Ashburn
Davis, D.
Davis, J.
Galbaugh
Langel
Martin
McCoy
Pesce
Phillips

John R. Ashburn, Jr.
Department of Medical and Clinical Psychology

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Doctor of Philosophy
2006

Major Advisor: Wendy Law, Ph.D., Department of Medical and Clinical Psychology

Dissertation Title: Neuropsychological Construct Structure of a Brief Computerized Neuropsychological Battery: Windows Spaceflight Cognitive Assessment Tool (WinSCAT)

ABSTRACT

Computerized performance assessment of neurocognitive functioning has increased tremendously over the past several years. However, little is known about how well these measures assess neurocognitive constructs they are purported to evaluate, especially in healthy, non-clinical populations. The Windows Spaceflight Cognitive Assessment Tool (WinSCAT) is a five-subtest battery derived from the larger Automated Neuropsychological Assessment Metrics (ANAM) computerized battery for use in evaluation and assessment of spaceflight crew members during space missions. The WinSCAT subtests also have been applied for assessing neurocognitive functioning in clinical populations. Findings indicate the WinSCAT subtests evaluate the cognitive domains of attention, executive functioning, memory, and possibly visuospatial processing. To determine the cognitive content structure of the WinSCAT in healthy non-clinical samples, two studies were performed based on both archival and prospectively-collected data sets. A battery of widely used, traditional clinical neuropsychological tests was administered with the computerized WinSCAT. Bivariate correlation and multiple regression data analyses were utilized to evaluate the extent to which the WinSCAT subtests were associated with specifically-hypothesized cognitive domains. Statistically significant demographic, general ability, and motor functioning variables were covaried to control for their potentially confounding contributions to relationships between the traditional and computerized testing measures. The WinSCAT tasks in the first (archival) study were found to predict performance on traditionally-derived index scores of attention, executive functioning, and memory. In the prospectively-collected data for study 2, the WinSCAT tasks were found to predict performance on traditionally-derived scores of executive functioning, memory, and visuospatial processing. The combined results across the two studies overall support the four-domain structure of the WinSCAT, with some differences in the specific domains supported in the separate studies. The neuropsychological domain of memory was supported in both studies, whereas the domains of attention, executive functioning, and visuospatial processing were supported in one of the two studies. Differences between the samples are believed to have contributed to the failure to obtain full support for all four domains across both studies. These results have implications for the appropriateness of future use of the WinSCAT to evaluate neurocognitive functioning for application with healthy and clinical samples.

Dawnavan Scott Davis
Department of Medical and Clinical Psychology

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Doctor of Philosophy
2006

Major Advisor: Tracy Sbrocco, Ph.D., Department of Medical and Clinical Psychology

Dissertation Title: Beauty is in the Eyes of the Beholder: Definitions of Attractiveness
Among African American and Caucasian Women

ABSTRACT

Obesity is a national epidemic affecting more than 127 million people (CDC, 2003). Nearly 78% of African American women in the U.S. are currently overweight or obese. Despite the negative health consequences associated with obesity, culturally-mediated views of attractiveness and body image may serve as risk factors for obesity among certain ethnic groups. The traditional body image literature has been constrained by entirely focusing on body thinness as the only component of attractiveness.

There is evidence to suggest that some African American women hold a multi-component definition of attractiveness (Harris, 1990, Parker, 1995). A culturally sensitive silhouette assessment method is needed to assess these components. The current study used a Model Rating Task (MRT) that extended previous silhouette measures to include: 1) dressed models; 2) models of heavier BMI categories, and 3) shaded models to represent African America stimuli. With the MRT, the impact of attire, body size, and model ethnicity on definitions of attractiveness could be examined. Participants were 80 African American and 80 Caucasian women with a mean age of 41.40 years, SD= 11.25, a mean body mass index of 28.30 kg/m2, SD= 6.78, an average education level of 15.43 years, SD= 2.51; and average yearly income of $50,000 (20,000). Mean attractiveness rating scores (ranging from 1 to 8) were compared between ethnic groups for dressed and undressed models across five BMI categories ranging from underweight to class II obesity. Participant ethnicity did not affect overall attractiveness scores, F(5,148)= .89, p=.49. However, model presentation did with both African Americans and Caucasians rating dressed models more attractive than undressed models, F(5,148)= 3.08, p<.01. Contrary to expectations, participant ethnic identity and SES did not differentially impact the effects of participant ethnicity or model attire status on attractiveness. Using regression analysis, model dress, F(5,152)= 3.15, p= .01, was the only significant predictor of attractiveness with higher ratings for dressed models.
Findings from this study provide some evidence for the utility of the MRT as a culturally appropriate assessment for attractiveness. Future studies should examine the validity of the MRT, the relationship of body image and attractiveness, and extend investigations of attractiveness to include other salient components such as facial features and skin complexion. Expanding our understanding of the construct of attractiveness and body image among African American women and other ethnic minority subgroups is a necessary step in developing culturally sensitive weight loss and weight gain prevention programs. It is crucial that researchers and health care providers understand that attractiveness may differ by culture and that the appearance based drive for thinness can not and should not be relied upon to motivate certain subgroups to achieve healthy body weight.

