Dissertations: 2003

Austin-Lane                              Crowder                      Kalupa
Bossart                                     Geisbert                      LeBaron
Byrnes                               Hakre                     Quigley
Ceremuga                          Hook                       Steele
Chavez                              Hughes                   Vest
                                                                    Williams

Joy Austin-Lane
Department of Preventive Medicine and Biometrics

Doctor of Public Health
2003

Major Advisor: Galen L. Barbour, M.D., Department of Preventive Medicine and Biometrics

Dissertation Title: Influences on Tobacco Control Funding Decisions:
Explaining State Variation in Appropriations

ABSTRACT

This policy research on state tobacco control funding decisions provides a conceptual framework and quantitative model for explaining this outcome. Studying resource allocation for tobacco control is important because of the impact increased funding can have on health outcomes. One published study on state variation in tobacco control appropriations reported little explanatory value of tobacco burden of disease factors. The current research used both qualitative and quantitative methods to identify factors that were useful in explaining this outcome following the state settlements with the tobacco industry.

Key informant interviews with state policy experts yielded a comprehensive list of 26 influences on state tobacco control funding decisions. In a modified Delphi process, experts completed ratings of importance and the 11 factors that received the highest ratings were: budget situation, state priorities, tobacco industry economic and political activity, the role of the governor, the role of a legislator who champions tobacco control, public opinion, dedicated tobacco control funds, tobacco control advocacy, leadership, and coalitions. A conceptual diagram was developed to portray the relationships of these factors with state tobacco control funding.

To test the explanatory value of these factors, a multiple linear regression model was developed with existing cross-sectional data on all 50 states. Five measures were significantly associated with the outcome and explained 49% of the variance. The 26 states with Gross State Product from tobacco had lower allocations for tobacco control (Tobacco Economy). The 27 states with laws preempting stricter local ordinances were less likely to allocate funds for tobacco control (Tobacco Industry Political Activity). States that had Democratic governors with a high degree of executive authority allocated more funding for tobacco control (Role of Governor). Citizen liberalism was positively associated with higher allocations for tobacco control (Public Opinion).

These results imply that a strong scientific case for tobacco control is not sufficient to secure prevention funding at the state government level. Attention to political and economic aspects of the state budget process could lead to increased tobacco control funding. Further research is needed to determine whether these factors are predictive of appropriations in future years and how the factors can be used to influence future funding decisions.

Katharine Nina Bossart
Department of Microbiology and Immunology

Doctor of Philosophy
2003

Major Advisor: Dr. Christopher C. Broder, Department of Microbiology and Immunology

Thesis Title: Structural and Functional Studies on the Fusion and Attachment Envelope Glycoproteins of Nipah Virus and Hendra Virus

ABSTRACT

Nipah virus (NiV) and Hendra (HeV) virus are emerging, biosafety level 4 paramyxoviruses responsible for fatal zoonotic infections of humans from pigs and horses, respectively, and are the prototypic members of a new Paramyxovirinae genus called Henipavirus. These enveloped, negative-sense RNA viruses infect cells through a pH-independent membrane fusion event mediated through the actions of their attachment (G) and fusion (F) envelope glycoproteins, which are also the principal antigens to which neutralizing antibodies are directed. Understanding the biological and functional features of the viral glycoproteins will help define the characteristics and properties of these novel viruses, and may provide insights into membrane fusion mechanisms, the virus infection process, and towards the development of therapeutics. Here, recombinant vaccinia virus vectors were generated to express the NiV and HeV glycoproteins. Glycoprotein functions and their cellular tropism characteristics were examined with a quantitative assay for membrane fusion. NiV and HeV glycoprotein-mediated fusion could be blocked by virus-specific antisera or synthetic peptides corresponding to the C-terminal a-helical heptad repeats of NiV or HeV F. Both F and G glycoproteins were required for membrane fusion and a broad species and cellular tropism pattern was observed for both HeV and NiV. Further, protease treatment of receptive host cells abolished viral glycoprotein-mediated fusion activity, suggesting a cell-surface protein serves as a receptor for these viruses. In addition, interactions between the glycoproteins of the paramyxoviruses have not been well defined, but studies reported here show the NiV and HeV glycoproteins are capable of a highly efficient heterotypic functional activity amongst themselves, but not with other related paramyxoviruses. Finally, although closely related, these viruses were also distinguished from known paramyxoviruses by possessing a strong physical interaction between either viral F glycoprotein and the HeV G glycoprotein, while little interaction was noted between NiV G and either viral F. These later findings may provide a crucial new system for elucidating the paramyxovirus binding and entry process.

