The Science Poster Day 2007 Dean's Prize Winners!
Alex Babayan
Rachel Cohen
Ni Feng
Jun Gong
Kelly Havens
Aria Hong
Jason Kang
Jennifer Kaplan
Kyeong Kim
William Kim
Ashley Koegel
Kunal Mehta
Michelle Palacios
Alexander Patananan
Edward Pham
Sacha Prashad
Dominique Richardson
Michael Rome
Aswin Sekar
Govind Shah
Aida Sun
Darcy Wanger
Michael Zhang
Dagny Zhu
Mr. Alex Babayan
Mentor:
Patricia E. Phelps
Title: Neuronal positioning errors in reeler spinal cord contribute to alterations in nociceptive function
We recently showed that reeler mice have incorrectly positioned neurons in the dorsal horn laminae I-II and that they exhibit profound sensory defects including hyperalgesia and relative insensitivity to mechanical nociception (pain). We reasoned that these sensory alterations in reeler mice may result from anatomical abnormalities in either dorsal root ganglia (DRG) or dorsal horn neurons, since DRG nociceptors conduct noxious information into laminae I-II of the dorsal horn. Nociceptive stimuli induce transcription of c-fos, and subsequent accumulation of Fos protein in the nuclei of stimulated dorsal horn cells. To test the hypothesis that Fos expression is altered in reeler mice, we applied thermal or mechanical stimuli to the left hindpaw of wild-type and reeler mice and subsequently analyzed the number of Fos-immunoreactive nuclei. Following thermal stimulation, reeler mice have more Fos-positive cells in laminae I-II than wild-type mice, an observation that is consistent with reeler mice displaying hyperalgesia. Additionally, we observe fewer Fos-labeled cells in laminae I-II of reeler mice than wild-type mice following mechanical stimulation; consistent with the observed insensitivity of reeler mice to mechanical nociception. In contrast, we found no differences in the size, number, or distribution of the DRG peptidergic and nonpeptidergic nociceptors between wild-type and reeler mice. These findings suggest that positioning defects in the dorsal horn, rather than the DRG, contribute to the alterations in reeler nociceptive processing.
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Ms. Rachel Cohen
Mentor: Daniel T. Blumstein
Title:
The effects of insect noise on the song characteristics of the little greenbul (Andropadus virens)
Noise from insect song constitutes a large proportion of the ambient noise in the environment, which may drive changes in bird song structure. The little greenbul (Andropadus virens) is a common passerine in central African rainforests, and its song consists of four distinct song types. Populations of little greenbuls were recorded in Uganda, and the maximum frequencies of each song type were measured. We correlated a measure of insect song frequency with little greenbul song frequency. We focused on the times of day when low frequency insect song was dominant (8 AM) and when high frequency insect song was dominant (10 AM). We extracted principle components from a variety of remote sensing and bioclimatic data and correlated these with the insect song frequencies. We found a positive correlation between song type 4 and insect song. We also found negative relationships between song types 1, 2, and 3 and insect song. Thus, little greenbul populations may have modified their maximum frequency in order to utilize frequency windows available to them. Furthermore, insect song was positively correlated with habitat and climate data. Thus, insects in different habitats vocalize at different frequencies, which could drive changes in little greenbul song frequencies across populations.
Ms. Ni Feng
Mentor: Barney A. Schlinger
Title: Androgens and muscles in a tropical bird with an elaborate courtship display
Testosterone is responsible for a variety of courtship and aggressive behaviors in temperate bird species, but little is known about its influence on complex courtship displays commonly seen in tropical species. The golden-collared manakin (Manacus vitellinus) of Panamanian rainforests performs a visually and acoustically stimulating display. To attract females, male manakins produce loud firecracker-like sounds generated by the rapid and forceful contraction of their wings. Since these “wingsnaps” are male-specific and require substantial use and coordination of their wings, we hypothesize that wing muscles are androgen sensitive, and that circulating testosterone must be converted to 5α-dihydroxytestosterone by 5α-reductase in order to bind androgen receptors in wing muscles. We quantified 5α- and 5β-reductase activities in the limb muscles of male and female manakins as well as in the zebra finch (Taenopygia guttata), a non-wingsnapping bird. Enzyme activities were measured in the supracoracoideous (SC), a wing muscle previously found to have morphological adaptations for greater force generation in male manakins, and the gluteal (GL), which is not involved in the wingsnap. Although we observed no sex-specific differences, both male and female manakins had significantly higher 5α-reductase activity in the SC than in the GL. Furthermore, male manakin SC had significantly higher 5α-reductase activity than male zebra finch SC. Our results suggest androgens may have direct control over manakin wing muscles to influence wingsnaps in their courtship display.
