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Research

Research at Juniata

Patrick Harris looked at the soil chemistry affecting the American Chestnut Orchard in Raystown

Faculty Research Initiatives and Projects

Dr. Douglas Glazier. Ecology of freshwater springs, including effects of temperature and fish predation on the physiology, behavior, and life histories of amphipod and isopod crustaceans. Theory of biological scaling (how various traits and processes vary with body size in organisms). The ecological consequences of colonizing land by aquatic ancestors, as revealed by comparative studies of aquatic, semi-terrestrial, and terrestrial amphipods and isopods in various regions of the world. http://jcsites.juniata.edu/faculty/glazier/

Dr. John Matter. Life-history and demography of squamate reptiles (lizards and snakes); reproductive biology and effects of pesticides on reproductive development in vertebrates. http://jcsites.juniata.edu/faculty/matter/

Dr. Vince Buonaccorsi. GCAT-SEEK, coordinator. Incorporating massively-paralleled sequencing technology into the undergraduate curriculum.

Dr. Norris Muth. Research on invasive plants, plant pests, and plant pathogens. Citizen science programs for early detection and rapid response to invasive species. Long term ecological monitoring (Ecological Research as Education Network).

Dr. Chris Grant. Mercury (Hg) bioaccumulation in Salvelinus fontinallis (brook trout) and Hg cycling in aquatic ecosystems. Genetically divergent lineages of Pennsylvania fishes: the Juniata Redbelly Dace.

Dr. Jill Keeney. Yeast as a model system for the study of DNA mobility (retrotransposition).

Dr. Gina Lamendella. “Using High-throughput Illumina Sequencing to measure the microbial community response to fracking in aquatic ecosystems” Collaboration with Environmental Protection Agency, and Dr. Christopher Grant (Juniata College); “Temporal analysis of gut microbial community dynamics within an Inflammatory Bowel Disease Cohort”  Collaboration with Lawrence Berkeley National Laboratory and New England Bio Labs; “Genome sequencing of hydrocarbon degrading bacteria isolated from biotrap amended beads in the Gulf of Mexico” Externally funded by DOE: Joint Genome Institute; “Impact of Resistant Starch Diets on Gut Microbial Community dynamics" Externally funded by General Mills Inc and Ingredion LLC

Student Research Experiences, Summer 2012

Kathryn Brown, 2014. Kate Brown at Iowa
The University of Iowa.  Dr. Steven J McElroy.
The effects of Paneth cell disruption on the immature small intestine.
      Necrotizing enterocolitis (NEC) is a severe disease of the small intestine that affects 7% of infants with a birth weight of 1500g or less, and has a mortality rate of almost 40%.  While the pathophysiology of this disease is not completely understood, past studies have indicated diminished, or an absence of, Paneth cell function as a determining factor in the contraction of NEC.  Paneth cells are located in the crypts of the small intestine and are important in immune response, microbiota maintenance, mucosal development, and maintenance of the small intestinal stem cells.  The McElroy lab is investigating many different aspects of the Paneth cell-NEC relationship; my project centered on the effects of Paneth cell ablation on the intestinal stem cells.  More information can be found at the McElroy lab website (http://www.medicine.uiowa.edu/mcelroylab/) or my blog from the summer (http://katesiowasummer.blogspot.com/)!

Clay Cooper 2013
Penn State Hershey Medical Center Department of Emergency Medicine, Dr. Thomas Terndrup
Effectiveness of an Internal, Computer-Based Chest Compression Simulator on CPR Training: A Longitudinal Study
     I worked with 11 medical students in Penn State Hershey College of Medicine’s Emergency Department Summer Research Program. My project was a one-year follow-up study to determine retention of a new method of teaching CPR using the mannequins in the Simulation Center. We retested over 100 nurses, EMTs, and Emergency Medicine residents who participated last year, using compression rate, depth, recoil, and hand placement as determining factors. The second year medical students will be retested in October, and we hope to submit for publication before Winter break.