Jon M. Davis
Emerging Infectious Diseases

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Doctor of Philosophy
2006

Major Advisor: Alison D. O’Brien, Ph.D., Department of Microbiology and Immunology

Dissertation Title: The Modulation of Polymorphonuclear Neutrophil Function by
Cytotoxic Necrotizing Factor Type 1 - Expressing Uropathogenic Escherichia coli

ABSTRACT

Uropathogenic Escherichia coli (UPEC) cause more than 85% of all urinary tract infections (UTI). These infections primarily affect women, and over half of all women will experience at least one UTI in their lifetime. Animal models of UTI pathogenesis have provided some insight into the role of various UPEC virulence factors. In these animal studies, the toxin Cytotoxic Necrotizing Factor type 1 (CNF1) has been shown to have a significant role in the pathogenesis of UTI. One of the most striking features of CNF1-expressing UPEC infection in the in vivo models was magnitude of the acute inflammatory response. Compared to a cnf1 isogenic mutant, CNF1-expressing UPEC elicited an acute inflammatory response characterized by an extensive infiltration of polymorphonuclear leukocytes (PMN) into tissue infected with CNF1-expressing UPEC. In spite of the enhanced acute inflammatory response, the CNF1-expressing UPEC had a significant survival advantage compared to the cnf1 isogenic mutant. CNF1 is an AB toxin that deamidates the catalytically-active glutamine residue in the Rho family of small GTPases. The Rho family GTPases are intracellular signaling molecules responsible for the control of many cellular function in eukaryotic cells such as PMNs. In PMNs, Rho GTPases control the processes of phagocytosis and chemotaxis in addition to the generation of reactive oxygen species (ROS). These observations formed the foundation of the hypothesis that CNF1-expressing UPEC modulate PMN function. This hypothesis was tested in vitro with elicited mouse PMNs and showed that CNF1-expressing UPEC modulate the antimicrobial response of PMNs through several mechanisms. First, CNF1-expressing UPEC alter the capacity of PMNs to remodel their plasma membrane and cluster CD11b in response to serum-opsonized UPEC which leads to a diminished PMN phagocytic capacity. Second, CNF1 expressed by UPEC results in an increase in the amount of Rac2 in PMNs. Third, PMNs co-incubated with CNF1-expressing UPEC generate an enhanced intracellular ROS. Finally, CNF1-expressing UPEC release CNF1 via outer membrane vesicles that interact with the PMN membrane. These results support the hypothesis that CNF1 is a UPEC virulence factor and lead to a new molecular model of UPEC interaction with the host innate immune system.

 

Traci Galbaugh
Pathology

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Doctor of Philosophy
2006

Major Advisor: Dr. Mary Lou Cutler, Ph.D., Associate Professor, Department of Pathology

Dissertation Title: The regulation of lactogenic differentiation in mammary epithelial cells by Ras-dependent and -independent signal transduction.

ABSTRACT

Epidermal growth factor (EGF) stimulation of mammary epithelial cells (MECs) inhibits differentiation and apoptosis. Excess activation of signaling pathways downstream of the EGF receptor has been directly linked to breast cancer development and chemotherapeutic resistance. Therefore, the dissection of these pathways in normal MECs is critical to the understanding of signaling events that impact breast cancer outcome. HC11 mouse MECs differentiate in response to lactogenic hormone resulting in expression of milk proteins including -casein. Previous studies have shown that EGF blocks differentiation through activation of the Ras/Mek/Erk and the PI-3-K pathway. Therefore, specific chemical inhibitors of signal transduction pathways, activated Ras (RasV12), dominant negative-Ras (DN-RasN17), dominant negative-Akt adenovirus (DN-Akt-adeno) and a conditionally active-Akt (CA-Akt) were used to analyze the role of the Ras and PI-3-K pathway in blocking HC11 lactogenic differentiation.
 