Mr. Jun Gong
Mentor: Ellen M. Carpenter
Title: Motor neuron loss in the lumbar spinal cord of Hoxc10 mutant mice
Hox genes encode transcription factors that are critical for the organization and development of the vertebrate body plan. In particular, Hox genes are involved in establishing the rostrocaudal patterning of the central nervous system via the generation and specification of neuronal cell types in discrete regions in the developing hindbrain and spinal cord. Our current study addresses the role of the Hoxc10 gene in patterning the motor neurons of the lumbar spinal cord. Hoxc10, along with its paralogs Hoxa10 and Hoxd10, is expressed in the lumbar spinal cord and surrounding mesoderm as well as in the hindlimbs. Hoxc10 knockout mice appear behaviorally normal, but demonstrate a significant loss of motor neurons in the lumbar spinal cord as determined by cell counting studies on hematoxylin and eosin-stained serial tissue sections. The loss of motor neurons is apparent in all motor column divisions, including the medial and lateral motor columns (MMC and LMC), which innervate the axial and appendicular musculature, respectively. A loss of motor neurons is also evident in the medial and lateral components of the LMC, which innervate the ventral and dorsal musculature, respectively. The loss of motor neurons in Hoxc10 mutant mice ranges from 54% to 74%, with an average loss of 64% in Hoxc10 mutant mice. These observations support a role for Hoxc10 in the generation and/or specification of lumbar motor neurons.
Ms. Kelly Havens
Mentor: Abby Kavner
Title: High pressure electrochemical behavior of AgI
In an attempt to understand how pressure influences charge transfer behavior, electrochemical reactions were generated in-situ in the diamond anvil cell. The ionic solid Silver iodide (AgI) was used both because its phase diagram has been extensively studied and because its phase boundaries are visible under the microscope. Electrochemical dissociation and resistance measurements were performed. A diamond anvil cell was loaded with AgI, and electrodes were introduced between the diamonds to allow an electrical current to pass through the sample. Pressure was measured using ruby fluorescence. The resistance was measured using a Fluke multimeter. In a previously published study, AgI was found to dissociate at 4 to 6 GPa to Silver metal and Iodine. In this study, we were able to electrochemically dissociate AgI at lower pressures by applying a voltage (2 to 7 volts) across the electrodes in the diamond cell. Reaction rates were found to be sensitive to pressure, voltage, and phase, with Iodine and Silver metal formation occurring exclusively in different phases. The resistance was found to decrease by more than three orders of magnitude between 4 and 8 GPa. The potential applications of these experiments are both scientifically and industrially important, including understanding charge transfer rates in the mantle and producing more efficient batteries and fuel cells.
Ms. Aria Hong
Mentor: Kent Hill
Title: Determining the function of growth arrest specific 11 (GAS11) and the dynein regulatory complex in Danio rerio
Flagella and cilia play diverse and essential roles in many organisms. Members of the recently identified trypanin family of proteins have been identified in cilia-containing organisms ranging from Giardia lambia to Homo sapiens, but not in organisms that lack a motile cilium such as nematodes and vascular plants. These data suggest that trypanin-family proteins are required for ciliary motility. Two members of this protein family, trypanin from Trypanosoma brucei, and its Chlamydomonas reinhardtii homologue, PF2, are both components of the dynein regulatory complex (DRC). The DRC is an integral component of the flagellar axoneme that regulates flagellar motility in these organisms. The mammalian trypanin homologue, growth arrest specific 11 (GAS11), which localizes to areas requiring dynein regulation in cultured cells, is hypothesized to be a component of the mammalian DRC. Very little is known about GAS11 or the mammalian DRC. Here, we show that a single GAS11 homolog in Danio rerio localizes to areas that require cilia, including the developing ears, the pronephric ducts, and the neural tube. Morpholino antisense oligonucleotide-mediated knockdown of GAS11 in Danio rerio results in phenotypes characteristic of ciliary defects. This result suggests that GAS11 plays a critical role in ciliary function. Gaining a greater understanding of GAS11 function will help elucidate the function of DRC, a vital cellular structure; it may also offer valuable insight into human diseases caused by defective cilia, including Polycystic Kidney Disease (PKD), infertility, situs inversus, and certain types of hearing impairments.