Kimberly R. Byrnes
Neuroscience Program

Doctor of Philosophy
2003

Major Advisor: Juanita J. Anders, Ph.D., Associate Professor of Anatomy, Physiology, and Genetics, and Neuroscience

Thesis Title: 810 NM Light Treatment of Acute Spinal Cord Injury Alters the Immune Response and Improves Axonal Regeneration and Functional Recovery

ABSTRACT

Spinal cord injury (SCI) results in substantial and often permanent impairment of function due to the lack of regeneration of damaged axons. Despite vigorous research, no cure for SCI has been found. Light therapy (LT), through the absorption of light by target tissue, improves healing in a number of injury models. However, no study to date has assessed the ability of LT to facilitate the regeneration of specific spinal cord tracts. Our hypothesis was that transcutaneous application of 810 nm light promotes axonal regeneration and functional reinnervation following transection of the corticospinal tract (CST) by changing the extracellular milieu of the spinal cord. Three studies were implemented to investigate this hypothesis. First, anterograde and retrograde tract tracing techniques were used to investigate axonal regrowth after SCI and LT. LT (810 nm) was applied at the site of acute injury to the CST of adult rats. Anterograde tract tracing demonstrated that LT improved axonal regrowth after injury, with significant increases in axon number (199 +/- 12) and distance of regrowth (8.7 +/- 0.8 mm) as compared to controls (p<0.01). Double label retrograde tract tracing revealed that transected axons regrew and reinnervated motor neurons in the lumbar spinal cord in the light treated group only (p<0.05). Functional analyses revealed that this regeneration was coupled with significant improvement in 2 tests of CST performance, angle of rotation and ladder beam cross time (p<0.05). Second, to explore the effect of LT on the spinal cord cellular environment, we investigated the inflammatory response after SCI, using quantitative immunohistochemistry techniques. This study revealed that LT suppressed the invasion/activation of macrophages, microglia and T lymphocytes after SCI (p<0.001) and delayed the activation of astrocytes. The third study explored gene expression after SCI and LT. A number of cytokines and chemokines were assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of interleukin 6, monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS) was suppressed at 6 hours post-injury by LT (p<0.01). These results demonstrate that LT has an anti-inflammatory effect on the spinal cord after injury and significantly improves axonal regeneration and functional recovery.

Major Thomas E. Ceremuga
Neuroscience Program

Doctor of Philosophy
2003

Major Advisor: Joseph T. McCabe, Ph.D., Vice-Chair of Department of Anatomy, Physiology & Genetics; and Professor of Molecular and Cellular Biology, and Neuroscience

Thesis Title: Cullin 5 Expression in the Rat: Cellular and Tissue Distribution, and Changes in Response to Water Deprivation and Hemorrhagic Shock

