Student Abstracts A-C

Student Thesis Abstracts A - C

AstroPhysics

  • Fronefield Crawford, III
  • James W. Partan
  • Alex Kristian Wong

    Biology

  • Rachel Covey Brown
  • Daniel Casey
  • Deborah Golda Feiner
  • Shaw Cartin Henderson
  • John Roland Hering
  • Paul Hohenlohe
  • Scott R. Martin
  • Neil Mehta
  • Katharine M. Nash
  • Brian Rho
  • Deborah Edin Schein
  • David M. Scholle
  • Cynthia Sharpe
  • Bradley Theodore Smith
  • Dana Tomasino
  • Rajnish Tandon
  • Erik L. White
  • Sara White

    Chemistry

  • Adam S. Carroll
  • Jon F. Fielder
  • Carolyn Flesner
  • Amy M. Gehring
  • Doron Greenbaum
  • Nina D. Raoof
  • Barbara A. Roe
  • Joseph P. Sadighi
  • Tanya L. Schneider
  • Chu Ri Shin

    Computer Science

  • Daniel P. Fasulo
  • Sean D. Sandys
  • Angela M. Schuett

  • ASTROPHYSICS

    Fronefield Crawford, III

    Predictions of Glitch Behavior from Pulsar Models

    Current neutron star theories use a two-component model of the star interior, which is believed to consist of a degenerate neutron superfluid core surrounded by a rigid crust of iron nuclei in a sea of free electrons.

    At the extremely high densities near the center of the star (1011 to 1015 g/cm3), the characteristics of the matter are largely unknown. We have created several models of the neutron star interior employing different equations of state in order to place restrictions on the possible equations of state of the matter. We have been able to set limits on the mass, radius, and central density from these models. We have also been able to discredit one of the models as unrealistic due to observational constraints on the mass.

    It is also possible to use the model parameters, particularly the crust parameters, to determine the feasibility of the starquake model as the mechanism by which some pulsars exhibit glitches, which are sudden spin-ups in a pulsar's extremely steady rotation rate. Both the starquake model and its alternative, the vortex creep model, are explained in the paper.

    We have obtained all available pulsar glitch data for the Crab and Vela pulsars, for which the most glitch data has been obtained. We hoped to establish which glitch mechanism model is more realistic by comparing the crust parameters from the models with the glitch data, but were unable to complete a thorough comparison due to time constraints and the sparseness of the glitch data. We have however, noticed a trend in the data, and have calculated a preliminary crust shrinkage using the available data and the model results.

    James W. Partan

    Progress Towards a Magneto-Optic Trap for Rubidium

    (See Physics section for abstract.)

    Alex Kristian Wong

    The Effects of Dark Matter on the Dynamics of Spiral Galaxies

    We investigate the effects of non-luminous material on the orbits of stars in spiral galaxies. As the existence of non-luminous material becomes accepted, it is important to investigate how this material affects astronomical systems. We will examine how so called "Dark Matter" affects the orbits of stars in spiral galaxies, by creating and examining numerical simulations of a simple test particle in a potential field. The potential field accounts for the following spiral galactic components: the nuclear bulge, the disk, the spherical halo, and the extended non-luminous corona. We report the current state of the computational simulations of orbits in the potential field with different initial conditions, and we also describe numerous problems encountered with the numerical methods and the simulation in general.

    BIOLOGY

    Rachel Covey Brown

    The Effects of Prenatal and Early Postnatal Stress on the Adult Neurochemical Stress Response

    The stress response is known to be affected by a number of different variables, including prenatal alcohol exposure and early postnatal experience. This set of experiments was designed to examine how pre- and postnatal experiences interact to alter the neurochemical response to stress in adulthood. Two neurochemical indices were examined: 1) levels of allopregnanolone in blood plasma and brain tissue, and 2) levels of catecholamines in several regions of the brain.

    Allopregnanolone is a neuroactive steroid known to be involved in the neurochemical response to stress. Whether administered or endogenously produced, allopregnanolone has anxiolytic behavioral effects that are mediated by its action as a positive allosteric modulator of the GABAA receptor. Other anxiolytic neuroactive steroids have been found to affect the stress-induced activation of the mesocorticolimbic dopamine system, suggesting that allopregnanolone might have similar effects.

    We found that prenatal alcohol exposure combined with early maternal separation resulted in an increase in the endogenous production of allopregnanolone in response to adult stress in females only. This increase in production of allopregnanolone was mirrored by a decrease in the level of dopamine released in the prefrontal cortex, indicating that this pathway is affected by allopregnanolone. Prenatal alcohol exposure was also associated with increased levels of dopamine in the nucleus accumbens and striatum, indicating that alcohol may slow development of these dopaminergic pathways, ultimately resulting in upregulation of GABAA receptor expression at the pathway's origin. Finally, there was some suggestion that pre- and postnatal condition may have some effect on the development of noreadrenergic pathways in the brain that are responsive to stress.

    Daniel Casey

    Genetic Analysis of unc-45 Mutants in Caenorhabditis elegans

    The unc-45 gene in Caenorhabditis elegans is an essential muscle-affecting gene. The gene has been located in the worm genome and cloned. Recently, the gene has been sequenced. The purpose of this study was to perform a series of genetic tests on two of the lethal alleles of unc-45, st601 and st604, to gain a better understanding of unc-45's role and interactions. Tests performed were a null phenotype test and two tests of the maternal rescue observed in the lethal alleles. The results did not conclusively prove that st601 was a null mutation, but did not rule out that possibility either.