Mr. Jason Kang
Mentor: Genhong Cheng
Title: The structural characterization of TRAF3 in the production of type I interferons
Understanding the way in which the innate immune system recognizes and responds to the presence of virus has been the focus of many recent studies. Type I interferons (IFNs), a family of cytokines, are produced and secreted by mammalian cells upon viral infection. These type I IFNs are created through several different signaling pathways. A great deal of progress has been made in elucidating Toll-like receptor (TLR) pathways and their responses to viral infection; however, the exact mechanisms of other pathways remain unclear. Tumor necrosis factor receptor-associated factor 3 (TRAF3) was identified as a critical molecule required for the induction of type I IFNs in response to viral infection. Nevertheless, the way in which TRAF3 regulates the transcription of type I IFNs remains unclear. The focus of this project was to identify the structural regions of TRAF3 that are required for the production of type I IFNs. Also, the study attempted to characterize the structural features of TRAF3 that distinguish it from other related molecules in the TRAF family, such as TRAF2. The questions were answered by creating a library of chimeric proteins consisting of TRAF3 and related molecules. Knowing the critical regions of TRAF3 will further clarify the pathways used by the immune system to fight viral infection, and may ultimately contribute to the development of vaccines and treatments for viral infection.
Ms. Jennifer Kaplan
Mentor: Patricia E. Phelps
Title: The presence of noradrenergic axons in the lumbar enlargement of paraplegic adult rats does not provide evidence of axon regeneration
The combination of olfactory ensheathing glia (OEG) implantation and treadmill step training shows promise in promoting axon regeneration and functional recovery after a complete spinal cord transection. Our previous study demonstrated that OEG implantation has the capacity to stimulate axonal growth across the lesion site, and that the addition of step training augments kinematic performance. In that study, we noticed noradrenergic axons in the caudal spinal cord of both OEG- and media-injected (control) rats and therefore wondered if they are a valid marker of regeneration. We then questioned where and how these axons enter the spinal cord caudal to the lesion site. In our analysis of the L4-L5 spinal cord level, we determined that equal densities of noradrenergic axons are present in both OEG- and media-injected adult rats. Although a small percentage of these fibers associated with the spinal cord vasculature, the majority traveled independently and densely populated the dorsal horn. We suspect that most of these noradrenergic fibers enter the spinal cord from the meninges and represent sympathetic innervation to the lumbar cord. Thus, we conclude that the presence of noradrenergic axons in the lumbar levels should not be used as a measure of axonal regeneration across the lesion site. Further studies will seek to determine the functional significance of this sympathetic population in the caudal spinal cord.
Ms. Kyeong Kim
Mentor: Ren Sun
Title: Conserved herpesviral kinase plays a critical role in viral persistence by inhibiting IRF-3 mediated type I interferon response
Herpesvirus establishes a persistent infection throughout the lifespan of the host by bypassing the host immune surveillance and causes opportunistic diseases upon malfunction of the host immune system. Therefore, it is essential to elucidate the mechanism of herpesviral immune evasion to control herpesviral diseases. In this study, we used murine gamma-herpesvirus 68 (MHV-68) as an animal model based on its relevance to tumorigenic human gamma-herpesviruses. For the systematic analysis of herpesviral immune evasion mechanism, we screened the previously generated mutant library of MHV-68 for genes that antagonize type I interferons, the first line of host immune defense. Our initial screen identified ORF36, a conserved kinase in all subfamilies of herpesvirus, to be critical for viral growth by inhibiting interferon regulatory factor 3 (IRF-3)-mediated production of interferon beta. In-depth biochemical analysis revealed that ORF 36 localizes to the nucleus, sequesters IRF-3, therefore inhibiting IRF-3 from binding the cellular co-transcriptional activator CBP, and thus preventing the recruitment of RNA Polymerase II to the interferon beta promoter. Inhibition of IRF-3 activity by MHV-68 ORF36 is crucial for viral replication, and virus with dysfunctional ORF36 is significantly attenuated. Therefore, identification of the viral genes responsible for immune bypass, such as ORF36, will contribute to the development of cure against herpesvirus in the future.
