Undergraduate Research Fellows 1998-1999

Priya Banerjee

Superantigens (SAgs) are immunogenic proteins capable of stimulating a large percentage of T cells (up to 20% of the entire population). The magnitude of T cell activation resulting from SAg exposure is far greater than that seen with conventional antigens. In human disease, SAgs have been linked with sever biological reactions such as food poisoning, toxic shock and autoimmune diseases. They stimulate T cells primarily by crosslinking the variable region of the beta chain of the TCR (VB) with the MHC II molecules on antigen presenting cells. Thus, T cells expressing a particular VB are specifically selected for proliferation followed by anergy and apoptosis (programmed cell death). One of the best characterized SAg is staphylococcal enterotoxin B (SEB). It is a 24.8 kD monomeric protein with 5 a-helices and 12 B-pleated sheets. Recently, inhibition of SEB-induced T cells proliferation was demonstrated using synthetic peptides to the 141-156 amino acid region. Moreover, it contains the first five of the ten highly conserved amino acids among SAgs, KKKVT. Thus, the 141-156 peptide provides a suitable basis for creating a SEB neutralizing antibody. The process of creating a neutralizing antibody entails 1) propagation of the antigen in an artificial system, 2) immunization and purification of the antibody to the antigen, and 3) in vitro analysis of the antiserums ability to block SEB effects on immunocompetent cells.

Natalie Bucheimer Incorportation Of Radioactive Galactose Into The LOS Of Neisseria gonorrhoeae

Neisseria gonorrhoeae is a human pathogen responsible for gonorrhea in humans. This organism expresses a surface antigen, lipooligosaccharide (LOS), on its outer membrane. This molecule, composed of lipid A and a core oligosaccharide, is immunogenic and therefore thought to be involved in Neisseria pathogenicity and virulence. A method for tracking LOS synthesis and expression would be useful in studying modifications and variability of LOS involved in pathogenesis. The gonococcus is unable to utilize galactose as a carbon source, yet galactose is a key sugar in LOS structure. Successful introduction of a radiolabeled galactose into the gonococcus should yield expression specifically in the LOS and not in other sites of biosynthesis, making galactose a good target for labeling LOS. E. coli possesses a galactose operon consisting of three genes, E, J, and K, that have been previously cloned and sequenced. This operon will be inserted into a plasmid expressed in E. coli that is capable of mobilizing during conjugation. Conjugation of this donor strain with the gonococcus will produce conjugates capable of expressing radioactive galactose from the media. In this way a strain of Neisseria gonorrhoeae will be created specifically for the study of LOS synthesis and expression. This strain will hopefully be useful in studying the pathogenicity of the organism in humans.

Andy Grollman Investigating Premature Cell Death in Yeast

I am doing my work with Dr. Marco Colombini on mutant yeast cells that are missing both genes that code for the Voltage Dependent Anion Channel (VDAC). This channel is located on the outer membrane of the mitochondria and is responsible for the passage of certain metabolites used for ATP synthesis. Yeast cells without VDAC were shown to have an increased lag phase when transfered from storage in glucose to lactate which was not seen for the wild type yeast cells. I will be using a flowcytometer to measure the mitochondrial membrane potential of the mutant cells during their growth curve to determine the cause and effect relationship of the missing VDAC on these cells.

Brian Higgins

Eashen Liu Comparative Study of Sound Localization Between Congeneric Species

Jeremy Michalke Distribution of Lymphohematopoietic Cells In Feline Fetuses

he formation of blood, called hematopoiesis, begins in the early yolk sac but as embryogenisis proceeds, this function is taken over by the fetal liver and finally by the bone marrow. The hematopoietic stem cell(HSC) gives rise to all of the formed elements of the blood, from platelets to the immune system. Consequently it is possible to completely reconstitute the blood of an irradiated animal with new stem cells.

Headed by Dr. Peter Gasper, our lab is focusing on testing the efficiency of using fetal hematopoietic cell (FHC) transplantation as a vaccine for HIV. Specifically, we will be transfecting genes that endow lymphohematopoietic cells with lifelong protection against lentivirus infections. The model we are using is feline immunodeficiency virus (FIV). FIV is a naturally occurring retrovirus of domestic cats that provides valuable resources for understanding mechanisms of pathogenesis and for the development of effective antiviral therapy and vaccines with direct relevance to HIV.

