coding regions proximal to the telomere. Telomerase, the enzyme responsible for lengthening telomeres, is tightly regulated by a host of telomere-binding proteins, one of which, POT1, serves as a negative regulator. By obtaining and sequencing a clone of Xenopus POT1, a better picture of the evolution and mechanism of telomere regulation may be attained. This clone can then be used as a molecular reagent and in further expression studies to determine its mode of action. In this study, the Xenopus POT1 gene was amplified by reverse-transcription PCR in two pieces and cloned for comparison to the POT1 genes of other organisms ranging from humans to yeast, and with the putative Xenopus POT1 transcript submitted to GenBank by Ishikawa et al. (Accession Number: AY207366). This study was funded in part by a grant to JS from the National Science Foundation.

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The Enteropathogenic Escherichia coli Global Regulator Ler is Critical to
Pathogenesis in the Nematode Caenorhabditis elegans

Alexander Michael Barron

(Advisor: Mellies)

The development of effective therapies and vaccines to combat bacterial pathogens requires in vivo study of the host-pathogen relationship. The development of animal models that effectively mimic the conditions encountered by the bacterial pathogen in the host is of the utmost importance in the search for identifying potential therapies. Enteropathogenic Escherichia coli (EPEC), a diarrheal pathogen that kills millions of children each year in the developing world, does not yet have an established animal model that can be used for in vivo studies. EPEC causes diarrhea by colonizing the epithelium of the human small intestine where it uses a type III secretion system (TTSS) to inject effector molecules that subvert host cell signaling directly into human intestinal cells, and a type IV pilus termed the bundle forming pilus (BFP) to mediate the spread of disease in the host. Mechanistically, however, the process by which EPEC causes diarrhea is not fully understood. Recently, the nematode Caenorhabditis elegans has emerged as a useful animal model for several bacterial pathogens including Pseudomonas aeruginosa, a pathogenic bacteria that utilizes a TTSS homologous to that employed by EPEC. We have attempted to establish C. elegans as an animal model of infection that can be used to study EPEC disease in vivo. By fluorescent microscopy we have visualized EPEC expressing green fluorescent protein (GFP) in the intestine of the worm in greater numbers than non-pathogenic controls. Survival and colonization of the nematode gut by infecting EPEC required expression of the global virulence regulator Ler, which controls expression of the TTSS which is necessary for virulence in humans.

Interestingly, the EPEC's TTSS did not appear to play a role in colonization or survival in the worm intestine. While the BFP did not appear to serve a function in EPEC colonization of the C. elegans alimentary track, aggregates of EPEC have been visualized in the worm gut by fluorescent microscopy. Finally, we establish that although the TTSS does not appear necessary for colonization of survival of EPEC in the C. elegans gut, elements of the secretory apparatus of the TTSS are expressed in the worm alimentary track. Together these results support the effectiveness of C. elegans as an animal model for EPEC infection in humans. This study was funded in part by an HHMI Undergraduate Research Program grant.

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In vivo and in vitro Reduction of 1,2-diacetylbenzene Neurotoxicity by N-acetyl-L-cysteine

Amir Bashar

(Advisor: Black)

Certain hydrocarbons present in soil/water are metabolized to gamma-diketones that react with cellular proteins. 1,2-diacetylbenzene (DAB) is a known active metabolite of 1,2-diethylbenzene, a minor neurotoxic component of many aromatic solvents. It has been reported that 1,2-DAB produces discoloration of animal tissue (including neural tissue) and induces axonal pathology in anterior horn cells of spinal cord (Kim et al, 2001). Neurofilament proteins appears to be the preferred target of 1,2-DAB, since neurofilament accumulates in the axonal swellings, thereby causing hind limb weakness. Biochemical studies have shown that 1,2-DAB reacts with epsilon amino groups of lysine moieties of proteins to form isoindoles. 1,2-DAB also forms chromogenic high molecular weight adducts when incubated with pure bovine serum albumine. However, when glutathione (GSH) is added to the mixture, 1,2-DAB does not produce chromogenic high molecular weight adducts. Therefore, it is believed 1,2-DAB is more reactive towards GSH, and treatment with N-acetyl-L-cysteine, a precursor of intracellular GSH, may act as an inhibitor to 1,2-DAB toxicity. This study shows that boosting GSH levels delays the onset and degree of axonal pathology and shows that the GSH precursor NAC plays a role in inhibiting 1,2-DAB toxicity. Although the mechanism of 1,2-DAB toxicity is not fully understood, I hypothesize that GSH plays a role in 1,2-DAB detoxification. This study was funded in part by an HHMI Undergraduate Research Program grant.