Ashley Greenawalt, 2013
University of Maryland, Baltimore. Paul Wilder, Ph.D.
Developing an in-vivo assay for Hoxb13 in an ER positive breast cancer cell line
   
My project was to develop an assay and collect data for a PO1 grant that was investigating the protein Hoxb13. Over-expression of Hoxb13 is found in estrogen-receptor positive (ER+) breast cancer with aromatase-inhibitor resistance (AI-R), which later develop Tamoxifen (the current drug treatment) resistance. It is also suspected that after cancerous cells become AI-R, Hoxb13 is an upregulator in metastatic breast cancers.  I was collecting preliminary data on potential “hits” that would selectively kill cells expressing Hoxb13 in a ductal breast cancer cell line. I used a BIOFX robot to add drug compounds from several libraries in a serial dilution. SYBR green was used to analyze cell growth and determine appropriate “hits” which were included in the grant.

Alyssa Grube, 2014Alyssa Grube summer research 2012
University of North Carolina School of Medicine, Chapel Hill. Rita Tamayo, PhD.
“Regulation of the colonization factor GbpA by a cyclic-di-GMP sensing riboswitch in Vibrio cholerae.”
     Vibrio cholerae
is a Gram-negative bacterium that causes the diarrheal disease cholera. V. cholerae is found naturally in the aquatic environment, often attached to surfaces containing chitin, a polymer of the sugar N-acetylglucosamine. It can also colonize the gastrointestinal (GI) tract by attaching to epithelial cells of the small intestine. Integral to the attachment of V. cholerae to both chitin and the intestinal epithelium is an N-acetylglucosamine binding protein, GbpA. Because of the importance to GbpA to the lifecycle of V. cholera, we are interested in how the bacterium regulates GbpA production. GbpA may be regulated by Vc1, a predicted cyclic-di-GMP sensing riboswitch in the 5’ untranslated region of the gbpA transcript. Riboswitches are regulatory elements that, when bound by a specific ligand, undergo a conformational change that affects transcription or translation of the downstream gene. Our lab has previously shown that Vc1 RNA binds cyclic-di-GMP in vitro. This suggests a potential regulatory pathway in which cyclic-di-GMP directly controls expression of GbpA via Vc1. However, it is unclear whether the interaction positively or negatively regulates gbpA expression. Therefore, we sought to elucidate the regulatory role of cyclic-di-GMP and Vc1 in regulating GbpA. We mutated nucleotides in Vc1 that are predicted to be important for sensing or responding to c-di-GMP, then assessed GbpA production by western blot. Because mutations in Vc1 resulted in reduced GbpA levels, the Vc1 ribsoswitch likely functions as an “on switch” in response to c-di-GMP. Unexpectedly, GbpA production in strains with mutated Vc1 was still affected by changes in intracellular c-di-GMP levels. These findings suggest that c-di-GMP regulates GbpA through an additional mechanism distinct from Vc1. These results point to complex control of GbpA during the lifecycle of V. cholerae. Furthermore, our findings add to the understanding of the in vivo function of c-di-GMP sensing riboswitches.

Jessica Scales, 2015
Juniata College.  Dr. Jill Keeney
"Identifying Host Regulators of Ty1 Transposition in Saccharomyces cerevisiae" Jessica Scales at the microscope
   