Mr. William Kim
Mentor: Utpal Banerjee
Title: Characterization of the role of hypoxia-inducible factor-1a in lineage specification during Drosophila larval hematopoiesis
Hif-1α, a key component of the hypoxia-induced pathway, has been implicated in the maintenance of several types of mammalian stem-cells, including hematopoietic stem-cells. Mis-regulation of this pathway has been implicated with the onset of various blood-related illnesses, such as leukemia. However, due to limitations in in vivo approaches along with the inaccessibility of the mammalian hematopoietic stem-cell niche, the role of the hypoxia-induced pathway and its regulators remains to be fully deciphered. In Drosophila, hematopoiesis parallels mammalian blood-cell development in that several molecular aspects have been evolutionarily conserved, allowing Drosophila to be used as a relevant model system. Recently, the Notch and Hedgehog signaling pathways have been implicated in the maintenance of Drosophila hematopoietc stem-cell-like precursors in the medullary zone, which have a higher level of ROS than the more differentiated cortical zone. I have used gain/loss-of-function strategies to characterize the role of Hif-1α in Drosophila hematopoiesis. Immunohistochemical staining for Similar, the Drosophila homolog of mammalian Hif-1α, in larval lymph glands indicates expression in crystal cells, the Drosophila counterpart of mammalian melanocytes. Reduced Sima function results in fewer crystal cells, which are derived from precursors in the medullary zone. Converse experiments, using gain-of-function alleles of Sima results in the expansion of crystal cell number and loss of medullary zone markers. These preliminary results indicate a novel role for Sima in lineage specification during larval hematopoiesis.
Ms. Ashley Koegel
Mentor:
Dennis Slamon
Title:
Identification of protein interacting partners of H37/RBM5 lung cancer tumor suppressor gene product
One of the first genetic alterations in lung cancer is deletion at chromosome 3p21.3 (>90% of small cell lung cancer and 50-80% of non-small cell lung cancer). Due to its location within the 3p21.3 region, H37/RBM5 is likely a tumor suppressor gene. Using human A549 NSCLC cells transfected with H37, it has been shown that H37 is involved in G1 cell cycle arrest and increases apoptosis, significantly inhibiting human lung cancer growth both in vitro and in vivo. In order to further elucidate the cellular and molecular functions of H37, a primary yeast two-hybrid screening of 16,000 clones from the human testes cDNA library was performed. 75 positive clones were identified by low stringency plating criteria. I am currently validating these potential targets for true interactions by purifying individual “prey” plasmids in E. coli and co-transforming them with H37 “bait” plasmids into yeast cells on the most stringent plating criteria. Utilizing β-galactosidase as a reporter assay, the interaction of the “bait” and “prey” plasmid protein products generates a blue colored yeast colony. Based on subsequent DNA sequencing of positive colonies, protein interactions will be further validated in vitro by co-immunoprecipitation and in-vivo by immunoprecipitation. This research may uncover the H37 TSG mechanism in human lung cancer and ultimately lead to earlier detection in lung cancer as well as the eventual development of novel therapeutics.
Mr. Kunal Mehta
Mentor: Jacob Schmidt
Title: Nucleotide sequencing and orientation detection of single-stranded DNA with a biological nanopore
Recent research has shown the promise of using protein nanopore-based sensors for rapid and sensitive detection of a wide range of analytes. There is significant interest in using these sensors to sequence single molecules of DNA. Although previous work has shown that protein nanopores can discriminate among individual nucleotides in single-stranded DNA, sequencing at single-base resolution has not yet been realized. We are investigating an approach to achieve single-base resolution by trapping the ssDNA in the nanopore between a dsDNA hairpin on one end and either a conjugated magnetic bead or streptavidin molecule on the other and measuring the resulting ionic current when the DNA is driven through the pore. Our preliminary experiments using the pore protein alpha-hemolysin and conjugated magnetic bead have been encouraging; we have measured distinct current magnitudes that may suggest single-molecule detection of adjacent polyhomonucleotide regions 40 bases long (3’–hairpin–A 40C 40–bead–5’). We are also exploring a related phenomenon where the trapped nucleotides appear to tilt toward the 3’- or 5’- end of the DNA strand under the application of an electric field. This behavior suggests the existence of an energy of activation for the orientation transition that can be affected by the applied field. Ultimately, we hope that this work will establish the feasibility of nanopore DNA sequencing and provide insight into the physics of the trapped ssDNA-nanopore assembly.