Activated RasV12 expression resulted in reduced tyrosine phosphorylation of Stat5 and -casein expression in response to prolactin. However, the expression of DN-RasN17 enhanced Stat5 tyrosine phosphorylation, Stat5 DNA binding and -casein transcription. The expression of DN-RasN17 also blocked the activation of the Mek/Erk pathway by EGF and prevented the block of lactogenic differentiation induced by EGF. Stimulation of HC11 cells with prolactin resulted in the association of the SHP2 phosphatase with Stat5, and this association was inhibited by DN-RasN17 expression. Furthermore, the expression CA-Akt blocked lactogenic differentiation of HC11 cells. In contrast, treatment with LY294002 or infection with DN-Akt-adeno enhanced -casein transcription and rescued -casein promotor driven luciferase activity in the presence of EGF, demonstrating that the EGF block of HC11 lactogenic differentiation is, in part, dependent on PI-3-K/Akt. Moreover, the inhibition of either PI-3-K (LY294002) or mTOR (Rapamycin) abolished the activation of p70S6 Kinase (p70S6K) by EGF in HC11 cells, suggesting that PI-3-K signaling via p70S6K contributes to the EGF block of lactogenic differentiation. Additional investigation determined that EGF activates p70S6K resulting in the phosphorylation of RPS6, eIF4E and 4E-BP1 via PI-3-K/Akt/mTOR dependent mechanisms.
 
In conclusion, these results demonstrate that in HC11 cells, DN-Ras inhibits the Mek/Erk pathway and enhances lactogenic hormone-induced differentiation, in part by inhibiting the association of the SHP2 phosphatase with Stat5. In addition, this data demonstrates that the EGF-induced activation of PI-3-K stimulates the Akt/mTOR/p70S6K pathway to influence the regulation of lactogenic hormone-induced differentiation.

Felicia D. Langel
Microbiology and Immunology

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Doctor of Philosophy
2006

Major Advisor: Brian C. Schaefer, Ph.D., Assistant Professor, Department of Microbiology and Immunology

Dissertation Title: Molecular Mechanisms of Bcl10-Mediated NF-?B Signal Transduction

ABSTRACT

Bcl10 is a key signaling intermediate in the TCR-to-NF-?B pathway in T lymphocytes. It is currently believed that, once activated, Bcl10 functions within a multi-protein signaling complex that activates the IKK complex. Bcl10 is thought to regulate this signaling complex, but how it transmits its signal through the complex is unknown. A thorough knowledge of Bcl10 biology is critical to understanding how Bcl10 functions and how it regulates its binding partners. In this study, we used mutational analysis, molecular imaging, biochemistry, and computer/bioinformatics modeling to elucidate a structure and function for Bcl10. From our data, we identified a novel binding site for MALT1 within the Bcl10 protein, hypothesized that this site is completely separate and distinct from the binding sites of other Bcl10 signaling partners, and proposed two regulatory functions for the Bcl10 C-terminus. These findings suggest that Bcl10 has multiple functional domains and, hence, that Bcl10 molecular biology is more complex than previously thought. These observations serve to emphasize Bcl10's role as a crucial regulator of the TCR-to-NF-?B signaling pathway.

 

Nicole C. Martin
Emerging Infectious Diseases

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Doctor of Philosophy
2006

Major Advisor: LCDR Timothy H. Burgess, M.D., Department of Virology, Infectious Diseases Directorate, Naval Medical Research Center

Dissertation Title: Differential Dengue Tropism & Neutralization: Potential Mechanisms of Pathogenesis

ABSTRACT

The mechanisms underlying the pathogenesis of dengue hemorrhagic fever (DHF) remain poorly understood, but a clear correlation appears to exist between dengue viral load and disease severity. While antibody-dependent enhancement and T-cell-mediated pathogenesis theories suggest an immunopathologic basis for the development of severe disease, intriguing evidence suggest a role for viral strain differences. Consistent genetic differences exist in the envelope glycoproteins of dengue 2 strains associated with DHF epidemics (Asian genotype) and dengue 2 strains only associated with DF (American genotype). It has also been established that dengue virus infection can be mediated by C- type lectins DC-SIGN and L-SIGN. DC-SIGN is found on dendritic cells, a presumed target in human infection. L-SIGN is found on liver sinusoidal endothelial cells. Such cells have been shown to tolerize naïve T cells. To avoid the pitfalls associated with current measures of infection and neutralization, we developed an assay that uses cells expressing these relevant lectin receptors and low-passage viral isolates that yields results within 24 hours. Using this assay, we examined whether Asian and American genotype dengue 2 viruses exhibit differences in utilization of these two receptors. Our results showed that American strains infect DC-SIGN bearing cells to a greater extent than LSIGN bearing cells while Asian strains preferentially infect L-SIGN bearing cells. A single mutation in the envelope glycoprotein of an American strain at E390 from aspartic acid (American) to asparagine converted the C-type lectin binding phenotype from an American strain to an Asian strain by the observation that the E390 amino acid (aa) in the Asian strain is also asparagine. Furthermore, Asian and American strains differed in their sensitivity to antibody neutralization. The neutralizing capacity of mAbs 3H5 and 4G2 for Asian virus was significantly decreased when infection was measured in L-SIGN bearing cells compared to DC-SIGN bearing cells. We also found that serum from Venezuelan DF patients had much greater neutralizing capacity for Asian virus in LSIGN cells than serum from patients who progressed to DHF. Further, the magnitude of neutralization of L-SIGN-mediated Asian virus infection was inversely associated with disease severity. Taken together, our studies suggest that differences in receptor utilization and neutralization sensitivity between Asian and American dengue 2 strains may contribute to our understanding of the role that viral strain differences play in dengue pathogenesis.