Ty1, a retrovirus-like retrotransposon, undergoes replication in Saccharomyces cerevisiae by relying on host proteins. The mobile element, similar to the HIV retrovirus, can be assayed by utilizing either an endogenous element or a galactose inducible promoter located on a plasmid (pGTy1). The host gene RTT105 has been identified to regulate Ty1 retrotransposition in S. cerevisiae, promoting Ty1 mobility in the galactose-induced assay, but inhibiting endogenous Ty1 mobility. Another host gene, Ere2 (RTT10), has been found to share a similar phenotype to that of RTT105, as determined through a transposition assay. The novel WD40-domain protein is responsible for endosomal recycling within the retromer pathway. Prior studies suggest a link between Ere2 and Ty1 transposition. With the use of deconvolution fluorescent microscopy, examination of Ty1 Gag-foci in ere2 cells reveals that in the absence of Ere2, Ty1 Gag-foci form congregations towards the plasma membrane and linear patterns across the cell. Deletion of rtt105 negatively impacts the quantity of foci per cell, but not location. Fluorescently tagged TyA does not colocalize with Ere2. Construction of a double gene deletion to assay for Ty1 mobility resulted in increased regulation of Ty1 mobility, suggesting a possible relationship between RTT105 and Ere2.

Keiko Sing , Dec 2012
University of California, San Francisco: Dr. Śaunak Sen
Finding a Candidate Gene for Agenesis of the Corpus Callosum in a Cross between I/LnJ and C5BL/6J Mice Strains.
     Previous research has determined a correlation between agenesis of the corpus callosum (AgCC) and developmental deficits including Autism Spectrum Disorder. My project analyzed genotypes and corpus callosum measurements of a cross between a mouse strain with AgCC, I/LnJ, and strain exhibiting normal corpus callosum development and behavior. After an extensive data cleaning process, phenotypes were analyzed in conjunction with genotypes using R programming language and the R/qtl package. We found a quantitative trait locus for AgCC on chromosome 5 and after comparison with a previous backcross, we narrowed the list of candidate genes for AgCC in the I/LnJ strain from 695 to one.

Nathan Smith, Class of 2013 (center in photo)
Massachusetts Institute of Technology. Peter C. Dedon, PhD. Nathan Smith summer 2012 (center)
A Novel Approach to Genome-Wide DNA Damage Mapping
     As of yet, no successful method has been devised for mapping DNA damage across whole genomes.  Mapping DNA damage is an incredibly complex task that requires researchers to overcome many challenging obstacles, most notably identifying and tagging sites of damage without inducing artificial damage.  I helped develop a method utilizing both wild type and mutant endonucleases that convert AP sites or single-stranded nicks into double-stranded breaks in conjunction with traditional deep sequencing methods and high-throughput Illumina sequencing.  This method was analyzed via plasmid and oligonucleotide models. 

Steven Strutt 2013
The Pennsylvania State University, Ed Dudley Ph.D.
Does spacer acquisition occur in Salmonella?
     CRISPR spacer acquisition in Salmonella enterica, much like E. coli, has never been shown in a natural system. We thus examined the possibility of environmentally active bacteriophage to induce acquisition. We isolated and purified Salmonella-specific bacteriophage from swine and poultry environmental samples. After characterization, these phage were used to challenge the CRISPR system in S. enterica for spacer acquisition.

Aaron Womer 2014
Juniata Colege, Jill Keeney, PhD
Retrotransposition in yeast
      I work with Saccharomyces cerevisiae, studying a gene called Rtt105 gene.  Rtt105 is a gene of unknown function which has been shown to be linked to the regulation of the activity of the Ty1 retrotransposon, which is a DNA element which operates like a retrovirus with the inability to infect other cells, copying and inserting itself inside the genome of single cells.  My work so far has focused on isolating Rtt105 ortholog genes in four other non-Saccharomyces yeast species.  I copy these genes using PCR and then ligate them to plasmid DNA so they can be replicated in E. coli and sequenced using the Sanger sequencing method.  After I know the sequence of a new homologous gene, I transform that gene into a strain of S. cerevisiae without the Rtt105 gene so that I can determine its power of regulating Ty1 activity and compare its functionality to the S. cerevisiae Rtt105.  By comparing the gene sequences of Rtt105 homologs, my lab mates and I hope to determine putative active residues in the protein.  In the future, site-directed mutagenesis of these important residues may help us find the RTT105 protein’s active site and help us begin to understand the mechanism of the protein’s function.