In order to use FHC, we must first understand the distribution and characteristics of lymphohematopoietic cells in fetuses. How is FHC distributed in the fetal liver? When does FHC develop histocompatibility complexes? When is FHC most active? My current and planned research attempts to answer these questions.

Anjalee Mitra The Distribution of GABAergic Fibers Along The Spinal Cord Of The Lamprey

Iris Ng Type III Secretion System Analysis- The Study of Type III Secretion System in Aeromonas spp.

Aeromonas spp. was found to be associated with infections of human wounds especially those sustained in aquatic environments. These infections include localized extensive muscle necrosis and septicaemia that accompanied by skin lesions, fever, chills, and hypotension. It was found that Aeromonas spp. is also an etiological agent of human diarrheal diseases that can be either self-limiting or a life-threatening cholera-like disease. The pathogenicity of Aeromonas spp. is mainly caused by a number of extracellular proteins it secreted into infected host cells. Many of the genes for these virulence proteins have been cloned and sequenced. They include aerolysin, lipase, chitinase, and amylases. Other toxins that were identified include hemolysins, enterotoxins and a variety of different proteases. Among these, aerolysin and GCAT (a lipase) has been shown to be secreted by the general secretory pathway. Since many of the pathogenic mechanism of Aeromonas spp. remain unknown, our study is to identify a newly proposed virulence factor secretion machinery - Type III Secretion System in Aeromonas spp.. We hope to find out the translocated bacterial proteins in the host cells and to determine the distribution and concentration of these secreted protein.

The Type III Secretion System is a specialized protein secretion machinery that export virulence factors delivered directly to host cells. These factors subvert normal host cell functions in ways that are beneficial to invading bacteria. In contrast to the general secretory pathway, type III secretion system is triggered when a pathogen comes in contact with host cells, and the export of extracellular protein is independent of the sec-machinery. To examine the type III secretion system in Aeromonas spp., chromosomal DNA of different clinical and environmental strains of Aeromonas spp. were isolated and purified. The purified DNA was then blotted onto a nylon membrane and was probed with radioactive fragments of the hrp gene from Pseudomonas syringae and the Locus for Enterocylic Effacement (LEE) region from Enteropathogenic E. coli (EPEC). Both of these regions were found to encode components of type III secretion systems. The complementarity of Aeromonas spp. genetic material to these genes can also be assayed by PCR. Using a conserved type III apparatus sequence to construct primers, we can potentially clone the type III genes in Aeromonas spp. , and subsequently analyze the protein sequence. The detection of translocated bacterial proteins in the host cells can eventually be done by immunofluorescent staining with antibodies, confocal microscopy and optical sectioning. The distribution and concentration of the secreted protein can also be studied by Western immunoblotting.

Kyong Pae Pak Characterization Of Novel Hrp-1 Regulated Genes In Pseudomonas syringae

The hrp gene cluster found in Pseudomonas syringae strains, along with avr genes, have been shown to control the pathogenicity and host range of the bacterium. It has also been demonstrated that an alternative sigma factor known as Hrp-L positively regulates both hrp and avr genes. Using this knowledge, a simple plate assay was developed to look for novel Hrp-L regulated genes in the genome of P. syringae strains that might also function in pathogenicity. The plate assay relies on a unique two promoter based system involving the lactose operon and the arabinose operon. A genomic library of P. syringae strain PSS61 will be screened for Hrp-L dependent promoter fragments. These fragments will be characterized by using DNA sequence analysis. The PSS61 genome will then be probed for the Hrp-L regulated gene and mutated to determing the effect on hypersensitive response and pathogenicity in host plants.