Ms. Michelle Palacios
Mentor:
Catia Sternini
Title:
Effect of chronic opiate treatment on agonist--induced µ opioid receptor (µOR) trafficking in enteric neurons
Patients suffering from chronic pain often rely on the use of opiate analgesics. However, prolonged opiate treatment induces undesirable side effects including tolerance and severe impairment of gastrointestinal motility. The µ opioid receptor (µOR) is the target of opiates like morphine and fentanyl, which differ in their efficacy to induce µOR endocytosis -- a mechanism regulating cellular responsiveness to opiates. Here we determined the effect of chronic treatment with fentanyl, an efficacious internalizing agonist, or morphine, which has low internalizing efficacy, on agonist-induced µOR trafficking in enteric neurons. Guinea pigs received subcutaneous injections of fentanyl, morphine or saline for 4-7 days. Ileum specimens were exposed to DAMGO (an internalizing ligand) or morphine, and processed for µOR immunohistochemistry. µOR immunoreactivity was observed at the cell surface in unstimulated neurons from each group, and in the cytoplasm of neurons from each group following DAMGO. Morphine caused strong µOR internalization in neurons from animals chronically treated with morphine, but only a small level of internalization in neurons from chronic fentanyl-treated animals. This study shows that chronic activation with morphine but not fentanyl increases morphine ability to induce µOR internalization, but does not affect DAMGO internalizing efficacy. This suggests that opiates differ in their ability to induce intracellular adaptations, which are likely to be the basis for the development of tolerance, a major limitation in their clinical use.
Mr. Alexander Patananan
Mentor: Utpal Banerjee
Title:
A novel cell lineage tracing method in Drosophila melanogaster
For complex organisms to develop, an array of time-dependent genetic events must occur to establish various tissues. To understand the formation of these specific tissues from precursor cell types, cell division patterns controlled by gene expression programs during morphogenesis are studied. Currently, X-gal staining, antibody marking, and in-situ hybridization are among the many methods used to monitor these patterns in Drosophila melanogaster. However, multiple generations are usually required to achieve desired genotypes and immunohistochemistry is often needed to visualize tissues. This proof of principle study describes a novel single cross cell lineage tracing method in Drosophila that allows for the association of specific developmental processes to genes expressed in particular cells. Previously characterized genes were monitored at the third-instar larval stage via a gal-4 enhancer trap system and genetic constructs using upstream activator sequences, flippase, and Flippase Recognition Target sequences. Fluorescence microscopy was used to visualize current and pre-third instar gene expression in the brain, lymph gland, eye-antennal and wing discs, using red (RFP) and green (GFP) fluorescent proteins, respectively. The majority of samples expressed both RFP and GFP with varying degrees of intensity and maintained expression patterns similar to those obtained from other techniques. Overall, this system saves time and resources and has the potential to accelerate the characterization of genes in a comprehensive lineage tracing scheme.