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One in the Hand or Two in the Bush? Worldwide Bird Introductions
Have Favored Species Using Human-modified Habitats

Laurel Kruse Black

(Advisor: Yezerinac)

Invasion biology seeks to interpret past introductions to predict the successes of future introductions and to find general patterns of introduction success. Introductions of birds are much more fully described than those of other taxa, and as such are well-suited to such analyses. This study investigates the effects of human-modified habitat use on bird introductions at three levels: introduction success, introduction effort, and introduction probability. I used a global dataset of all known bird introductions, including total number of introductions and number of successful introductions for each species transported outside of its native range. Results were significant for all levels investigated: human-modified habitat use increased introduction success and number of introductions, and the habitat use distribution of introduced and non-introduced birds differed significantly. Overall these results demonstrate that anthropogenic effects on introductions and invasions are far-reaching.

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Toward Understanding Ler-DNA Binding in vitro

Anna Laura Brown

(Advisors: Glasfeld/Mellies)

Enteropathogenic Escherichia coli (EPEC) is the leading cause of diarrheal death in infants in developing countries. The genes that allow EPEC to differ from non-virulent forms of E. coli are located in a 36kb pathogenicity island, termed locus of enterocyte effacement (LEE). The LEE is negatively regulated by a prevalent nonspecific DNA binding protein called H-NS, and is positively regulated by LEE-encoded regulator (Ler). Ler and H-NS share a 17% sequence conservation over the whole protein, and a 39% sequence conservation over the DNA binding domain identified in H-NS. H-NS binds intrinsically curved DNA and is estimated to regulate between 5 and 30% of all genes in E. coli. We hypothesize that both Ler and H-NS bind to the minor groove of DNA, and to test this hypothesis a number of competitive binding assays were developed utilizing a fragment of DNA known to bind these two proteins. Using fluorescence anisotropy and agarose gel shifts, we were able to show that Ler and H-NS will bind to dI-dC significantly more than dG-dC indicating that an alteration in the minor groove is able to select for protein binding. The minor groove binding drug distamycin was able to disrupt Ler and H-NS binding to a fragment of LEE DNA using fluorescence anisotropy, and netropsin was also able to disrupt Ler binding using agarose gel shifts. A suitable major groove-binding drug as a control was unable to be located. These results support the hypothesis that Ler binds to the minor groove, however, further experimentation is required.

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Processing of GFP-labeled Prohormone ELH in Aplysia Bag Cells

Julie Vanessa Corbett

(Advisor: Arch)

Aplysia californica bag cells produce proELH, a prohormone processed in the secretory pathway to produce nine different peptides including the carboxy-terminal, biologically active egg-laying hormone (ELH) and so-called acidic peptide (AP). These two peptides have been shown to secrete in different stoichiometric ratios when exposed to different secretagogues despite concurrent processing. This suggests that bag cells may have a mechanism by which to sort the peptides for unequal release, either by trafficking the peptides into distinct vesicles or by controlling the secretion at the point of exocytosis.

Surrogate cells have been investigated to address this issue and bypass the problems associated with primary cell culture. Herman (2002) constructed DNA plasmids for an ELH-EGFP fusion protein. This plasmid was subsequently transfected into PC12 cells (Herman, 2002; Phillips, 2003) and INS-1 cells (Tinker, 2003). It was found that PC12 cells are not a suitable surrogate as they do not possess all the required endoproteolytic enzymes for correct proELH processing. INS-1 cells may be a proper surrogate but more analysis is required. This thesis investigates the prohormone processing of the ELH-EGFP construct in Aplysia californica bag cells. Aplysia cells contain the appropriate proteolytic enzymes to cleave the fusion protein, and correctly, sized intermediates were found using anti-GFP western blots. There is the possibility of an additional cleavage of EGFP from ELH. The plasmid was composed so as not to contain any known cleavage sequence between ELH and EGFP. Cleaved EGFP is unexpected and the construction of the plasmid may need to be revisited. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Searching for PC1: Evidence of Kiss-and-Run Exocytosis
in Aplysia californica Bag Cells