ABSTRACT

Protein degradation by ubiquitination and the 26S proteasome is used to modulate the steady-state levels of proteins and to regulate cellular processes. Proteins become targets of the proteasome by covalent attachment of polyubiquitin chains, which requires three main enzymes (E1, E2, and E3). It is the E3 ubiquitin ligases that control the selection and specificity of substrate ubiquitination. Cullin-5 (Cul-5), a member of the cullin family of E3 ubiquitin ligases, remains obscure. The goals of this research project were to characterize Cul-5, and investigate its response to cellular stresses of water deprivation and hemorrhagic shock in the rat.
Northern blotting of poly(A)+ RNA from various rat tissues demonstrated the cul-5 transcript is approximately 6.3 kb. Reverse transcription-polymerase chain reaction (RT-PCR) indicated cul-5 mRNA is present in twelve tissues examined: brainstem, cerebral cortex, cerebellum, hypothalamus, aorta, gastrointestinal tract, heart, kidney medulla, liver, lung, skeletal muscle, and spleen. Quantitative realtime PCR confirmed RT-PCR results that Cul-5 mRNA is ubiquitously expressed and that levels are similar in all tissues. Cellular specificity examination showed cul-5 mRNA expression in rodent neuronal, glial, and vascular endothelial cells in the central nervous system (CNS) via RT-PCR. We corroborated these data by immunocytochemical techniques demonstrating Cul-5 protein presence in neurons, astrocytes, blood vessels, and choroid plexus in rat.

Functional assays measured cul-5 mRNA expression responses to water deprivation and hemorrhagic shock. Quantitative realtime PCR showed significant cul-5 mRNA elevations in the rat cerebral cortex (3 fold, p<0.001), hypothalamus (2 fold, p<0.007), and kidney (1.5 fold, p<0.04) following 48 hours of water deprivation. Water deprivation for 24 hours or rehydration (24 hours access to water following 48 hours of water deprivation) also increased kidney cul-5 mRNA levels (1.5 fold, p<0.04 and 3 fold, p<0.001 respectively). Hemorrhagic shock was used as a second in vivo cellular stress model. Rats were subjected to volume controlled (27 ml/kg) hemorrhage over 10 minutes and kept in shock for 60 minutes. Levels of cul-5 mRNA were significantly increased in the brainstem and cerebellum (1.6 fold, p<0.01 and 1.5 fold, p<0.05 respectively), and decreased in the hypothalamus (0.5 fold, p<0.05) compared to sham-treated rats.

We determined that Cul-5 is synthesized in all tissues and organs we examined, and in neurons, glia, and endothelial cells in the CNS. Using two paradigms of cellular stress, we found cul-5 mRNA levels in the CNS are altered by water deprivation and by hemorrhagic shock. However, much remains to be revealed concerning what precise physiological role(s) Cullin-5 plays in various cellular processes.

Bonnie Roberts Chavez
Department of Medical and Clinical Psychology

Doctor of Philosophy
2003

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

Thesis Title: Effects of Stress and Relaxation on Time Perception

ABSTRACT

Changes in time perception during and following experiences of stress and relaxation are commonly reported, but little is known about the direction and nature of any time perception changes. In this experimental study, men and women ages 18 to 79 were randomly assigned to one of three conditions: stress (n = 39) - speech preparation and presentation, relaxation (n = 38) - a progressive muscle relaxation exercise, or control (n = 39) - listening to a biography on tape. Multiple measures of time production, time estimation, perceived rate, and attention to time domains (past, present, and future) were made before and after the experimental phase. Retrospective time estimates were made after the experimental phase. Self report, physiological, and biochemical measures of stress were used to evaluate response to the experimental conditions, and results were consistent with increased stress in the stress condition and increased relaxation in the relaxation condition. Measures of duration judgment were relatively consistent within individuals with larger variance from person to person. Some of the individual variance was associated with gender and age with women having generally larger duration judgment ratios (subjective/objective time) than did men (t(110)=2.91, p<.01) and age was significantly correlated with duration judgment ratio. Time was reported to pass slower than usual during relaxation (t(34) = 5.37, p<.01), but time spent relaxed was remembered as significantly shorter than time spent in the control activity (F(2,108) = 9.88, p <.01). Stress led to increases in subjective to objective time ratio (t(37) = 2.34, p <.05), and time following a period of stress was reported to pass quickly. Attention to the present was related to lower reported distress, whereas attention to the past was positively correlated with self-reported distress. These data imply that changes in time perception may reflect changes in experiences of stress or relaxation. Also, interventions to increase focus of attention on the present may be helpful to reduce distress.