Mr. Edward Pham
Mentor: Daniel T. Kamei
Title:
Engineering transferrin for cancer targeting using kinetic modeling and site-directed mutagenesis
The major route of cellular iron uptake involves an endocytic pathway in which the interaction of iron-bound transferrin (Tf) proteins with cell-surface transferrin receptor 1 (TfR1) complexes causes transferrin to be transported into the cell. Since many cancer cells overexpress TfR1 (because they require more iron for their abnormally rapid proliferation), cytotoxic drugs can be preferentially delivered to cancer cells by conjugating them to Tf. Tf rapidly leaves the cell after it is internalized, however, thereby reducing the time allowed for these drugs to be released in the cell. We used mathematical modeling to identify a design criterion for engineering Tf to increase its time associated with the cell, and found it to be the Tf iron release rate. A modified Tf ligand with a lowered iron release rate was then generated by altering the iron binding sites of Tf. The trafficking properties of wild-type and modified Tf molecules were compared by radiolabeling each ligand with iodine-125, incubating the ligands with HeLa cells, and measuring how much radioactivity was associated with the cells. The amount of modified Tf associated with the cells was 51% greater than the amount of native Tf over a two hour period. In addition, diphtheria toxin conjugates of modified Tf were significantly more cytotoxic than conjugates of native Tf (IC 50 = 0.02 nM for modified Tf conjugate vs. 0.1 nM for native Tf conjugate). The results of this study suggest that inhibiting iron release may improve the efficacy of Tf as a drug carrier by allowing Tf to remain associated with cells expressing TfR for longer periods of time.
Ms. Sacha Prashad
Mentor: Hanna Mikkola
Title:
Hematopoietic stem cell development in the human placenta
A hematopoietic stem cell (HSC) is a type of multipotent stem cell that retains the ability to self-renew as well as differentiate into all lineages of blood cells. HSCs sustain blood cell production throughout the lifetime of an individual. However, little is known about the microenvironment needed for the development and maintenance of HSCs. We recently discovered that in mice, the placenta is one of the first organs to harbor self-renewing, multipotent hematopoietic stem cells. We are now investigating whether the human placenta possesses a similar ability to support HSC development, and possibly even generate HSCs de novo. We have identified multilineage hematopoietic progenitor cells (putative HSCs) as early as 4 weeks developmental age in human placental tissue explant cultures, and we are in the process of determining the developmental timepoints when the population of these putative HSCs is expanding. We are also attempting to localize these putative HSCs further in the placental niche using immunohistochemistry. We will determine whether these putative HSCs are true HSCs by assessing their ability to self-renew and to reconstitute the entire hematopoietic system after transplantation into irradiated recipient mice. We hope that characterization of the placental niche of the HSC will ultimately allow us to recreate this environment ex vivo so that we may expand HSCs from cord blood or generate HSCs from embryonic stem cells for therapeutic purposes.
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Ms. Dominique Richardson
Mentor: Daniel T. Blumstein
Title: A quantitative analysis of yellow-bellied marmot pup fear screams
In addition to producing well-studied alarm calls, yellow-bellied marmot pups produce screams, which superficially seem different from alarm calls but have not been previously analyzed. The objectives of this study are to analyze and describe the screams and nonlinearities found within them, and to determine if information about the caller is contained in the screams. To characterize the tonal structure of the screams, we measured 16 acoustic features of 194 screams from 23 individual pups. Measurements were averafed and compared to respective measurements taken from juvenile alarm calls. Discriminant function analysis—used to quantify degree of individuality in individual screams—revealed that approximately 60% of the original group cases were correctly classified and that frequency characteristics were particularly important in discriminating individuals. The frequency of warbles and two types of nonlinearities—subharmonics and biphonation—were also quantified. We found that warbles occurred in approximately 75% of screams, subharmonics in% and biphonation in 13%. Results indicate that screams are significantly different from alarm calls in all measured dimensions and that information about the caller is contained within the screams. The complexities and nonlinearities present in screams may lead to increased individuality in screams. The ability to produce nonlinearities may also be advantageous to a marmot pup in other ways. Nonlinearities may prevent habituation or enhance the low frequency of screams, allowing pups to repel attackers. Both hypotheses required further observation and testing in future experiments.
Mr. Michael Rome
Mentor: Peter J. Bradley
Title: Identification of a novel dense granule protein GRA12 in Toxoplasma gondii
Toxoplasma gondii is an obligate intracellular parasite that is an important pathogen of immunocompromised individuals and congenitally infected neonates. Host cell invasion and intracellular survival are believed to be maintained in part by specialized secretory organelles named dense granules. In a recent proteomic analysis of the Toxoplasma secretory organelles, our lab identified 38 novel proteins. In this study, we characterized one of these novel proteins to determine its role in Toxoplasma infections. To assess localization, we expressed and purified a 253 amino acid portion of the protein. The purified protein was then injected into mice for polyclonal antibody production. Using the resulting antisera in an immunofluorescence assay, we found that this protein is a dense granule protein, which we now refer to as GRA12. To directly assess the function of GRA12, we used a gene disruption approach. The resulting ∆gra12 parasites were used in a competition growth assay to monitor growth differences in knockout parasites. We found that ∆gra12 parasites were not able to fully invade or survive in human foreskin fibroblast cells as efficiently as wild type parasites. These results indicate that GRA12 contributes to the parasite’s ability to invade and sustain infection in host cells. Finally, using co-immunoprecipication assays, we have identified candidate GRA12-interacting proteins that may provide important clues to how GRA12 aids in the intracellular lifestyle of this important pathogen.