    Deborah Golda Feiner

    Long-Term Recovery After Spinal Cord Crush in the Goldfish

    While the mammalian central nervous system (CNS) fails to regenerate following spinal cord injury, that of non-mammalian vertebrates does regenerate. Approximately 309 days after a spinomedullary level crush (SMLC), the goldfish, Carassius auratus, will regain normal swimming behavior (Bernstein, 1964). In addition the C-start, a rapid, evasive maneuver subserved by the brain stem escape network (Eaton, et al., 1982) returns after post-operative intervals of approximately three months or more (Northern, 1989; Freemer, 1990; Prendergast, 1993). The initially recovered C-start, however, is elicited less frequently than in control fish (Freemer, 1990; Prendergast, 1993). In addition, computer analyses used to quantify the parameters of the C-starts given by SMLC fish, have shown the recovered C-starts are less robust, with longer latencies than those of control (Freemer, 1990; Prendergast, 1993).

    In this study we investigated whether the probability of eliciting a C-start, and C-start kinematic parameters change over time after initial recovery. We conducted behavioral testing of the SMLC group until 15 months post-operatively, and found that in two fish in our experimental group (n = 5), not only did the probability of response increase dramatically but also certain C-start parameters: stage one latency, stage two latency, stage two angle, angle 70 ms after start, and center of mass displacement, changed significantly over time. In these parameters, these fish improved sufficiently to be within control ranges at 12 months post-operatively.

    We conclude from this study that regeneration in the goldfish is a dynamic process, which continues for long post-operative intervals, and we suggest that this may provide insight into why the CNS regeneration seen in non-mammalian vertebrates is successful as compared to the abortive regeneration observed in the mammalian CNS.

    Shaw Cartin Henderson

    Characterization of N-Acyltransferase in Bean and Corn

    Ceramide synthesis in bean and corn was studied in vitro to determine the requirements of the reaction and the substrate specificity and selectivity of the sphinganine N-acyltransferase ceramide synthesizing enzyme. An assay using [14C]-palmitoyl CoA or [3H]-sphinganine as substrate was used to monitor the incorporation of radiolabeled substrate into ceramides in bean and corn microsomal membrane fractions. Ceramide synthesis in a bean and corn did not require ATP and was not stimulated by the addition of DTT. Activity was found to be localized in the endoplasmic reticulum from cell fractions isolated by ultra-centrifugation. N-acyltransferse activity was greatest at a pH of 7.4 in bean and approximately 7.2 in corn, both as a temperature of 37�C. Maximum ceramide synthesis was obtained with sphingosine in both bean and corn. N-acyltransferase in bean showed a strong preference for 16:0 and 24:0 CoA. Competition between 16:0 and 24:0 CoA demonstrated that both substrates apparently compete for the same site on the enzyme. In corn, acyl-CoA specificity differed slightly in product formation increased with increasing carbon chain length (24:0 CoA as substrate resulted in highest activity; 16:0 CoA was lowest). The specificities of N-acyltransferase for various acyl-CoA's in bean and corn corresponded to the ratio of fatty acids found naturally in bean and corn sphingolipids. Sterylamine and ethyl alcohol both inhibited ceramide synthesis as did the addition of detergents. These results suggest that N-acyltransferase may have a role in the synthesis of ceramides in vivo and account for the distribution of fatty acids found in sphingolipids of plant tissue.

    John Roland Hering

    The Role of the Mauthner Cell in the Recovery of the Fast Startle Response in Goldfish

    The Mauthner cells (M-cells) are a pair of large, identifiable neurons in the goldfish brain that are known to be involved in the fast startle response known as the C-start. The M-cells, while not necessary for the C-start, are believed to be important in the rapid initiation of this response in normal fish.

    Goldfish have the ability to regenerate neurons in their central nervous system (CNS). The C- starts that are recovered after a spinal cord crush, however, are not the same as in normal fish. We hypothesized that the M-cells would not be necessary for the recovery of C-starts after a spino- mecullary level crush (SMLC). To test this hypothesis, three groups of fish were compared. Group 1 had an SMLC and a selective double ablation of their M-cells, Group 2 had an SMLC and a sham ablation of their M-cells, and Group 3 received only the SMLC.

    The M-cells are not necessary for the return of the C-starts after an SMLC. All Group 1 fish gave C- starts. There were no significant differences in most of the kinematic parameters of the C-starts when Group 1 was compared to Groups 2 and 3. Two of the Group 3 fish showed improvement in their C- start parameters over time approaching control values. When Group 1 was compared to these two "improvers" from Group 3, significant differences in C-start parameters were found. Group 1 latencies were longer, and the angles 709 msec after start, angle 2 magnitudes, center of mass displacements, and velocities were all smaller than these values in "improvers."

    We conslude that the M-cells may be important in the speed and robustness of the recovered C- starts. However, they do not seem to play a role in determining the frequency of response, and we were unable to determine whether they play a dominant role in determining the side of the C-start.

    Paul Hohenlohe

    Niche Separation, Morphology, and Performance in Two Species of Peromyscus

    Gause's principle of competitive exclusion dictates that two species cannot occupy identical niches, but the ecological differences between two similar, sympatric species of deer mice (Peromyscus maniculatus gracilis and Peromyscus leucopus noveboracensis) are unclear. This study examines three aspects of ecological separation in these species: niche separation, morphology, and performance. Using Hopkins Memorial Forest in northwest Massachusetts where both species are found, it relates these three aspects of ecology in two dimensions, altitude and in arboreality, and also examines feeding performance.