A. Tamara Crowder
Neuroscience Program

Doctor of Philosophy
2003

Major Advisor: Thomas E. Cote, Ph.D., Department of Pharmacology

Dissertation Title: Co-expression of Regulator of G Protein Signalling 4 (RGS4)
and the mu opioid receptor in regions of rat brain:
Evidence that RGS4 attenuates mu opioid receptor signalling

ABSTRACT

Regulators of G protein Signalling (RGS) proteins influence G protein-coupled receptor signal transduction by enhancing the intrinsic GTPase activity of G proteins. The RGS-enhanced GTPase activity of G proteins may be responsible for the desensitization of certain G protein-coupled receptors, including the mu opioid receptor. The goal of this research was to evaluate the ability of recombinant RGS4 to affect mu opioid receptor-mediated cellular signalling and to identify regions of the rat brain in which both RGS4 and the mu opioid receptor are co-expressed.

We evaluated the ability of recombinant RGS4 to affect [D-Ala2, N-Me-Phe4, gly-ol] enkephalin (DAMGO)-mediated inhibition of adenylyl cyclase activity in membranes of SH-SY5Y cells, a cell line that express endogenous mu receptors. Recombinant RGS4 caused a concentration-dependent attenuation of DAMGO-mediated inhibition of adenylyl cyclase activity.

RGS4 diminished the efficacy, but not the potency, of DAMGO in inhibiting adenylyl cyclase activity. In contrast, RGS4 had no effect on the ability of GTP S, a nonhydrolyzable analogue of GTP, to inhibit adenylyl cyclase activity. RGS4 also had no effect on DAMGO stimulated [35S]GTP S binding in SH-SY5Y membranes. Additionally, RGS4 was tested for its ability to affect [3H]DAMGO binding to the mu receptor. RGS4 failed to affect either the KD of the Bmax of [3H]DAMGO in saturation binding experiments.

Antibodies generated against rat RGS4 and the rat mu opioid receptor were used in immunohistochemical staining to identify specific regions of rat brain where the two proteins are co-expressed. Both RGS4 and mu opioid receptor proteins were present in many of the same regions of the brain. Further, we demonstrated that RGS4 is primarily localized to the nucleus, but that administration of fentanyl, a potent mu opioid agonist, induces translocation out of the nucleus, to the cytoplasm in the hippocampal CA3 pyramidal neurons.

Together, these findings are consistent with the proposal that RGS4 can desensitize mu opioid receptor by increasing the intrinsic GTPase of Gi-type G proteins associated with the mu opioid receptor and that, in vivo, RGS4 and the mu opioid receptor are co-expressed in many of the same regions of the rat brain.

Thomas W. Geisbert
Department of Pathology

Doctor of Philosophy
2003

Major Advisor: Elliott Kagan, M.D., F.R.C.Path., Department of Pathology

Thesis Title: "Pathogenesis of Ebola Hemorrhagic Fever in Primate Models
In Vivo and In Vitro