Samantha Buckley 2014
University of Maryland School of Medicine in Baltimore City.  Dr. Wilson and Juniata alumni, Christina Ross.
Gene expression in cancer cell lines
      The overall interest of the Wilson lab included measuring transcriptional activity and gene expression through mRNA regulation (mostly degradation) and the effects of this on the protein levels.  I worked with Christina and her project involving two breast cancer cell lines, MCF7 and MDA-MB-231, and the transcription factor Tristetraprolin (TTP).  The two aims of the project included learning more about the affects of TTP on the two cell lines and the mechanisms by which TTP is suppressed in the cells.  For the first aim, the effects of TTP, the MDA cells (don’t naturally produce TTP) had a FLAG-TTP vector inserted into some cells and the protein levels of certain cell cycle regulators were observed through western blotting procedures.  For the second aim, the mechanisms by which TTP is suppressed, Luciferase assays were used to predict transcriptional activity in both cell lines of the TTP gene and amounts of MAPK proteins were observed through western blots.  Through out the summer I was able to maintain cells using sterile techniques and follow protocols involving western blots, cloning, transfections, and luciferase assays.

Nathan Wilson, 2013
Juniata College. Dr. Jill Keeney
Characterization of Functional Domains of RTT105
      The goal of my research this summer was to find functional domains of a protein that is encoded by a non-essential gene in S. cerevisiae, RTT105. This gene was found as a regulator of Ty1 mobility (Ty1 being the most common of transposition events in yeast). RTT105 encodes for a relatively short protein of 209 amino acids. To characterize the functional domains of the protein, I have been assaying transposition of randomized mutants and site-directed mutants. Based off of this information, I then created more site-directed mutants and am assaying their effect on transposition now. Successful identification of functional domains may lead to finding interacting proteins or specific amino acid sequences that regulate Ty1 mobility. 

Clarissa Diniz, 2014
Univeristy of Pennsylvania. Dr. Jonathan Katz
      I was part of the Undergraduate Student Scholars Program (USSP) in the NIH Center for Molecular Studies in Digestive and Liver Diseases at the University of Pennsylvania. My research was to identify the role of epithelial IKKβ signaling in the local esophageal microenvironment and in epithelial-stromal interactions in the esophagus. I worked with the mice model EBV-ED-L2/Cre; IKKβL/L . They were sacrificed one, three, six, and twelve months of age, their esophagus removed and analyzed. I was able to perform several staining techniques such as b
romodeoxyuridine (BrdU), CD-31, CD-45, and alpha smooth muscle actin in the esophageal samples. My analysis showed how the loss of IKKβ in the esophageal epithelial causes an increase in cellular proliferation, a change in the vasculature architecture, and an expansion of myofibroblasts.

Ryan Trexler, 2013
Juniata College. Dr. Regina Lamendella and Dr. Chris GrantRyan Trexler water sampling
The response of freshwater aquatic microbial communities to Marcellus Shale natural gas extraction
     
Recently, natural gas extraction has dramatically increased in Western Pennsylvania. Minimal research has been conducted in regards to studying the impact of natural gas drilling on microbial community structures of freshwater ecosystems. During the summer of 2012, various microbial samples were collected from headwater streams found in watersheds of Marcellus shale natural gas wells. We employed the power of Next-generation sequencing and the 16S rRNA gene in order to study potential shifts in the structure of microbial communities. In aim to determine potential community changes, this fall we will analyze the sequence data using various bioinformatics and statistical tools.