    Significant results were achieved in all three aspects of ecological separation but could not be firmly correlated to each other. A significant change in proportions of the two species was found along the 1300 foot elevation gradient, with P. maniculatus more common at higher elevations, corresponding to P. maniculatus' more northerly distribution. The species overlapped at all scales, however, so horizontal distribution does not play a role in niche separation. No difference was found between the species in capture rates at different heights in trees, but P. maniculatus was found to run up trees more often when released. The two species also differed morphologically, with P. maniculatus having longer tails, feet, and ears. They differed in feeding strategies on hazelnuts given to them in the lab, making holes in the shell of different sizes and different locations. Planned experiments on climbing performance and aggressive interactions were prevented by the risk of hantavirus infection.

    The nature of the ecological separation between these two species seems complex and geographically variable. This study indicates P. maniculatus as more arboreal in morphology and one measure of niche distribution, and suggests that feeding strategy may separate the species. Differences along other ecological dimensions or aggressive interactions may also allow these two species to coexist.

    Scott R. Martin

    Structure-Function Mutational Analysis of the Vaccinia Virus dUTPase

    Vaccinia virus is a eukaryotic DNA virus that replicates in the cytoplasm of its hosts. Because of the cytoplasmic location of its life cycle, vaccinia carries and transcribes its own replicative machinery. One protein involved in DNA replication is dUTPase, which catalyzes the hydrolysis of dUTP to dUMP, which then serves as a substrate for dTMP synthesis. dUTPase is also necessary to prevent accumulation of dUTP, which can be incorporated into DNA and cause excision-repair and DNA fragmentation if present at high concentrations. dUTPase exists as a trimer with 5 highly conserved motifs, which are thought to be important in trimerization and as the active site of the enzyme.

    An overexpression plasmid coding for a truncated enzyme lacking motif 5 was constructed, but was found to express a protein slightly larger than the normal dUTPase. This occurred because the stop codon was removed from the mutant, and another was not immediately encountered. This mutant did not show activity in an enzyme assay; this may have been because of the importance of motif 5, or it may have been due to structural changes caused by the extra amino acid residues. In the future, site- specific mutagenesis and photo-affinity labeling studies will be necessary to show more clearly areas of structural and functional importance in vaccinia virus dUTPase.

    Neil Mehta

    The Importance of LMAN for the Redevelopment of Plasticity in the Zebra Finch Song

    Male zebra finches are critical period learners, meaning that they pass through the stages of song development only once, and song learning is completed at approximately 90 days when the male bird reaches sexual maturity. It was originally thought that once the song has been developed, or crystallized, the song cannot change. But in 1992, Williams & McKiggen demonstrated that injuring the right ts (tracheosyringeal) nerve resulted in some changes in syllable syntax, or temporal pattering of the song. Consequently, some form of vocal plasticity remains even after song learning is complete.

    This experiment determined that LMAN, the lateral magnocellular anterior nucleus of the neostriatum, is necessary for redevelopment of plasticity that results from injuring the ts nerve. Adult male zebra finches that were given LMAN lesions followed by ts nerve injuries had significantly fewer changes in syllable syntax than birds that were given LMAN sham surgeries, in which no lesion was induced, followed by ts nerve injury. Thus LMAN must be intact in order for these changes in syllable syntax to occur.

    The LMAN is part of a secondary neural pathway from HVc (high vocal center) to RA (nucleus robustus of the archistratium) that is necessary for the original development of the bird song, but it is not necessary for song production in the adult. Lesioning of the secondary pathway, namely LMAN, in the developing bird results in severe song deficits in the adult song, but lesioning of the secondary pathway, namely LMAN, in the adult has no effect on song production (Bottjer, Meisner & Arnold, 1984). Thus the secondary pathway was not thought to be used for song maintenance after song development is complete.

    Since the LMAN is necessary for the redevelopment of plasticity following ts nerve injury, this pathway must return to a more immature and plastic state that was present during the initial learning of the song.

    Katharine M. Nash

    The Role of Dispersal Mechanisms on the Colonization of Spring Herbaceous Flora in Secondary Woodlands

    Dispersal mode affects the ability of some herbaceous species to colonize secondary forests in Williamstown, Massachusetts. The canopy layer of secondary forests 50 to 85 years old did not differ significantly from that of primary forests with respect to species frequency, basal area, and light availability. However, in the herbaceous layer, overall differences in species nighness and total abundance of the spring flora were significant between forest types (p<.001). The species dispersed by ants, the myrmecochores, were particularly limited in their distribution to primary forests (p<.05). Nonetheless, dispersal mode was not the only limiting factor. The species which were severely limited to primary forests also shared common life history traits and ecological niches. Species which were dispersed by the wind also were restricted to certain forests, but their distribution did not correlate with the successional history of the sites. In this case, the limitation may have been due to another factor such as the occurrence of large gaps in the canopy layer.