ABSTRACT

Ebola virus (EBOV) causes severe hemorrhagic fever (HF) with high mortality in humans and nonhuman primates. Despite progress made during the last decade to identify key modulators of EBOV pathogenesis, cultural mores, and a range of logistical problems, have hindered the systematic pathogenetic analysis of human EBOV infections. Nonhuman primate models of EBOV HF were developed, but with few exceptions, previous investigations examined animals naturally infected or killed when moribund, and shed little light on the pathogenesis of infection during times before death. In this study, we investigated the process(es) triggering the coagulation abnormalities characteristic of primate EBOV infections and attempted to identify the sequence of key morphologic, virologic, and inflammatory events. This study examined tissues of 21 nonhuman primates over time and also temporally evaluated EBOV infection of primary human monocytes/macrophages (PHM) and endothelial cells in vitro. Results showed that tissue factor plays an important role in triggering the hemorrhagic complications that characterize EBOV infections, and dysregulation of protein C exacerbates disease. Increased levels of TF were associated with lymphoid macrophages, while analysis of peripheral blood mononuclear cell RNA showed increased tissue factor transcripts by day 3. Analysis of PHM RNA at 1, 24, and 48 hours showed increased tissue factor transcripts while levels of tissue factor protein were dramatically increased by day 2. A rapid drop in plasma protein C levels was evident in all monkeys by day 2. Moreover, replication of EBOV in endothelial cells was not consistently observed until the latter stages of disease, well after the onset of disseminated intravascular coagulation, suggesting that the characteristic coagulation abnormalities are not the direct result of EBOV-induced cytolysis of endothelial cells. Dendritic cells in lymphoid tissues were identified as early and sustained targets of EBOV implicating their role in the immunosuppression characteristic of EBOV infections. Bystander apoptosis and loss of NK cells was a prominent finding suggesting the importance of innate immunity in determining the fate of the host. Accordingly, primate models have been invaluable in identifying several new targets for chemotherapeutic interventions that may ameliorate the effects of EBOV HF.

Shilpa Hakre
Department of Preventive Medicine and Biometrics

Doctor of Public Health
2003

Major Advisor: Donald Roberts, Ph.D., Department of Preventive Medicine and Biometrics

Thesis Title: The Epidemiology of Malaria in Belize, 1989-1999

ABSTRACT

Retrospective analyses were conducted to describe the epidemiology of malaria in Belize over a 10-year period and to determine if environmental factors influenced the incidence of malaria at macro- and micro-scales. The purpose was to contribute to the current body of knowledge regarding malaria transmission in Belize, which may aid in region-specific malaria control efforts.

Malaria data were obtained from the National Malaria Control Program's National Malaria Database. Malaria rates were calculated using the 1991 population census of Belize (Central Statistics Office). Other data were obtained from the National Meteorological Service (Belize), National Center for Environmental Prediction (NCEP), National Weather Service (U.S.A.), Global Land One-Kilometer Base Elevation (GLOBE), National Geophysical Data Center, Land Information Centre (LIC, Belize), village and vector surveys, Ministry of Health (MOH), SPOT and Landsat multi-spectral images. Most of the data from these sources were collected for purposes other than the specific aims of the studies in this dissertation.

Over a 10-year period, malaria incidence rates varied temporally and spatially; southern and central areas of Belize had consistently higher rates of malaria than northern areas. Toledo District had the highest Plasmodium vivax incidence; whereas, Stann Creek District had the highest P. falciparum incidence. Malaria incidence was highest during 1993 through 1996. Plasmodium falciparum incidence was highest in the transitional months preceding the wet season in Stann Creek.
Vector surveys conducted in villages in the districts of Cayo, Stann Creek, and Toledo indicated that Anopheles darlingi was most common and abundant in Stann Creek District whereas, An. albimanus was most common and abundant in the other two districts. The epidemiology of P. falciparum incidence among very young children in Stann Creek, along with the common occurrence and seasonal abundance of An. darlingi, indicates that malaria transmission occurred locally and An. darlingi was the likely vector of P. falciparum in that district.

Preliminary results indicated malaria incidence differed geographically by season, type of vegetation, and proximity of villages to rivers or streams. Examination of associations between weather and malaria incidence indicated that precipitation was associated with malaria transmission. Higher total rainfall was associated with a higher malaria risk in villages. An assessment of the relationship between rainfall and malaria incidence in microenvironments, represented by districts, indicated that the relationship seen for the country was especially significant in Cayo and Toledo Districts, where higher rainfall increased malaria risk in villages; whereas, the opposite was seen for Corozal and Orange Walk. Examination of the relationship between vegetation and malaria incidence indicated that more forest cover was associated with higher risk of malaria in villages. This relationship was specifically seen in Belize, Cayo, and Stann Creek Districts.