Mr. Aswin Sekar
Mentor: Desmond Smith
Title: Identification and validation of copy number associated expression loci using radiation hybrids
Radiation hybrid cell lines contain genetic material from more than one species. Donor cells are lethally irradiated with x-rays, causing random breakages in the cells’ chromosomes, and some of the DNA fragments are rescued upon fusion with a host cell line. A commonly used mouse/hamster radiation hybrid panel, T31, consists of 100 cell lines with an average mouse marker retention frequency of 30% and an average donor fragment size of 10 Mb. As a system with a well-defined genetic perturbation, the panel presents an excellent opportunity to look at the effects of abnormal copy numbers as they pertain to gene expression, on a genome-wide level. In this study, microarray analysis of the T31 panel was performed and dense genotype data for each cell line was obtained using Comparative Genomic Hybridization (CGH) arrays. Linkage analysis was done whereby the copy number of a mouse marker on the CGH array was correlated with the mRNA transcript abundance levels of every probe on the expression array. Above a LOD threshold of 3, tens of thousands of trans regulatory interactions were identified. This approach, thus, affords the ability to map the regulatory landscape of the mammalian genome at a high resolution and allows for the identification of regulatory loci for virtually any desired gene in the mammalian genome.
Mr. Govind Shah
Mentor: Steve E. Jacobsen
Title: Discovering novel factors in the de novo DNA methylation pathways in Arabidopsis
Cytosine DNA methylation in Arabidopsis thaliana is an important mechanism for maintaining genome stability and silencing transposons and other harmful DNA sequences. In addition, DNA methylation in A. thaliana also regulates the expression of some developmentally important genes. The misregulation of DNA methylation is also linked to genetic instability and cancer in mammalian cells. The protein DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) is responsible for all de novo methylation in A. thaliana, as well as the maintenance of CHH methylation. As there are several other factors known to be responsible for establishing de novo DNA methylation, we suspect DRM2 exists in a complex with other proteins. In order to identify novel factors in the putative DRM2 protein complex, we tagged and purified DRM2-associated proteins through a tandem affinity approach using a biotin-streptavidin affinity system. We have acquired preliminary mass spectrometry data on the DRM2 protein complex purified using biotin-streptavidin affinity. The identification of factors in such a complex can potentially provide the missing link in current pathways.
Ms. Aida Sun
Mentor: Gregory Grether
Title: Mate choice analysis of hue preference as a form of countergradient variation in the guppy, Poecilia reticulata
Countergradient sexual selection may occur due to sensory bias that constrains condition dependent sexual traits across populations with variable resource availability through genetic divergence. Female guppies (Poecilia reticulata) use the orange spots of males in sexual selection. These orange spots are composed of two pigments: carotenoids and drosopterins. The amount of carotenoids found in guppies is dependent upon their environment as they obtain carotenoids solely from diet. Conversely, drosopterins can only be synthesized and are highly heritable. Their drosopterin pigment level matches the carotenoid availability within their stream, resulting in a uniform pigment ratio. This may be the result of female preference for a specific orange pigment ratio i.e., hue preference. This project tests the hue ratio hypothesis in guppies through mate choice trials. We hybridized two guppy populations that were divergent for orange coloration. F2 males were assigned a pretrial relative drosopterin rank and matched with parental virgin females. Physical characterization of the males’orange spots followed the trials. Preliminary results indicate significant differences between female receptivity and male courtship between populations. However, no evidence for drosopterin ranking effect has been identified yet. These results are based on relative drosopterin measurements taken prior to the trials. Further analyses are in process to incorporate pigment content into the mate choice data. These results will further elucidate the behavioral significance of the drosopterin pigment.