    Brian Rho

    Characterization of Naturally-Occurring Daily Torpor in Peromyscus maniculatus

    Deer mice, Peromyscus maniculatus, were captured in the Hopkins Memorial Forest and implanted with temperature sensitive telemeters to enable remote measurement of body temperature of animals in an outdoor enclosure. Body temperature measurements were recorded to characterize the daily torpor cycle of mice subjected to native environmental conditions. Both and ambient temperature measurements were taken at 1 or 2 minute intervals for 24 or 12 hour periods, respectively. The average euthermic Tb and depth of torpor were found to be 34.2�C and 24.1�C, respectively. The mice typically enter torpor a few hours prior to sunrise and had bouts that lasted an average of 4.9 hours.

    Deborah Edin Schein

    Genome Mapping in Tetrahymena thermophila Using Homologous Recombination and Chromosome Fragmentation

    Tetrahymena thermophila is a ciliated protozoan that has two nuclei: a germ-line micronucleus and a somatic macronucleus. The somatic macronucleus is generated during conjugation of two cells, through replication and alteration of micronuclear DNA sequences. We have initiated a new experimental approach for mapping genes in the macronuclear genome of Tetrahymena using a technique that has already been developed in yeast. This technique involves the creation of stable chromosome fragments through recombination of endogenous DNA sequences with cloned homologous sequences on a linear DNA vector. The size of the resultant fragments will determine gene location on an endogenous chromosome. The first stage of this project involved the design and construction of two DNA vectors containing a chimeric neo-gene to allow for the selection of transformed cells, as well as a single telomere sequence in two orientations. Tetrahymena DNA sequences in these plasmids will be located at a free end when the plasmids are linearized, and are expected to recombine with the host chromosomes to produce two new stable fragments that are maintained by the cell. Included in these vectors are sites at which additional segments of macronuclear gene sequences as well as replication origins, which offer a replication advantage to the exogenous fragments, can be introduced. The second stage of this project strove to determine appropriate conditions needed for the efficient transformation of Tetrahymena cells using electroporation.

    David M. Scholle

    Insect Diversity and Variability Within the Understory and Canopy of a Temperate Deciduous Forest and a Tropical Rain Forest

    Forest insect communities are distributed in space and time according to factors whose combined action we do not well understand. Consistent characteristics unique to temperate and to tropical forests give us some indication of which factors may be most important. The right and diverse communities of emergent trees, vines, epiphytes in the tropical rain forest canopy are thought to maintain the documented concentrations of insect abundance and diversity there. In the temperate forests insects do not appear to survive well in the canopy and are normally found in higher concentrations near the ground. In the rain forest the preference for the canopy may be related to the relatively lower year round growth in the dark understory. In addition to the physical characteristics of a habitat, the strength and character of patterns among insect communities depend n the scale in space and time over which one takes measurements. At an annual scale, communities of insects appear to vary in both number and makeup, and their variation may be due to density-independent factors such as weather or to density-dependent factors such as competition and predation. On an hourly scale, changes may reveal distinct natural history patterns of insects and their immediate response to atmospheric changes such as temperature and light fluctuations, or an afternoon storm.

    Data from this study suggest that though previous studies have round tropical insects to show specificity for the canopy, concentrations of tropical insects can occur near the ground possibly in correlation with steep topographic slope. Among heights different orders and families may show characteristic preference for ground or canopy survival. The diurnal patterns of insect activity may differ among forest heights; in the canopy a greater proportion of insects appear to fly during the early hours of the morning. Results from this study remain suggestive. While all orders show largely unpredictable fluctuation of a uniform degree, some orders may sustain distinguishing variabilities. Relative variability rankings among orders may be conserved between both temperate and tropical areas. Further studies of this nature should include a test of the efficiency of the traps used during the study, replication of catches through time and space, and measurement of tropical and temperate communities during analogous seasons.

    Cynthia Sharpe

    Further Molecular Characterization of unc-45, A Muscle-Affecting Gene in Caenorhabditis elegans

    The unc-45 gene in the nematode Caenorhabditis elegans is an essential gene, involved in body wall muscle thick filament assembly. Sequencing of the unc-45 region was begun prior to the initiation of this project, and the goal of this project was to continue sequencing the gene and, if time permitted, further characterize the gene. In November 1993, the C. elegans Genome Sequencing Project joined the effort to sequence unc-45, and they have provided us with incomplete sequence. That information allowed us to choose the best probe for a new attempt at cDNA library screening. Additionally, in an effort to determine how unc-45 interacts with the myosin isoforms of the pharyngeal muscles, transmission electron microscope studies have begun.

    Bradley Theodore Smith

    The Purification and characterization of the Bacillus subtilus SOS Repressor

    The repressor protein of the Bacillus subtilis damage inducible DNA repair system (SOS system) was identified as the product of the B. subtilis dinR gene. The dinR gene was amplified with PCR and cloned into the pET-21 a plasmid where its expression was under the control of a bacteriophate T7 promotor; DinR protein was overexpressed in E. coli BL21(DE3) cells containing the pET- 21a/dinR plasmid. The DinR protein was purified to homogeneity using heparin-agarose column chromatography and Superose 12 gel filtration. Purified DinR was shown to undergo autodigestion at alkaline pH in a reaction that followed first-order kinetics over a two pH unit range (pH 8.5 to pH 10.5); the pH of the reaction was estimated to be approximately 9.6. Amino-acid sequence analysis of the autodigestion products revealed that the primary cleavage site in the reaction is at Ala91-Gly92. Three secondary cleavage sites were found in the amino-terminal fragment of DinR. These secondary cleavage events appear to occur after the primary event, which implies that the carboxy-terminal fragment of DinR presumably contains the proteolytic active site and can retain its proteolytic activity even after the primary cleavage event has occurred. At physiological pH, B. subtilis RecA promoted DinR cleavage. However, the kinetics of RecA-mediated cleavage in vitro were not consistent between trials. These results suggest that the mechanisms of SOS repressor inactivation have been highly conserved between E. coli and B. subtilis.