Alex Sickler 2014
Juniata College. Vincent Buonaccorsi, PhD.
Genome Annotation of Sebastes rubrivinctus and Sebastes nigrocinctus
     Two Pacific Rockfish (Sebastes rubrivinctus and Sebastes nigrocinctus) were sequenced using next-generation sequencing methods. I worked with a group of collaborators from the University of Southern California to annotate both genomes. The main reason for sequencing and annotating both genomes was to analyze the phenotype of negligible senescence (lack of old aging) in S. nigrocinctus which can live up to 160 years and to compare S. nigrocinctus to S. rubrivinctus to determine any differences that may account for negligible senescence. My specific role was to install and test the software used for genome annotation and train the software so it would be optimized for use in Sebastes rockfish. After the software was installed and optimized, both genomes were annotated.

Alexander Weimer, 2013
Juniata College, Dr. Chris Grant
Assessing Biodiversity and Environmental Mercury Accumulation in Remote Streams Associated with Marcellus Shale Development
      This past summer I worked with Dr. Grant and five other students to assess any impacts of Marcellus Shale natural gas extraction (i.e. fracking) on localized mercury levels. It is well documented that operations such as clear cutting for the installation of a well pad can increase mercury levels in aquatic ecosystems. However, to date no research has been done detailing the effects of frack water contamination on mercury bioaccumulation and biomagnification in stream ecosystems. In order to assess these ecological interactions on mercury levels, fish were sampled via standard electrofishing protocols and analyzed for mercury concentrations in muscle tissue. While this methodology was highly useful for assessing current levels of impact, electrofishing proved to be an inefficient way to address our project's secondary goal: the identification of wild populations of brook trout (Salvenlinus fontinalis) and brown trout (Salmo trutta) as a means to reclassify streams for protection. I was given the opportunity to explore novel detection methods of aquatic organisms as a project that would add value to the overall goals of the research. I settled upon developing a methodology for collecting free floating DNA from the water column (eDNA) and using that as a template for species specific polymerase chain reactions (PCR) to positively identify what species are present in a body of water. The cytochrome oxidase region of the mitochondrial genome was chosen as the target for amplification as its intrinsic rate of mutation is slow enough to design highly specific primers. This project was so fascinating that I have decided to pursue this topic for my senior thesis. I have done a lot of research in my four years at Juniata, but this summer will be one I will remember for the rest of my life.

Nicole Marks, 2014
Juniata College.  Dr. Chris Grant.
The impact of crayfish on the biomagnification of mercury across trophic levels of stream ecosystems.Nicole Marks in lab
      The purpose of my research with Dr. Grant this summer was to gain a better understanding about the controlling factors of mercury cycling and biomagnification within headwater stream ecosystems.  Furthermore, we were interested in learning about any potential impacts of Marcellus shale on bioaccumulation and biomagnification of mercury.   This summer we visited 22 remote forested streams in northwestern Pennsylvania experiencing Marcellus shale drilling activities within their watersheds.    My research focus was on crayfish collection in order to determine the role they play in the mercury cycle of headwater stream ecosystems, with a special interest in the trophic transfer of mercury to top predatory fish (e.g. Salvelinus fontinalis).  From the 22 streams, 3-5 crayfish were collected in 3 size classes to total about 90 crayfish.  The crayfish were collected along with sediment, moss, other macroinvertebrates, and trout over the summer in order to be analyzed for mercury concentration by the Direct Mercury Analyzer-80 (DMA-80).  The results of these analyses are being used to assess the trophic transfer of mercury in streams.  Additionally, species identification of the crayfish and individual-specific measures including weight, total body length, carapace length and width, tail length, and chela length and width will be used in order to determine if any of these characteristics are controls on mercury concentrations in crayfish.