Katrina Pei Construction Of Self-Assembled Protien-DNA Nanostructures

This project proposes methods to construct two and three dimensional nanostructures through biological self-assembly and protein-DNA interactions. The construction of these nanostructures involves the use of DNA designed to have lac operator site in orientation controlled by cyclization constraints. Due to the cooperative interaction between bivalent lactose repressor proteins and operator DNA sites, which are target sites for the repressor, DNA "squares" are brought together to form structures in the shape of a two-dimensional grid, a three-dimensional cube, and a sandwich. The assemblies are expected to have dimensions of 20 to 30 nm.

Alexandra Pratt Exploration Of Sexual Mating Of The Unicellular Algal Genus Haematococcus/H3>

The alga Haematococcus is unique in that it accumualted red pigments called cartenoids in the cytoplasm when placed under stress (high light or nitrogen deficiency). As a member of the Volvocales family and as a close relative of the widely studied Chlamydomonas reinhardtii, it is assumed that Haematococcus is capable of sexual mating, and the genetic relatedness of various H. pluvialis strains. The project will ultimately contribute to the growing body of knowledge of Haematococcus that will be helpful in making the organism a uselful transformation system for studying carotenoid snythesis.

Erica Rosenbaum Interaction Among Pre-mRNA Splicing Factors

Kamyar Sartip Crystallization Of The VDAC Channel

Elaine Sit Investigation Of The _imp_ Gene As A New Anti-Bacterial Drug Target

Bacterial infections once easily combated with antibiotics have become resistant to standard treatments. During the past decades, formerly reliable antibiotics have become increasingly ineffective. Moreover, some of these "superbugs" have developed resistance to the actions of later-generation antibiotics once reserved for extreme situations. What can be done to create new drugs against these new super-resistant microbes?

One approach is to identify new targets for antibiotics. A novel and promising candidate is the imp gene. This gene was identified in the common bacteria, Escherichia coli. Mutations in this gene significantly increase the permeability of E. coli to various detergents, dyes and antibiotics; hence the gene's name: increased membrane permeability. A deeper understanding of the gene and its protein product may facilitate or advance the discovery of novel antimicrobial drugs. Presently, E. coli strains that carry imp mutations are used by several pharmaceutical firms to screen natural products for potentially effective anti-bacterial treatments.

My project is directed at investigating imp's role in cell physiology. Currently, this is not known. I will approach this problem by determining why overproduction leads to death. An attractive hypothesis is that imp is involved in a biological pathway for membrane growth. A classic way to probe a biochemical pathway is to isolate mutations that compensate for an alteration to the pathway. I will attempt to test my hypothesis by isolating mutations that suppress the overproduction-lethality phenotype. If imp is involved in a membrane growth pathway, I should be able to isolate mutations in known membrane pathway genes that suppress the imp overexpression-lethality phenotype and gain a great deal of insight on the inner workings of this gene.

Ali Tabatabai Sequencing And Phylogenetic Analysis of Dinoflagellate RuBisCo

Kevin Tsai Enhanced Production of Biologically Active Protiens In Drosophila

Anjali Kaushiva Determination of the Structural Interaction Between the RecD and RecB Subunits of the RecBCD Enzyme from Escherichia coli

Syed Ali

Chung Cho Studying Roles of HMG-I/Y in neoplastic transformation

Meng-Hsiu Wu Interaction of Ethylene Hormone Receptors of Arabidopsis

Mary Torregrossa Effects of Oxytocin on Female Reproductive Behavior in the Prairie Vole

Grace Han Structural Determinants of DNA Modification Under UV Light

Anne Silva Synthesis of Photoactive Probes to Map the Active Site of Glycoprotein Remodeling Enzyme, Glucosidase I

Syed Zeyad Identifying Genome Organization in Pseudomonas syringae pv. tomato DC3000 A possible pathogenicity island have been shown to be located in the Pseudomonas syringae genome called the hrp cluster that might have evolved through horizontal transfer. Many of the effector proteins, host range determinants, and virulence factors have not been located and proposed to be dispersed in various locations in the genome. Although there is little information about the P.syringae chormosomal genetics, no funding have been successful to map the whole genome due to the availability of the P.aeruginosa genomic map. Using a specifically designed promoter trap assay, this work screened the P. syringae pv. tomato DC3000 genome for promoter active fragments and ran a base pair comparison of these fragments with the known gene database in general and P.aeruginosa database specifically. It was found that almost two thirds of the seqeunces obtained have unidentified genes or unknown similarities to the P.aeruginosa database. It is concluded that the Pseudonomas syringae genome remains a mystery and that a mapping project of the genome of this organism will add important insights on the biology of this organism.