    Dana Tomasino

    Structure-Function Analysis of the B. subtilis SOS Repressor

    This investigation undertook a structure-function analysis of the B. subtilis SOS repressor protein with the aim of further characterizing its interaction with B. subtilis RecA protein and with damage- inducible (din) gene operators. E. coli LexA and RecA were used as model proteins in an effort to optimize conditions for cross-linking B. subtilis repressor to B. subtilis RecA using a bifunctional, photoreactive cross-linker, sulfosuccinimidyl 7-adiso-4-methylcoumarin-3-acetate. E. coli LexA and RecA were cross-linked with less than 10% efficiency; cross-linking efficiency was not significantly increased by any of the alterations in assay conditions tested. conditions used in attempts to cross-link LexA to RecA proved more conducive to cross-linking LexA repressor to itself. B. subtilis repressor was also cross-linked to itself with approximately 15% efficiency. Such studies may eventually prove useful in identifying the dimerization domains of both SOS repressors.

    Conditions for cross-linking B. subtilis repressor to a photo-affinity-labeled DNa probe containing its operator site were optimized. The probe was constructed using 5-azido dUTP to replace the two thymines in one of the operator half-sites with 5-azido uracil. Cross-linking efficiencies of roughly 25- 30% were obtained in assays conducted at 4�C at pH 9.0 with 5 µM repressor and 3.9 nM photoprobe. A 24-amino acid peptide comprising residues 161-184 of the repressor was cross-linked to the din operator consensus sequence. This peptide appears to overlap the repressor's putative catalytic domain. In addition, the peptide is located towards the repressor's carboxy-terminal domain, while the DNA binding domains of analogous repressor proteins have been found to occur within the amino terminus. control experiments must be performed before the identification of the cross-linked peptide may be considered definitive. The results of repressor:operator cross-linking and mobility shift studies also suggest an extension of the consensus sequence required for optimal repressor binding. Two A-T- rich regions flanking the previously determined consensus sequence, 5'BAAC-N4-GTTC-3', have been identified in all characterized B. subtilis din operators and appear also to be important for efficient repressor:operator binding. Based on these results, we propose the new consensus sequence: 5'- AAAA-N2-GAAC-N4-GTCC-N2-TTTT-3'.

    Rajnish Tandon

    The Effect of Fumonisin B1 on Ceramide Synthesis in Plant Tissue

    Fumonisin B1, a mycotoxin produced by the mold Fusarium moniliforme, has been demonstrated in animal systems to inhibit sphinganine N-acyltransferase, a key enzyme in the sphingolipid biosynthesis pathway. As this mold is found predominantly on corn and other grains, this study was designed to characterize the inhibition of this enzyme by fumonisin B1 concentration of 100 nM with 50% inhibition at a concentration of 4 nM. In bean, complete inhibition of sphinganine N- acyltransferase was observed at 1 µM with an IC50 value of 30 nM. The plant microsomes exhibited decreased sensitivity to hydrolyzed fumonisin B1 with 50% inhibition occurring at 400 nM in corn and 2 µM in bean. Fumonisin B1 and hydrolyzed fumonisin B1 are non-competitive inhibitors of both palmitoyl- CoA and sphingosine. Treatment of corn shoot tips with 1 µM fumonisin B1, sufficient to cause tissue necrosis, caused a 45-fold increase in the level of free phytosphingosine to a concentration of 630 nM/gram of fresh weight. Levels of free sphinganine increased 10-fold to a level of 38 nM/gram of fresh weight. These results indicate that fumonisin B1 disrupts ceramide synthesis in vitro and in vivo and leads to the accumulation of free long-chain bases which can derange cellular processes leading to tissue necrosis.

    Erik L. White

    Patterns of Deoxyuridine Triphosphate Nucleotidohydrolase Expression and Activity in vivo in Cells Infected with Vaccinia Virus

    Vaccinia Poxfiridae is a DNA virus with a cytoplasmic site of replication. Since viral DNA replication occurs autonomously from that of the host cell, the viral genome codes for many enzymes are analogous to, but distinct from, their host cell counterparts. Typically, the host cell enzymes which are replaced by viral copies are down-regulated during infection. Furthermore, copynumber and activity level of specific viral enzymes are regulated by post-translational modification throughout the replication cycle. One enzyme that is a member of both categories is deoxyuridine triphosphate nucleotidohydrolase (dUTPase). The method for determining the in vivo activity of vaccinia dUTPase as a function of time post infection by thin layer ion exchange chromatography is described, and the activity profile for the enzyme over a replication cycle is presented. Preliminary experiments designed to elucidate the mechanism for viral control of host enzymes by immunoprecipitation of radio- labeled dUTPase are also described.