Gabrielle Cannon, 2014
Juniata College, Dr. Norris Muth
Assessment of plant community restoration following Tree-of-Heaven (Ailanthus altissima) control by Verticillium albo-atrum
      Biological control of the highly invasive Tree-of-Heaven (Ailanthus altissima) by the fungus Verticillium albo-atrum resulted in 100% mortality in a small Pennsylvanian study conducted by Don Davis and colleagues (2009). I worked with Dr. Norris Muth on a research project assessing the revegetation of the Davis study sites following Ailanthus mortality. I conducted and synthesized background research as well as participated in field data collection. We visited the Davis study sites and nearby, non-infected control Ailanthus stands to measure percent cover of both native and invasive grasses, forbs, shrubs, vines and tree recruits for each site. We found the vegetation composition of control and infected Ailanthus stands was statistically indistinguishable and that site size and shape had no discernible impact on the success of community restoration. This information is useful from a land management perspective, as landowners who wish to selectively remove Ailanthus may use V. albo-atrum with great success, but those who desire a more general elimination of invasive species may prefer other methods. The study has been submitted for publication as a Note in the journal Biological Invasions.

Jacob Oster, 2015
Juniata College, Dr. Chris GrantJacob Oster collecting stream samples
      My research with Dr. Grant involved a variety of field collection techniques for gathering samples across various trophic levels in forested stream ecosystems. Methylated mercury is a potent neurotoxin and endocrine disrupter with the potential for biomagnification in food webs. Marcellus Shale drilling has the potential to influence the concentration of methylmercury in remote forested stream ecosystems.  These impacts are of particular concern in Northwestern Pennsylvania as they raise concerns for the safety of consumption of game fishes, such as brook trout (Salvelinus fontinalis) Our research focused on the bioaccumulation and biomagnification of methylmercury in these aquatic ecosystems. More specifically, my ongoing individual project focuses on identifying macroinvertebrate species collected this past summer and studying species diversity and biomagnification of mercury within the different trophic levels of macroinvertebrates. Macroinvertebrates were identified to the family or genus level using taxonomic features.  Further, feeding strategy was used to determine functional groups and trophic position. Once the samples were identified, macroinvertebrates were freeze-dried, homogenized, and prepped for total and methylmercury analysis.  Total mercury concentrations were determined using a Milestone DMA 80  with atomic absorption spectrophotometry.  Methyl mercury concentrations were determined at the U.S. Geological Survey Mercury Lab using cold vapor atomic fluorescence spectrometry.  I am hopeful this research will lead to a better understanding of the mechanism by which macroinvertebrates metabolize methylmercury, and differences in bioaccumulation among functional groups and trophic position.

Mitchell Dunklebarger, 2015
Juniata College. Dr. Regina Lamendella
The
aim of my research is to learn more about how dysbiosis of the gut microbial community influences the etiology of Crohn’s disease. To observe changes in the gut microbial community of Crohn’s patients over time, we are using high throughput sequencing  to analyze  gut microbial communities. By performing a longitudinal study of changes in gut microbial communities at these points as compared to when the patients felt “healthy” , we hope to identify potential biomarkers for the onset of the disease and learn more about it’s complex etiology.

Elliott Perow, 2014
U.S. Fish and Wildlife Service/Juniata College, Dr. Christopher Grant and Mark Roberts
Stream restoration to increase habitat for native fishes
       
Native Pennsylvania fish such as Salvelinus fontinalis (brook trout) are negatively impacted by a reduction of habitat and non-point source pollution as a result of anthropogenic activities.  This past summer I worked for Chris Grant and Mark Roberts as part of an ongoing 4-year elliot perow stream nettingcollaboration (between Juniata College and the U.S. Fish and Wildlife Service) that is seeking to protect the long-term sustainability of native Pennsylvania fishes, such as the brook trout.  This collaboration seeks to identify streams in need of restoration, assess current stream conditions (e.g. macroinvertebrate and fish biodiversity), identify causes of decreased biodiversity, design and implement stream/watershed restoration plans, and monitor stream recovery/reintroductions of fishes.  I helped with multiple aspects of this work including identifying potential restoration sites, assessing biodiversity, implementing restoration plans, and documenting conditions post restoration.  When implementing stream restoration plans, I assisted in building in-stream structures such as log vanes, rock vanes, mudsills, and grade logs, which decreases erosion, and in-stream sedimentation, while optimizing fish habitat.  It is very rewarding to go back to the streams and see native fish using the structures that were built, and knowing that I had a positive impact on their survival feels great.  I am hopeful that the continuation of building in-stream structures and identifying the causes of decreased biodiversity will not only bring about more efficient and effective ways of protecting the sustainability of native fishes, but will also raise awareness among the public.    