Samantha Ruth Haberman

(Advisor: Arch)

Over the past decade, there has been an increasing interest in the occurrence of kiss-and-run exocytosis in synaptic terminals. This alternative form of exocytosis may be a mechanism behind neural cell plasticity and a process that contributes to learning and memory. Exocytosis can be studied in model systems such as the bag cells of the sea hare, Aplysia californica.A. californica creates a neuroendocrine peptide called the egg laying hormone (ELH) that provokes egg laying behavior upon secretion from the bag cells. ELH is initially formed in a large precursor that gives rise to other potentially active peptides, including acidic peptide (AP). During the last steps of proteolysis, ELH is cleaved from AP by the enzyme PC1, but the intracellular location of this cleavage is unknown. Either the proteins are cleaved in the trans Golgi network and packaged into vesicles or the precursor and PC1 are co-packaged and cleaved in the vesicles. If the ELH prohormone and PC1 are packaged into the same vesicle, one would expect ELH and AP to be released in stoichiometric amounts. However, results from earlier studies show that under some conditions, AP is released in larger quantities than ELH. If PC1 is found in the vesicles, it can be assumed that cleavage of ELH from AP occurs in the vesicles and the preferential release of AP may be accomplished by kiss-and-run exocytosis. In this study, Aplysia californica bag cells were analyzed for the presence of PC1 within vesicle fractions and the cell secretion to identify where PC1 resides. The strong presence of PC1 identified in the presynaptic terminals coupled with the fact that PC1 is secreted from the cells suggests that PC1 is in the vesicle and ELH and AP are cleaved in the vesicles, supporting the hypothesis that bag cells undergo kiss-and-run exocytosis. To confirm these findings, isolation of the vesicle fraction was attempted. While unsuccessful, the attempt yielded useful methodologic insights into effectively improving the yield of intact vesicles. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Detection of Antioxidant Gene Expression in Soybean Root Nodules Using Real-Time PCR

Jack Robert Kidney

(Advisor: Dalton)

The production of reactive oxygen species is a common theme in many important cellular processes found in plants. ROS can damage cells in a variety of ways including triggering cell death. Life forms employ antioxidants, specialized proteins and molecules, to detoxify unwanted ROS. Antioxidants play a particularly important role in plant nitrogen fixation. ROS has been implicated in both the natural and stress induced senescence (death) of nitrogen-fixing legume root nodules. Quantitative reverse-transcription PCR using real-time PCR is a fairly new and powerful technique for determining the quantity of specific segments of RNA within unpurified RNA extracts. Q-rtPCR can be used to determine gene expression by measuring the amount of a gene's target RNA transcript within a solution of RNA. In this study, q-rtPCR was used to determine the gene expression of two antioxidant genes in soybean (Glycine max) root nodules over the course of nodule life. The genes examined coded for cytosolic ascorbate peroxidase and cytosolic copper-zinc superoxide dismutase. The essential nodule gene coding for leghemoglobin A was also quantified for comparison, and as an indicator of nodule health. The expression of all three genes varied less than expected and none showed the expected drop in gene expression for the oldest nodules. The limitations of q-rtPCR are discussed, along with interesting results which may indicate that the type of ascorbate peroxidase expressed changed, even as its total expression stayed constant. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Estrogen Modulation of the Immune System: An Amphibian Model

Heidi Jean Reich

(Advisor: Ruben)

The regulatory actions of estrogen in the immune system have been studied for decades, but the diversity of estrogen's targets combined with the variability of its effects have hindered a comprehensive understanding of the role of this hormone in the immune system. In this paper, a new model is introduced for studying the immunomodulatory effects of estrogen. This is the first report of estrogen regulation of the immune system in Xenopus laevis, the South African clawed toad. Estrogens effects on Xenopus thymocytes and splenocytes were measured using hemagglutination to measure antibody production, staining with Annexin V and propidium iodide to measure apoptosis, BrdU assays to measure DNA synthesis, and staining with fluorescent, membrane-impermeable estrogen to measure estrogen binding at the plasma membrane. Estrogen appears to regulate Xenopus immunocytes in the thymus and spleen by enhancing antibody production, reducing apoptosis, and possibly through binding to plasma membrane receptors. Exploration of estrogen's regulation of the immune system in Xenopus offers information that may clarify the role of estrogen as an immunomodulator. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Effects of a Distal Viral Sequence and of an Overexpressed Host Gene on Barley
Yellow Dwarf
Virus Stop Codon Readthrough in Yeast