    Sara White

    The Ecology of Lonicera hirsuta (caprifliaceae) in Berkshire County, Massachusetts

    Lonicera hirsuta is found in only three locations in Massachusetts, all in Berkshire County, and is consequently listed as an endangered species in Massachusetts. L. hirsuta at the Pine Cobble site in Williamstown was studied for a period of five years to examine the ecology of the species, and L. hirsuta patches in Lee and on Bullock's Ledge in Williamstown were surveyed annually for growth as well. Surveys of L. hirsuta patches included ramet count and size, patch size and shape, canopy cover, amount of dates of flowering, and fruit production and disappearance. Species and size of vegetation found in or near L. hirsuta patches were analyzed, as were sizes and locations of stumps near patches. Historical research was carried out to provide histories of land use and disturbance on Pine Cobble, including its use as a woodlot and a recreational area, as well as periodic disturbance on the site. Greenhouse experiments were performed in conjunction with the fieldwork. It was found that L. hirsuta response to increased light levels by increasing its vegetative growth, and subsequently increasing its flower production. The disturbance caused by the clearings for roads, swales and house lots for the Pine Cobble development seems to have promoted increased vegetative and sexual reproductive output from many patches in the short run. It remains to be seen how the development directly or indirectly will effect the vigor of the plant in the long term. The long term conservation of L. hirsuta and its habitats on Pine Cobble may include permanently setting aside lots from development, limited transplantation from house lots to common areas, and controlled disturbance to form gaps periodically in the areas of L. hirsuta patches.

    CHEMISTRY

    Adam S. Carroll

    Partial Synthesis of 2-Azido-2'-Deoxyadenosine 5'-Triphosphate: A Novel Photoaffinity Labeling Reagent for Protein-DNA Photocrosslinking

    In 1984, the human immunodeficiency virus-1 (HIV-1) was determined to be the cause of the clinically identified syndrome AIDS. The enzyme reverse transcriptase (RT) catalyzes an essential step in the replication of the RNA genome of HIV. All the drugs currently available for treatment of AIDS are inhibitors of RT. These inhibitors are all nucleoside analogs that are incorporated by RT and terminate the growing strand through their lack of a 3'-OH group. These inhibitors are not flawless as anti- AIDS drugs because they can generate several harmful side effects following prolonged treatment. Also, RT is quick to develop mutations that confer resistance to the drug. The establishment of a more detailed mechanism for RT on the molecular level may allow novel approaches in the design of RT inhibitors. In order to understand in detail how RT binds primer/template, the synthesis of 2-Azido-2'- deoxyadenosine 5'-triphosphate (2-N3-dATP) was begun. This photoactive analog of 2'- deoxyadenosine 5'-triphosphate (dATP) is expected to act as a substrate for DNA polymerases. As such, the synthesis of photoactive DNA probes will be possible. The location of the photoactive group on the 2 carbon of the adenosine base places it in the minor groove of the DNA. Photoaffinity labeling of RT with the photoactive probe would allow crosslinking to RT and identification of amino acids that interact with the DNA in the minor groove. The synthesis of 2-N3-dATP was begun with the tributyl tin hydride reduction of 171 mg of 2-chloro-adenosine. The reduction steps involved protection of the 3' and 5'-OH groups with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TPDS- Cl2) in pyridine. The 2'-OH group was then activated for reduction with phenyl chlorothionocarbonate (PTC- Cl) in acetonitrile. The 2'-OH was then reduced with tributyl tin hydride in toluene and the protecting group removed by addition of tetrabutylammonium fluoride in tetrahydrofuran. The reduced nucleoside was purified by reverse phase HPLC. All the products of the above steps were characterized by thin layer chromatography (TLC) and UV absorbance spectroscopy. The pure 2- chloro-2'-deoxyadenosine was then hydrazinated by dissolving in pure hydrazine. Characterization by UV spectroscopy indicated complete conversion of the 2-chloro- to the 2-hydrazino- compound. However, treatment of 2-chloro-2'-deoxyadenosine 5'-monophosphate with hydrazine produced no detectable hydrazino product. The 2-hydrazino derivatives proved to be unstable, even in anhydrous methanol at -20�C. Attempts to diazotize 2-hydrazino-2'-deoxyadenosine with excess NaNO2 in acetic acid or trifluoroacetic acid were unsuccessful, suggesting the presence of residual hydrazine in the diazotization reaction, despite repeated co-evaporations with methanol and 2-propanol. These results suggest the need for reverse phase HPLC purification of 2-hydrazino-2'-deoxyadenosine prior to diazotization.

    Jon F. Fielder

    Synthesis of Novel Charged Ruthenium-Polypyridine Complexes - Work Towards an Electrically-Conducting Organic Polymer

    This work is concerned with the development of a model system for a ruthenium doped, electrically conducting organic polymer. Toward this end, several novel charged ruthe-nium-polypyridine complexes have been synthesized. [Ru(NH3)4(pre-IDP+)](PF6)3, where pre-IDP+ is 1-(2'-(1'- methyl- pyridinium))-2-(2''-pyridine)aminomethane, was prepared in a series of four steps. Oxidation of the ligand in air gave the imine analog, [Ru(NH3)4(IDP+)](PF6)3, where IDP+ is N-(2'-(1'- methyl)pyridinium)amido-2-pyridinecarboxaldehyde. Also synthesized was [Ru(bpy)2(pre- IDP+)](PF6)3. Substantial evidence was gathered to support the idea that this complex also underwent air oxidation to yield the IDP+ analog. Spectrophotometric studies indicated that in both cases putative oxidation was accompanied by a lower energy metal-ligand charge transfer absorption, which reflected increasing conjugation. In addition, initial work toward the development of an alternative model system was undertaken. Attempts to make the IDP and IDP+ variants, 2-(3'- pyridine)pyridinecarbox-aldimine and 2-(1'-methyl-3'-pyridine)pyridinecarboxaldiminium iodide, respectively, are reported.

