Stephens Curriculum Vitae With Abstract

7/27/2019 Stephens Curriculum Vitae With Abstract

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David B. Stephens, Ph.DCurriculum Vitae

7621 Leawood Blvd.Little Rock, AR 72205-2519

Work phone: 501-661-2801Home phone: 501-291-4140Cell phone: 501-772-7621

[email protected]

Education

1990 1996

University of Texas, Department of Chemistry and Biochemistry, Austin, TX .- Doctor of Philosophy in Biochemistry under the supervision of Professor Brent L. Iverson .- Title of Dissertation: "Polyclonal Catalytic Antibodies".- 5 years as Research Assistant and 4 years as Teaching Assistant.- Graduate-level coursework in Microbial Genetics and Computational Chemistry.

1983 1989University of Arkansas, Little Rock, AR.- Bachelor of Science in Chemistry.- Bachelor of Science in Biology.

Miscellaneous

1992 - Certified Electronics Technician Training, Austin Community College, Austin, TX1988 - Electronic Design Technology, NRI, Washington, D.C.

Research Experience

2001-2005

Research as a part-time postdoctoral fellow into the role of Rab small GTPase proteins in vesiculartrafficking, using the NIH model organismDictyostelium discoideum, in the laboratory of Dr. John M.Bush at the University of Arkansas at Little Rock. Investigations into the role of the contractile vacuole asa calcium homeostasis organ, implied by the presence in association with the CV of a proton pump, acalcium-proton antiport, calmodulin, and Rab11 bearing a possible pH-dependent CaM-binding site, forwhich I was a winner of an NSF Research Opportunity Award fellowship in 2002.Skills Acquired:

Dictyostelium cell culture.Transformation by electroporationGST-pulldownMutagenic PCR.Shuttle vector manipulation.UV confocal microscopy with digital interface.Research grant writing and submission.


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1995-1997Research as a postdoctoral fellow into the solubilization, purification, and reconstitution of the transporterassociated with antigen processing (TAP) in the laboratory of Dr. Matthew Androlewicz at the H. LeeMoffitt Cancer Research Center, University of South Florida. TAP is an ATP-hydrolytic peptide pumpsituated in the endoplasmic reticulum membrane, involved in the peptide charging of the majorhistocompatibility complex 1. The initial problem was the determination of conditions that would allow

for the solubilization of this integral membrane protein without loss of biological activity. Experimentalevidence has now been collected which demonstrates peptide binding by TAP solubilized under the properconditions and reconstituted into proteoliposomes; I determined that an important factor in maintenanceof peptide binding function was the replication of the proportions of different phospholipids found in theER membrane, leading to the publication of the first paper detailing the reconstitution of peptide-bindingactivity in solubilized TAP. Research was performed on the suitability of immunoaffinity chromatographyof solubilized TAP with competition elution utilizing the free TAP epitope, with inconclusive results.Hydrophobicity indices were generated for TAP primary sequences to aid in elucidation of transmembranedomains.Skills Acquired:

Mammalian cell culture.Radioiodination of proteins.Liposome and proteoliposome formation.Membrane protein solubilization and reconstitution.Autoradiography.Research grant writing and submission.

1990 1996

Research as a graduate student into production, isolation, and characterization methods for polyclonalcatalytic antibodies in the laboratory of Dr. Brent Iverson at the University of Texas at Austin. Thisresearch involved the organic synthesis of both transition-state-analog haptens and substrates, injection ofand blood collection from laboratory animals, isolation of pure IgG from sera, and a variety of techniquesfor catalytic and immunoassay. My initial work determined that hapten-affinity chromatography cannot

purify catalytic antibodies from whole IgG samples, a stumbling block to earlier attempts; bothmonoclonal and polyclonal catalytic antibodies failed to elute from a hapten-functionalized column undera wide variety of conditions. Whole IgG fractions were thus used, with preimmunization sera used as acontrol, and hapten titrations of catalytic reactions were used to estimate catalytic fraction. Studies of

both the maturation of the catalytic immune response and variation of response between individuals wereperformed. Mathematical models predicting the behavior of different distributions of pooledheterogeneous catalysts were constructed. Subsequently, a substrate column for catalytic elution ofabzymes was successfully developed, allowing for significant enrichment of the catalytic fraction in theIgG samples. Studies were performed to determine binding kinetics of catalytic fraction for hapten.Skills Acquired:

Antibody productionNMR spectroscopy.Affinity FPLC.Reverse-phase HPLC.Quantitative UV-Visible spectrophotometry.Enzyme-linked immunosorbent assay.Acrylamide and agarose electrophoresis.DNA ligation.