Iliyana V. Skorcheva

(Advisor: Russell)

Programmed stop codon readthrough is an unconventional mechanism by which ribosomes are directed to continue in-frame translation while bypassing the translational termination signal at a certain rate. This strategy is utilized by both plant and animal RNA viruses. Barley yellow dwarf virus (BYDV) is a positive-sense plant pathogenic RNA virus that uses programmed stop codon readthrough for the translation of an extended version of its coat protein. A previous investigation had characterized the BYDV sequences that program stop codon readthrough rate in plants, and had identified a proximal sequence immediately downstream, and a distal element (DE) 700 bases downstream of the leaky stop codon. The proximal sequence had been shown to have the same effect in the model host Saccharomyces cerevisiae. This investigation sought to test the hypothesis that the distal element will have the same effect on readthrough rate in yeast, as it does in plants. Readthrough efficiency by BYDV was compared in the presence and absence of the DE by using dual luciferase reporter plasmids. Quantification of the activities of the luciferase reporters gave evidence of a four-fold increase in readthrough rate when the DE was present. This finding supported the adequacy of yeast as a host organism for studies of BYDV gene expression. The benefit of using yeast for such research is it is tractable for genetic and molecular analyses and offers a powerful means of identifying host factors involved in a wide range of biological phenomena. Host factors are presumed to be involved in the stop codon readthrough event, because viruses are entirely dependent on their hosts for translation. An investigation by other researchers identified a number of host genes, the overexpression of which increased readthrough of a Tobacco mosaic virus (TMV) leaky stop codon. One such gene is STU2, which encodes a microtubule-binding protein. Another hypothesis of the current study was that overexpression of STU2 will result in increased readthrough efficiency by BYDV. Data from dual luciferase assays indicated a possible five-fold increase in readthrough rates. Further research using the dual luciferase reporter constructs is needed to prove that the product of STU2 affects readthrough in general, not just specifically in a TMV sequence context. This study was funded in part by a HHMI Undergraduate Research Program grant.

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Inhibit This! An Attempt at TRF1 Knockdown in Xenopus laevis

Tristan Michael Victoroff

(Advisor: Shampay)

Telomeres are nucleoprotein structures at the ends of chromosomes. They are fundamentally involved in essential cellular processes including DNA replication, programmed cell senescence, and overall maintenance of chromosome stability. Telomeres consist of short, tandemly repeated DNA sequences and associated proteins. TRF1 has emerged as one of the primary telomere regulatory proteins in vertebrates. In mammals, TRF1 controls telomere length and mediates interactions between telomeric DNA and the numerous other proteins present at telomeres. The human TRF1 protein has been extensively characterized in cultured cells. In vivo studies are largely lacking however, and geneticists will benefit from the expansion of TRF1 research to the African clawed frog, Xenopus laevis. This study represents part of a larger effort to develop Xenopus as a workable genetic system for studying telomere dynamics in vivo. Here I present an attempt to perturb TRF1 expression in Xenopus using short interfering RNA (siRNA). The principal aim of this investigation was to assess the function of the Xenopus TRF1 protein in the context of telomere length regulation. A more general goal was to establish a functional assay for studying Xenopus telomeric proteins using a short interfering RNA-based approach. Xenopus embryos were injected with siRNAs designed to interrupt TRF1 expression at the post transcriptional level, and TRF1 gene expression was examined using RT-PCR. A fluorescent dye tracer confirmed the successful delivery of siRNA to the embryos. RNA was extracted from individual embryos, and was successfully converted into cDNA. Variations in amplifiability of the cDNA precluded a conclusion about the effectiveness of transcript suppression, especially at earlier developmental stages. Nonetheless, the viability of RNA interference for studying gene function in an undergraduate research setting was confirmed. Strategies for improving the siRNA knockdown technique are discussed.

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