Jeremy Edwards Function of heteromorphic sperm in a species of stalk-eyed fly, Cyrtodiopsis dalmanni Cyrtodiopsis dalmanni produce two distinct sperm morphs based on length, a short and a long. By examining fertilized embryos I hope to determine which sperm morph is capable of fertilization. Also, by characterizing and quantifying sperm production in males of different ages, I hope to discover if sperm morphology or production changes with age.

Jennifer Young Protein-Protein Interaction Between Semushi, SUMO-1 and RanGAP in the Nuclear Transport Pathway Using a Yeast-Two Hybrid System

Marc Egeth Parasites and Sexual Selection in the Satin Bowerbird

Sin Kei Yeung The Effect of Neuropeptide Y on Hypothalamic Neurotransmitter Overflow

Intracerebroventricular (ICV) injections of Neuropeptide Y (NPY) stimulate orexigenic behaviors (food intake) in satiated animals. Several studies have shown that the ventromedial (VMH), paraventricular, and other specific hypothalamic sites mediate the action of NPY to stimulate feeding. Previous work by from the laboratory of Thomas W. Castonguay, Ph.D. reported that NPY promoted an increase in serotonin in the VMH from tissue punches taken from rats 15 minutes post NPY injection. Different dialysate collection techniques have been used to investigate the dynamics of NPY action. As a sequel to this prior work, in vivo microdialysis was used to describe the time course of the changes in VMH serotonin subsequent to NPY administration. The present investigation of NPY measured the effects of a single ICV NPY injection on the release of serotonin, as well as other neurotransmitters and their metabolites in the VMH. Dialysates were collected before and after NPY injection using a 1 mm CMA 12 microdialysis probe placed in the VMH and were analyzed with a HPLC equipped with an ESA electrochemical detector. Significant animal-to-animal variation in response to NPY has been observed.

David Klein

The Significance of Short-Term Postnatal Oxytocin and Arginine Vasopressin Manipulations in the Prairie Vole, Microtus ochrogaster

Oxytocin (OT) and arginine vasopressin (AVP) are mammalian neuropeptides. AVP and OT are made primarily in the central nervous system, but released into both the peripheral circulation and into the brain. OT in particular plays a major role in mammalian birth and lactation. These peptides also are manipulated at birth by humans, both accidentally and intentionally. For example, the common drug Pitocin, or synthetic oxytocin, is given to a large percentage of women in labor to increase uterine contractions. Human milk contains OT and the decision to breast feed may affect the peptide exposure of an infant. Little is known regarding either the behavioral or anatomic effects of such interventions during normal development.

Varying by only two amino acids, OT and AVP have extensive interactions and can affect each other's receptors. This is especially important because AVP has central effects on the hypothalamic-pituitary-adrenal (stress) axis and regulates the autonomic nervous system. I hypothesize that neonatal manipulations of OT or AVP will influence developing neural circuits and that these neural changes will have long-lasting effects on behavior. In addition, the level of alteration will be dependent on the animal's sex, the dose of hormone (or antagonist) injected, and the duration of time from the initial injection. Furthermore, pilot studies suggest that voles raised in isolation will have amplified results. As medical professionals continue to order synthetic forms of Oxytocin for pregnant females, this research will potentially portray additional positive and negative physiological effects of this peptide, emphasing neonatal changes.

Michael Atkin Determination of the Immunoregulatory Mechanisms Involved in IFN; Modulation of the Diabetic Process

Mihir Desai Characterizing in Vivo Chromatin Complexes and Binding Sites of DNA-binding Transcription Factors

Julia Redman A Molecular Genetic Screen for Factors That Regulate as-l-dependent Transcripton in Plants

Kevin Capps Toward Understanding The Mechanism of Oligosaccharyltransferase

Cary Pirone Patterns of Starch Accumulation in Legumes Injured By A Sap-Feeding Insect