    Carolyn Flesner

    Temperature-Dependent Fluorescence of Nitric Oxide

    The laser-induced fluorescence of nitric oxide (A2Sigma+, v=0 <-- X^2PI, v=0) is an important quantitative diagnostic technique for the study of combustion and atmospheric chemistry. Collisional electronic quenching poses a major limitation to the quantitative detection of nitric oxide. To correct laser- induced fluorescence measurements, quenching of NO(A2Sigma+) is quantified as a function of temperature and collision partner. Rotational analysis of fluorescence excitation spectra is used to non-intrusively probe temperature inside a vacuum cell. I simulate a spectrum of the NO(A2Sigma+, v=0 <-- X2PI, v=0) band with a C-language program. Although the simulation does not work perfectly, most spectroscopic transitions are accurately identified. Additionally, I confirm the NO self- quenching cross-section at 300K and 400K to be approximately 40 Ang^2.

    Amy M. Gehring

    Preparation of Lewis Acid Homologs of b-Lactamase II

    b-Lactam antibiotics such as penicillin and cephalosporin are rendered ineffective by the action of classes of bacterial enzymes called b-lactamases. b-lactamase II of Bacillus cereus is unique as a zinc metalloenzyme; a zinc(II) ion in this enzyme's active site is required for the hydrolysis of b-lactam antibiotics and appears to coordinate directly to the b-lactam portion of the molecule. The goal of this project is to synthesize a simple chemical model of the b-lactamase II active site that may then be used to study the coordination of carbonyl molecules in such a system. 1,5,9-Triazacyclododecane and hydrotris(3-tert-butylpyrazol-1-yl)borate were both used as zinc ligands to approximate this enzyme active site. A compound consisting of 1,5,9-triazacyclododecane and diphenylcyclopropenone coordinated to zinc(II) appears to have been isolated. In this system, the carbonyl coordinates weakly to zinc(II) in a s-mode. Molecular orbital calculations verify that with either of the zinc(II)-ligand complexes, if coordination of carbonyls is observed at all, it will be quite weak.

    Doron Greenbaum

    Synthesis of a New Ruthenium Lewis Acid Complex

    The goal of this project was to synthesize a ruthenium Lewis acid template that was capable of stably binding organic carbonyls. A new ruthenium Lewis acid complex [(h6-C6H6)Ru(h1- O=C(CH3)2)(P(C6H5)3)2]2+ was synthesized and characterized by 1H NMR. The chemistry involved in the synthesis of this type of complex should add to the knowledge of organometallic chemistry as well as help clarify the general mechanisms of transition metal catalysis involving organic carbonyls. On the basis of molecular orbital calculations [(h6-C6H6)Ru(h1- O=C(CH3)2)(P(C6H5)3)2]2+ should stably bind organic carbonyls and it is possible that a b- lactam complex could be synthesized in the future that would help elucidate the mechanism by which b- lactamases catalyze the hydrolysis of the b-lactam ring in cephalosporin and penicillin.

    Nina D. Raoof

    Late Steps in the Total Synthesis of Paeoniflorigenone

    Meaningful progress towards the synthesis of paeoniflorigenone, a heavily oxygenated, stereochemically complex tricyclic monoterpene has been made. Paeoniflorigenone is thought to be the primary pharmacologically active constituent of the paeony root, which is used in herbal remedies in East Asia.

    Earlier work on this project focused on three areas. First was the synthesis of an intermediate tricyclic lactone. This compound is the central point from which one may synthesize a number of different components of the paeony root. Second was a preliminary exploration of the specific route by which paeoniflorigenone itself might be synthesized, and an analysis of ways in which the problems encountered might be overcome. The third was an investigation of the benzoate ester pathway towards paeoniflorigenone production.

    This year's work focused upon developing more successful conditions for the reduction of the hydroxymethyllactone using diisobutylaluminum hydride, as well as on the reduction of the lactol moiety with sodium borohydride, while attempting to complete the synthesis of paeoniflorigenone. The final Swern oxidation was attempted, but was unsuccessful, and a more effective protocol for this step must be determined.

    Barbara A. Roe

    Work Toward the Synthesis and Isolation of Three Per-pyridinium Benzoquinones

    Progress was made toward the synthesis and isolation of three per-pyridinium benzoquinones: bis(1,1'-(2,2'-bypyridinium)) 1,4-benzoquinone tetrachloride, 2,3,5,6-tetrakis 4- (dimethylamino)pyridinium 1,4-benzoquinone tetrachloride, and 3,4,5,6-tetrakis 4- (dimethylamino)pyridinium 1,2-benzoquinone tetrachloride. The determination of the optimal reaction conditions for the substitution of 4-dimethylaminopyridine and 2,2-bipyridine onto 2,3,5,6- tetrachloro- p-benzoquinone and 3,4,5,6-tetrachloro-o-benzoquinone was attempted through testing a variety of changes in conditions. Spectroscopic studies found that a major side product of these reactions was the protonated form of the pyridine. The attempted inhibition of the formation of the protonated pyridines by the exclusion of water led to the proposal of a mechanism that included a radical generating step. The presence of radicals in some of the reactions was supported by NMR spectroscopy and observations of solvent effects.