 

 

Liberal Arts Symposium Research Presentations by Biology Students Spring 2012

http://www.juniata.edu/academics/research/symposium.html

Begley, Matthew (Hollidaysburg, Pa) [Jill Keeney]
     Kinetic Analysis and Structural Elucidation of MBD4
Bowman, Caitlyn (Seven Valleys, PA) [Dr. Vince Buonaccorsi]
     Sequencing and de novo assembly of the gopher rockfish transcriptome
Engle, Elyzabeth (Millheim, PA) R(P) [Dr. Norris Muth]
     Collection of Expert Knowledge on Productive Seed Cultivars Used on Small Farms in Huntingdon and Centre Counties
Greenawalt, Ashley (Clarion, Pa) [Dr. Jill Keeney]
     Colocalization with Fluorescence Microscopy of Ty1 Mobility in Saccharomyces cerevisiae
Gring, Jeffrey (Mechanicsburg, PA) [Dr. Douglas Glazier]
     The effects of temperature and body size on the metabolic rate of the freshwater amphipod Gammarus minus, a     comparison of two models
Grube, Alyssa (Lititz, PA) [Dr. Jill B. Keeney]
     Ty1 Retrotransposon and the Transposition Assay
Hoffman, Casandra (Coplay, PA) [Jill Keeney]
     Stress Granule Induction in Saccharomyces cerevisiae
Kump (State College, PA) [Douglas Glazier]
     The preferential feeding habits of the watercress snail
Leibensperger, Krista (Reading, Pennsylvania) [Chris Grant]
     The Juniata Redbelly Dace: divergent lineages in fish.
Reitman, Maxwell (Huntingdon, PA) [Jill Keeney]

     Characterization of RTT105 Orthologs in Yeast Species Closely Related to Saccharomyces cerevisiae
Smith, Nathan (York, Pennsylvania) [Dr. Jill Keeney]
     The Characterization of RTT105 Orthologs in Yeast Species Closely Related to Saccharomyces cerevisiae
Supenia, Emily () [Jeffrey Demarest]

     Macromolecular Crowding Affects the Diffusion of Calmodulin
Trexler, Ryan (Altoona, Pa) [Dr. Jill Keeney]
     Structural Analysis of S100B-P53 Inhibitors
Wilson, Nathan (Altoona, PA) [Jill Keeney]
     Characterization of Rtt105p: Finding Functional Domains via Transposition Assay
Yip, Christopher (Briarwood, New York) [Michael Boyle]
     Identification of a Putative Ovalbumin Binding Protein

2010-2011 Student Research Projects

Zac Cupler, Class of 2011
Faculty Advisor: Dr. Jill Keeney

Ty1, a retrotransposon element of the yeast Saccharomyces cerevisiae, has a life cycle biochemically similar to mammalian retroviruses.  The host RTT105 gene has been shown to regulate Ty1 transposition, but the host function of Rtt105 is unknown.  Recently, two reports have shown that the Ty1 life cycle involves cytoplasmic granules known as P-bodies.  P bodies are RNA-protein granules that reside in the cytoplasm and play a fundamental role in mRNA control by sequestering mRNA.  This localization potentially functions in a degrading mechanism to recycle the mRNA as well.  When translation is necessary, interaction with stress granules occurs, so that the appropriate mRNAs can be transferred to the stress granule where translation can proceed.
I am currently working to implement fluorescent in situ hybridization (FISH) of Ty1 mRNA to determine if Rtt105 protein and Ty1 mRNA colocalize in yeast cells.   My initial work with FISH will be to detect a fluorescence signal using tagged oligonucleotides complementary to Ty1 mRNA. I will then use fluorescent tagging of RTT105 protein to visualize if co-localization with Ty1 mRNA or P-body protein occurs.  This work will help to better understand the assembly and translational control P bodies and Ty1 mRNA play on retrovirus formation.