 

Andrea Jennifer McCoy
Microbiology and Immunology

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Doctor of Philosophy
2006

Major Advisor: Dr. Anthony T. Maurelli, Ph.D., Department of Microbiology and Immunology

Dissertation Title: Genetic and Biochemical Characterization of Peptidoglycan Synthesis in Chlamydia

ABSTRACT

The Chlamydiaceae family of bacteria are obligate, intracellular pathogens that cause significant diseases world-wide in both humans and animals. Despite having a cell envelope that resembles other gram-negative bacteria, the presence of peptidoglycan in the Chlamydia cell envelope has long been debated. Unlike other wall-less bacteria, chlamydiae synthesize penicillin-binding proteins, are sensitive to antibiotics that inhibit cell wall synthesis, and encode a nearly complete pathway for the synthesis of peptidoglycan. However, peptidoglycan has yet to be detected. In this work, the functionality of the peptidoglycan synthesis pathway in C. trachomatis was examined by genetically and biochemically characterizing key enzymes in the pathway.
 
Chapter 1 assesses the functionality of the chlamydial murA gene product as a UDP-N-acetylglucosamine enolpyruvyl transferase. MurA from C. trachomatis commits UDP-N-acetylglucosamine to the synthesis of peptidoglycan. The native chlamydial MurA contains an aspartate in the active site that confers resistance to fosfomycin, an antibiotic that inhibits MurA. Chapter 2.1 examines the D-alanyl-D-alanine ligase activity of the unique chlamydial MurC-Ddl fusion protein when the chlamydial enzyme is expressed in Escherichia coli. Recombinant C. trachomatis MurC-Ddl catalyzes the specific ligation of two D-alanine. Furthermore, the D-alanyl-D-alanine activity of MurC-Ddl is the target of the antibiotic D-cycloserine. Chapter 2.2 examines the phenotypes observed when the chlamydial D-alanyl-D-alanine ligase is expressed in a D-alanyl-D-alanine requiring mutant of the facultative, intracellular pathogen Shigella flexneri. Finally, Chapter 3 summarizes attempts to identify the source of D-alanine for peptidoglycan synthesis in Chlamydia. In the absence of an apparent alanine racemase gene, the source of D-alanine for peptidoglycan synthesis in Chlamydia is assumed to be either the mammalian host or a chlamydial gene encoding racemase activity. Taken together, the characterization of key enzymes in the PG synthesis pathway of Chlamydia suggests that these organisms synthesize PG and that the chlamydial PG structure is of the same composition as PG in other gram-negative bacteria. Furthermore, these findings pave the way for future research to answer the questions of how, when and why PG is synthesized in Chlamydia. The functionality of the PG synthesis pathway in Chlamydia opens the door to discovery of new and the use of pre-existing cell wall inhibitors for the treatment of chlamydial infections.

 

John T. Pesce
Molecular and Cell Biology Program

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Doctor of Philosophy
2006

Major Advisor: William Gause, Ph.D., Department of Microbiology & Immunology

Thesis Title: Early Events Leading to the Host Protective Th2

ABSTRACT

Events necessary in the development of Th2 immune responses are poorly understood. A popular model used to study the development of these responses involves intracutaneous inoculation with the intestinal nematode parasite Nippostrongylus brasiliensis. Using B7-1/B7-2-/- mice infected with N. brasiliensis, we have shown that Th2 effector cells are capable of developing in the absence of B7 signaling interactions, although a substantial decrease in B cell Ag-specific Ab production was observed. To examine the mechanism of T cell activation, OVA-specific DO11.10 T cells were transferred to recipient mice, which were then immunized with a combination of N. brasiliensis plus OVA or either alone. Only the combination of N. brasiliensis plus OVA triggered T cell differentiation to OVA-specific Th2 cells, suggesting that N. brasiliensis acts as an adjuvant to stimulate Ag-specific naive T cells to differentiate to effector Th2 cells.