Ms. Darcy Wanger
Mentor: Benjamin J. Schwartz
Title: Plastic solar cells: Molecular self-assembly and its electronic effects
Organic solar cells have great promise and intrigue in the area of energy production due to low bulk processing costs, but currently lack an acceptable efficiency for mass production. One component of the system that contributes to its inefficiency is the limited organization in electron transport. The goal is to synthesize fullerene compounds that arrange closely enough together such that an electron accepted by one fullerene has the energy, and ideally also the impetus through electrostatic forces, to “hop” from fullerene to fullerene. In a simplistic polymer and fullerene device, annealing causes islands of fullerenes to form, inducing intimate electron hopping within the islands, but no infrastructure present to ensure that they reach the electrode efficiently. On the opposing extreme, a molecule-to-molecule pairing keeps molecules within range, but does not facilitate appropriate charge separation. This work uses new fullerene materials that may thermodynamically assemble into stacks and therefore more effectively conduct electron current. Significant changes in electronic behavior have been observed; by noting and understanding how the behavior of molecules within a device affects its electronic properties, fabricating inexpensive and efficient solar cells for everyday use becomes a realistic possibility.
Mr. Michael Zhang
Mentor: Thomas M. Vondriska
Title: Characterization of the Tec tyrosine kinase signaling network in the murine myocardium
Members of the Tec family of non-receptor tyrosine kinases (Tec, Btk, Bmx, Emt and Txk) have well-established roles in regulating cell growth and differentiation. These proteins are known to be activated by growth factors and cytokines and are critically involved in T-cell activation. Recent studies have suggested an angiogenic role for one of the isoforms (Bmx) in ischemic injury to mouse heart and skeletal muscle. The specific roles that Tec family proteins play in mediating cardiac signal transduction pathways, however, remain largely unknown. In the present study, we used Western blotting to analyze the expression of Tec family kinases in whole heart and in isolated cardiac myocytes under normal, ischemic, and hypoxic conditions. We observed Bmx and Tec expression in both whole heart and myocyte lysates. In hearts subjected to ischemia/reperfusion (30 minutes ischemia, 24 hours reperfusion) and in myocytes subjected to hypoxia (2 hours), levels of Tec protein expression were comparable to that of sham controls and basal heart. We have also performed co-immunoprecipitation experiments in order to identify other components of the Tec signaling network in mouse heart. We have separated these Tec-associated proteins by SDS-PAGE, trypsin digested them and are now identifying them by LC/MS/MS. These studies are an important first step towards understanding the mechanistic role that Tec kinases play in myocardial ischemia.
Ms. Dagny Zhu
Title:Measuring osteoinductive variability across multiple lots of a single demineralized bone matrix (DBM) product
Mentor: Rick B. Delamarter
Spinal fusion surgeries require bone grafts or substitutes for optimal vertebrae fusion. Demineralized Bone Matrix (DBM), a common substitute, is an osteoinductive allograft derived from pulverized bone. While individual DBM lots from a single vendor would be presumed to possess equal osteoinductivity, we have found significant lot-to-lot variability. The goal of this study was to identify an assay that could accurately predict the osteoinductive potential of individual DBM lots. Using ELISAs, we measured levels of bone morphogenetic proteins BMP-2 and BMP-7 in different DBM lots. We also analyzed the ability of each lot to induce osteoblast differentiation in vitro by measuring alkaline phosphatase (AP) production. We then asked whether BMP and/or AP levels correlated with the ability of each DBM lot to promote fusion in rats that underwent posterolateral fusion surgery. Fusion success was determined using radiographs and physical examination of excised spines. Statistical analysis revealed significant lot-to-lot variability in BMP concentrations, extent of AP induction, and in vivo rates of fusion (p=0.04). Of the ten lots tested, two almost always promoted fusion, while five consistently failed. BMP-2 and BMP-7 correlated positively with each other (r=0.77) and positively predicted fusion success (p=0.01). AP levels did not correlate with fusion rates. Our results demonstrate the predictive ability of BMPs and encourage the use of BMP screenings to ensure a minimum level of osteoinductivity across DBM lots.