Protein purification.Enzyme kinetics.Phosphoramidite and phosphonium synthetic techniques.


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1988 1989Research as an undergraduate utilizing X-ray crystallography of halo-substituted picolines and nicotinatesin the laboratory of Dr. Alcuin F. Gremillion at the University of Arkansas at Little Rock.

1987 1988Research as an undergraduate involving the characterization of the quaternary structure of deer malate

dehydrogenase (EC 1.1.1.40) by starch gel electrophoresis in the laboratory of Dr. Alvan A. Karlin at theUniversity of Arkansas at Little Rock.

Employment Experience

2005-PresentHealth physicist for the Radiation Control Section, Arkansas Department of Health and Human Services.Responsible for inspection of sources of ionizing radiation, shielding calculations for installations, anddose assessment modeling. Training in hazardous materials and radiological emergency response,environmental sampling, and computer modeling of environmental contamination and biokinetics.

2002

Botany Instructor at the University of Arkansas at Little Rock.

2000-2002Instructor in Introductory Physical Science at Pulaski Technical College in North Little Rock.

1997-2002Event Horizon Development (http://www.eventhorizondev.com/html/ehd.htm).Proprietor of scientific and technical consultation business. Expert witness for attorney Hubert Alexanderin several cases. Development of web sites. Consultation and tutoring. Research into bioremediationtechniques, information retrieval and analysis, and computational chemistry. Independent contractor asInternet resource researcher for Hungry Minds, Inc. (http://www.hungryminds.com/) for a column calledScience in the News, StudyWeb (http://www.studyweb.com/) in the areas of Biology and ComputationalChemistry, and the Open Directory Project (http://dmoz.org) in the area of Molecular Evolution.

1999Visiting Assistant Professor of Chemistry, University of Central Arkansas, Conway, Arkansas.

1993-1994Volunteer high school chemistry teacher, Marywood Children and Family Services, Austin, TX.

1990Department of Radiochemistry, Arkansas Department of Health, Little Rock, ARRadiochemist working under Dr. George Dilbeck, responsible for the preparation and analysis of variousenvironmental solid and liquid samples for alpha, beta, and gamma radiation counts. Experience inscintillation count, alpha-beta emission count, and gamma spectroscopy. Developed a standard for swabsused to test GC detectors for63Ni leaks.

1988-1989

Quality Assurance laboratory technician for Tyson Foods of North Little Rock, ARMicrobiological assays of product and plant surfaces forEscherichia coli,Listeria monocytogenes,Staphylococcus aureus, and Salmonella, activity titrations of hypochlorite and quaternary ammonium saltantiseptic solutions, free fatty acid titration of cooking oil to determine extent of oxidation, and statisticalquality control.
http://www.studyweb.com/http://dmoz.org/http://www.studyweb.com/http://dmoz.org/


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Memberships

- American Chemical Society #2205461- American Society for Cell Biology #48130- Health Physics Society #34794- Mensa.

Publications

Stephens, D.B., and Iverson, B.L.(1993) "Catalytic Polyclonal Antibodies", Biochemical and BiophysicalResearch Communications 192:1439-1444.

Stephens, D.B., Wilmore, B.H., and Iverson, B.L.(1994) "Polyclonal Antibodies and Catalysis",Bioorganic and Medicinal Chemistry 2:1-6.

Stephens, D.B., and Androlewicz, M.J.(1997) "Reconstitution of Peptide-binding Activity by TAP inProteoliposomes", FEBS Letters, 416: 353-358.

Stephens, D.B., Thomas, R.E., Stanton, J.F., and Iverson, B.L.(1998) "Polyclonal Antibody CatalyticVariability", Biochemical Journal, 332:127-134.

Stephens, D.B., B.N. Mitra, and J.M. Bush (2003) "Analysis and function of Rab GTPases inDictyostelium discoideum: Key regulators of vesicular membrane transport and multicellulardevelopment", Recent Research Developments in Cell Biology, 1:203-236.

Presentations

August, 1993"Catalytic Polyclonal Antibodies", 206th ACS National Meeting, Chicago, IL.

October, 1996"Progress in the Purification and Reconstitution of Active Human TAP into Proteoliposomes", SuncoastBiomolecular Science Conference, Tampa, FL.