    Joseph P. Sadighi

    A 15N NMR Study of Azides

    A 15N NMR study was carried out on a series of twelve N-phenyl azoles. A new one-step synthesis of 1-phenyl-1,2,3-triazole was devised, based on the 1,3-dipolar cycloaddition of phenyl azide to the acetylene equivalent phenyl vinyl sulfoxide. This triazole and four tetrazoles were synthesized with enriched levels of 15N at specific sites to confirm the chemical shift assignments for each compound. The results of the labeling study confirm the earlier assignments for the 1-alkyl- and 1,5- dialkyltetrazoles, resolve a contradiction in the assignments for 2-alkyl- and 2,5-dialkyltetrazoles, and demonstrate the coincidence of two signals for 2,5-diphenyltetrazole.

    Tanya L. Schneider

    Bioactivity of Southeast Asian Dart Poison Components

    Dart poison from Southeastern Asia and its primary ingredient, Antiaris toxicaria sap, have long been a source of chemical interest. Early studies that investigated these materials and their toxic effect were crude and not definitive. Neither the structural identity nor the toxicity mechanism of the components of the sap and poison has been determined. An assay for Na+/K+ ATPase activity was established to test the inhibitory effect of the sap and poison on a simple level. Both the crude sap and poison mixtures were shown to inhibit enzyme activity. Using chromatography, particularly high pressure liquid chromatography, it was possible to separate the sap extract into fractions. Testing of these fractions in the enzyme assay revealed that the most polar and non-polar fractions demonstrated little inhibitory effect. The middle fractions showed varying effects on the enzyme; two fractions demonstrated a significant effect.

    Chu Ri Shin

    Determination of Chromium and Arsenic in Bridges and Moore's Ponds in Williamstown, MA and Hopkins Memorial Forest

    At present, the removal of fish from Bridges and Moore's Ponds, Williamstown, MA is prohibited due to the cancer risk of high levels of arsenic predicted by a 1987 Alliances Technologies study. A 1991 study by Nathalie Weicker reported low levels of chromium and undetectable levels of arsenic. In this study, hydride generation with a lower detection limit of 0.095 ppb As was used to analyze the concentration of arsenic and chromium in fish taken from Bridges and Moore's Ponds. Pond sediment, water, and vegetation, as well as soils from "untouched" Hopkins Memorial Forest were analyzed to investigate the possibility of bioaccumulation and biomagnification of these metals, and determine natural background levels for the Williamstown area. The sediment of Bridges and Moore's Ponds was found to contain levels of arsenic and chromium similar to the natural background occurrence for the Williamstown area. Bridges Pond sediment contained 7.1 ± 2.5 ppm As, and 34.2 ± 15.8 ppm and Moore's Ponds 4.2 ± 1.0 ppm As and 25.3 ± 14.1 ppm, and the natural background level determined from Hopkins Forest stream sediment was 6.2 ± 1.7 ppm As, and from Hopkins Forest surface soil, 28.9 ± 14.3 ppm. Vegetation samples from both ponds contained less than 5 ppm As. No bioaccumulation or biomagnification was observed in either pond. The water of both ponds contained less than 1 ppb As and the fish tissue of both ponds contained 0.0 ppm As. No cancer hazard due to potentially high levels of arsenic was found in the fish tissue of either pond, and the currently posted "Catch-and-Release-Only" signs at Bridges and Moore's Ponds can be removed.

    COMPUTER SCIENCE

    Daniel P. Fasulo

    Register Allocation in Functional Programming Languages

    Register allocation has traditionally been done by live-range analysis and graph coloring. However, researchers have begun to realize that this approach is not so useful in compilers for functional languages as in their traditional counterparts. Recently developed functional language compilers have therefore used different strategies that focus on reducing the overhead associated with function calls. Unfortunately, these strategies are often based more upon intuition than rigorous analysis.

    My thesis addresses this issue by describing a new optimization process in register allocation for functional language compilers. This process also focuses on reducing function-call overhead, but unlike its predecessors, it is based upon an abstract graph problem, which permits a more theoretical analysis of performance. In addition, the new optimization also makes use of the standard graph- coloring scheme, thus incorporating many of its benefits.

    Sean D. Sandys

    Support for Dynamic Itinerary-Based Communication

    Current technologies for programming massively parallel machines do not provide the programmer with the appropriate abstractions for communication. The Canister System was developed to allow programmers to compose point-to-point messages into communication patterns, called itineraries. While programmers may find this abstraction more appropriate for many algorithms, itineraries suffer from the fact that they must be statically determined at compile time.

    This work presents an extension to itinerary-based specification in the Canister System that allows the programmer to specify dynamic process instantiation and itinerary extension in a controlled manner. This mechanism, while not universal, allows programmers to specify more algorithms safely using a more appropriate level of communication abstraction.

    Angela M. Schuett

    Parametric Polymorphism in a Type-Safe Object-Oriented Programming Language

    This thesis describes the language PolyTOIL, a type-safe, polymorphic, object-oriented programming language. PolyTOIL is an extension of the object-oriented language TOIL, also developed at Williams College. The extensions to TOIL include support for bounded and unbounded parametric polymorphism. It is relatively unusual in using a "matching" relationship rather than subtyping to determine constraints on type arguments. Subtype polymorphism (through substitution) and inheritance are also features, though the subtype and inheritance hierarchies are not identified. A PolyTOIL interpreter has been implemented in ML and can be used to run PolyTOIL programs.


    Modified by: bbabcock
    Modification Date: Wednesday, March 8, 1995