Andrew Melber, 2011
Faculty advisor: Dr. Jill Keeney.
Characterization of the allele rtt105::mTn3 in Saccharomyces cerevisiae.  

Our lab is using Saccharomyces cerevisiae as a model organism to investigate genes determined to regulate the Ty1 retrotransposition system.  Specifically I work with understanding the gene RTT105 native function along with how it influences transposition. I have created several mutant strains deleting part or all of RTT105 from the yeast genome which are used in endogenous and over-expression transposition assays. I am also purifying Rtt105 protein, which we will then use for antibody production. With specific antibodies we will be able pursue an investigation of the protein dynamics of Rtt105. My last project is to use immunoprecipitation to identify other yeast proteins that interact with Rtt105; this work is done in conjugation with a mass spectrometry group at Pennsylvania State University, led by Dr. Hasan Koc. My work, along with the work of my lab mates, will hopefully unravel the cellular function of RTT105 and its role in regulation transcription.

Mark Weir, 2011
Faculty Advisor: Dr. Jill Keeney

I am currently doing research on how the yeast Saccharomyces cerevisiae gene, RTT105, affects the process of retrotransposition.  This is a process much like virus replication except that retrotransposition does not lyse the cell; instead the gene is able to copy and paste itself elsewhere else in the genome.  While there are many retroelements in yeast, the one of interest to us is the Ty1 element.  Rtt105 has been shown to be a regulator of Ty1 but not much else is known about it.  I am currently looking for functional domains, parts of the gene that are essential for the protein to function, by random PCR mutagenesis and site-directed point mutations (alanine scanning).  Mutants are assayed for transposition levels and compared to an rtt105 deletion strain (negative control) and a wild type strain (positive control).  The mutants that are chosen for sequencing fall phenotypically in between these two controls.  This will give us an idea into which parts of the protein are most important in its activity.  I am also studying genes similar to RTT105 in other Saccharomyces species.  We have cloned several of these genes, put them back into Saccharomyces cerevisiae, and assayed for retrotransposition efficiency.  To date, all are able to regulate transposition in S. cerevisiae.  I am also studying localization of a RTT105-GFP protein using confocal fluorescent microscopy. I have observed that the RTT105-GFP is a cytoplasmic protein that appears to be in specific punctate loci.  Recently, it has been shown that Ty1 RNA and proteins localize to processing bodies or P-bodies.  These are locations of mRNA decay and translation repression.  We are currently investigating whether RTT105 localizes to P-bodies by co-localizing RTT105-GFP with two fluorescently labeled proteins known to be localized to P-bodies, Dcp2-RFP and Edc3-mcherry.

Alexander Weimer, 2013
Faculty Advisor: Dr. Vincent Buonaccorsi

Using the Sebastes carnatus and S. chrysomelas species pair as the focus of my research, I am currently investigating the molecular mechanisms involved in ecological (sympatric) speciation.  These rockfish are ideal for a study such as mine because they are still in the early stages speciation, which means that areas of divergence between the two species are easily discernable against meager background levels of divergence.  The focal point of my research is to characterize one such island of genomic divergence surrounding the locus Sra. 7-2.  During my summer and semester research, I have been genotyping fifty-four individuals of each species at seven polymorphic microsatellite loci as well as numerous neutral loci for comparison.  From this data, I am able to approximate the time of divergence as well as determine the mode of speciation and whether natural selection is still acting.  At the time of this writing, the Sebastes rockfish genome is being sequenced and annotated in its entirety.  This new wealth of data will provide me with the raw materials to extend my research past one fragment of the genome, and enable me to generalize the mechanisms involved in ecological speciation.