The adjuvant-like properties of N. brasiliensis suggested an innate component of the immune response may be involved in Th2 development. Using microarray analysis, draining ear lymph nodes from N. brasiliensis infected mice exhibited significant increases in CCL2 which is known to be involved in the recruitment of Gr-1+ neutrophils. Flow cytometric and immunofluorescent
analysis of infected lymph nodes resulted in the observation of an increased presence of Gr-1+ cells. Depletion experiments, using anti-Gr-1 Ab, resulted in disruption of the polarized Th2 in vivo immune response, characterized by significantly increased levels of IFN-? gene expression, IgG2a elevations, and increased worm burden. CCL2-/- deficient mice infected with N. brasiliensis were used to determine if CCL2/CCR2 interactions were required for Gr-1 recruitment. CCL2 deficiency resulted in significantly decreased Gr-1bright cell recruitment. Absence of this population had an effect similar to that observed in anti-Gr-1 treatment experiments with increases in IFN-? and Th1 associated immunoglobulins. Flow cytometric sorting and mRNA analysis of Gr-1bright cells revealed that they consist of a purely neutrophil population which expresses high levels of TNF-a and TGF-ß. These studies show the integral role that the innate immune response plays in the development of a highly polarized Th2
immune response.

Overall, these studies have made significant contributions to the understanding of the development of Th2 immune responses. The adaptation of DO11.10 system into a Th2 context provides an essential tool which will allow the determination of specific factors that result in the activation of naïve T cells. As a direct result of developing this tool, we identified a neutrophil population that is essential for the proper polarization of Th2 responses. This finding is quite significant in that this is the first time that a neutrophil population has been implicated in the development of a Th2 immune response. While this work is still in its infancy, the work detailed in this thesis provides evidence that neutrophils may prove to be a significant target for future drug interventions in the field of allergy and asthma.

Jennifer M. Phillips
Department of Medical and Clinical Psychology

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Doctor of Philosophy
2006

Major Advisor: Neil Grunberg, Ph.D., Department of Medical and Clinical Psychology

Thesis Title: “Effects of Clozapine and Alprazolam on Cognitive Deficits and
Anxiety-like Behaviors in a Ketamine-Induced Rat Model of Schizophrenia”

ABSTRACT

Schizophrenia is a debilitating mental illness that affects approximately 2.2 million Americans (1% of the population) each year. Despite the large number of people affected by schizophrenia, there is no known cure for this disorder but antispychotics help to manage the symptoms of schizophrenia. Unfortunately, antipsychotic medications are frequently accompanied by debilitating side effects (e.g., extrapyramidal side effects, tardive dyskinesia) and low compliance. Alternative pharmacological treatment options are needed to improve the treatment and quality of life for individuals suffering from schizophrenia.
This doctoral research project examined the hypothesis that the cognitive deficits associated with schizophrenia can be medicated indirectly using anxiolytics, drugs that decrease anxiety, based upon a proposed relationship between anxiety and cognitive disruptions in schizophrenia. The current research also evaluated the validity and potential usefulness of ketamine administration to create a novel animal model that includes symptoms of schizophrenia and anxiety.
Two experiments were conducted to address these goals. Experiment #1 examined the effects of clozapine (an antipsychotic), alprazolam (an anxiolytic), and a combination treatment on ketamine-induced cognitive disruptions in prepulse inhibition (PPI) of the acoustic startle reflex and passive avoidance. Experiment #2 examined the effects of clozapine, alprazolam, and a combination treatment on ketamine-induced anxiety-like behaviors on the elevated plus maze, open field test, and social interaction test.
The major findings of the study were: (1) ketamine administration caused cognitive disruptions in PPI as well as passive avoidance; (2) only clozapine attenuated the cognitive disruptions caused by ketamine, and only in the PPI measure; (3) ketamine administration caused an increase in anxiety-like behaviors on the EPM, open field locomotor activity, and social interaction; (4) only alprazolam decreased the ketamine-induced increases in anxiety-like behaviors, and only in the measure of social interaction. The findings failed to support the hypothesis that ketamine-induced cognitive deficits could be attenuated with anxiolytic medications. In addition, these findings suggest that ketamine administration may provide a useful, but limited, model of concurrent symptoms of schizophrenia and anxiety.