December, 2003

"Null Mutation of the Dictyostelium Water Channel Gene AquaporinA (aqpA) Affects ContractileVacuole Function, Fluid Phase Transport and Phagocytosis", American Society for Cell Biology AnnualMeeting, San Francisco, CA

Awards

2002

NSF Research Opportunity Award

2005Finalist for a CDC Emerging Infectious Diseases grant under the auspices of the Arkansas Department ofHealth.


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Dissertation Abstract

Pointillism, a technique of painting in which small, distinct dots of pure color are applied in patterns toform an image, was developed by Georges Seurat in 1886; the most famous example of this technique may

be the painting La Parade de Cirque, a summertime view of a Parisian park. In the perception of thebeholder this myriad of distinct pigment points blends to form a smooth concerted view of the scene, a

bright happy tableau of picnickers, strollers and boaters. The immune response can be compared to thisvisual phenomenon; a multitude of responding B-cell clones, each producing a pure antibody moleculewith unique primary sequence and binding characteristics for the antigen, are brought to bear against thematerial stimulating the immune response, resulting in a smooth concerted response to this material. Asfirst noted by Linus Pauling years ago, the immune response develops exquisite binding affinity andselectivity of antibodies for their ground-state antigens, whereas enzymes possess the same bindingaffinity and selectivity for the transition states of reactions; this observation ultimately gave rise to theconcept of producing catalysts using the immune response to develop antibodies against transition stateanalogs.

The advent of catalytic antibodies opened a unique new window for the study of the immune response andbiocatalysis. The production of catalytic antibodies, with the use of the immune system as a molecularevolution engine to obtain antibodies possessing binding pockets with high transition state affinity,complements studies such as site-directed mutagenesis of enzymes admirably; the former works from the

theoretical direction towards enzyme-like catalysis while the latter is capable of incremental dissection ofknown biocatalytic sites. The vast majority of catalytic antibody studies have been done with monoclonalantibodies, harvested from immortalized B-cell clones grown in culture. Such monoclonal approaches are

preferred for mechanistic studies, but lack perspective as to the range of catalysts obtained and temporalcourse of response stretching the analogy given above a little further, these have been studies of each

point of color in isolation, in a highly artificial environment. Monoclonal studies can also be expensiveand time-consuming. Earlier attempts to isolate the full polyclonal catalytic response, in effect to look atthe entire panoply of colors in the portrait of the response, had met with failure and had been abandoned.By careful construction of controls and avoidance of hapten affinity chromatography, we were able tocharacterize a complete immune and catalytic response in mice and rabbits to the phosphoniumtransition-state analog for the hydrolysis of the triphenylmethyl (trityl) ether protecting group; this is agood choice as there is no known enzyme catalyzing the hydrolysis of this linkage. Our results indicatethat hapten affinity chromatography permanently removes and/or denatures the catalytic species present,hampering the earlier studies. Analysis of preimmunization serum for catalytic activity, along with

competitive inhibition of immunized serum activity with free hapten molecules, allowed forcharacterization of the catalytic fraction in the absence of hapten affinity isolation. Initial resultsindicated an apparent catalytic rate enhancement (kcat/kuncat) of 125 and a Km of 31 M, comparable withmonoclonals previously generated against the same moiety. The catalytic response exhibited a later andsteeper rise than the immune response, implying onset of catalysis as the immune response matures andantibody-hapten affinity increases. Subsequent experiments showed surprising levels of homogeneity ofcatalytic response both within and between individuals. Plasmon resonance techniques were used to study

binding kinetics, a substrate column was developed to effect catalyst enrichment by activity, and amathematical treatment of the behavior of pooled catalysts was developed.


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Contacts

Dr. Brent L. IversonUniversity of Texas at AustinDepartment of Chemistry and Biochemistry1 University Station A5300

Austin, TX 78712(512) [email protected]

Bernard BevillRadiation Control Section ChiefArkansas Department of Health4815 W. Markham, Slot 30Little Rock, AR 72205(501) 661-2107

[email protected]

Donald GreenePreparedness and Response

Arkansas Department of Health4815 W. Markham, Slot 30Little Rock, AR 72205(501) [email protected]

David SnellingsHealth Systems Licensing and RegulationArkansas Department of Health4815 W. Markham, Slot 30Little Rock, AR 72205(501) [email protected]
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]

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