Environmental risk factors and malaria incidence were assessed in households in San Martin, Cayo District and Red Bank, Stann Creek District. In San Martin, in 1997, proximity of a household to a stream, number of male occupants in a household, and having a history of malaria in a household were predictive of whether a household had malaria. In San Martin, malaria incidence was highest in males, especially in the 11 to 14 and 35 to 39 age groups. In Red Bank, in 1997, having a history of malaria in a household, construction of the outer walls, and the number of females in a household were predictive for malaria in a household. The 0 to 4 year-old age group had the highest malaria incidence in Red Bank.
Vector surveys conducted in 1997 and 1998 in both study villages showed An. albimanus was most common in San Martin; whereas, An. darlingi was most common in Red Bank. Anopheles darlingi and An. vestitipennis were also collected in San Martin, and An. albimanus, but not An. vestitipennis, was also collected in Red Bank in addition to An. darlingi. Malaria cases clustered by household in both villages. In San Martin during 1993 through 1998, only three to eight percent of households produced 50 percent or more of malaria cases. Similarly, in Red Bank during 1993 through 1998, only five to 12 percent of households produced 50 percent or more of malaria cases.

Malaria incidence varied by region, year, season, and populations in Belize during the study periods and these variations are linked to differences in environmental variables. Malaria control efforts might be more effective if environmental variables were accommodated in malaria control planning.

Gary L. Hook
Department of Preventive Medicine and Biometrics

Doctor of Philosophy
2003

Major Advisor: Philip A. Smith, Ph.D.

Title of Dissertation: Application of Solid Phase MicroextractionCoupled with Gas Chromatography/mass Spectrometry as a Rapid Method for Field Sampling and Analysis of Chemical Warfare Agents and Toxic Industrial Chemicals

ABSTRACT

The first question that must be answered prior to substantive quantitative exposure monitoring, regardless of the sampling and analysis method employed, is: What chemicals are present? In order to answer this question rapidly, there is increasing demand for field analysis of volatile and semi-volatile organic compounds with instrumentation that provides definitive identification. The military's interest in this capability stems from Presidential Review Directive 5 and other Department of Defense implementing instructions that have established the requirement for developing better means for operational exposure assessments and documentation of troop exposures during deployments.

Numerous methods have been developed for field analysis of organic compounds. However, these methods may have limitations such as lack of sensitivity, high false positive identification rates and provide only screening capabilities. Laboratory analysis, often with complex sample preparation requirements, is still required for confirmation of a chemical's identification.

The objective of this work was to evaluate the use of solid phase microextraction (SPME) coupled to gas chromatography/mass spectrometry (GC/MS) as a rapid method for field sampling and analysis to answer the important question of "What chemicals are present?" and other related questions that may arise.

To reach this objective, SPME sampling followed by analysis with a field portable GC/MS system was evaluated. The qualitative abilities of SPME-GC/MS were evaluated in an industrial workplace containing air contaminants from a poorly characterized paint and in an unplanned, emergency response situation. The qualitative abilities were further examined through development of field sampling and analysis methods to detect the chemical warfare agent (CWA) VX as a contaminant on clothing and soil. The quantitative potential for SPME-GC/MS was
examined through the development of a rapid method for estimating the airborne concentration of the CWA sarin.

This work demonstrated the robust nature and flexibility of the SPME- GC/MS system to rapidly detect, identify, and quantitate organic chemicals of widely varying volatility in the field while providing high quality data. This ability gives rise to wide application in industrial, emergency response, homeland security and military deployment operations for characterization of potential health, safety, and security hazards.

Capt Teresa M. Hughes
Department of Medical and Clinical Psychology

Doctor of Philosophy
2003

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

Dissertation Title: “Understanding the Regulation of Body Weight:
A Focus on Eating Patterns, Energy Intake, and Metabolic Rate”

ABSTRACT

The clinically observed eating pattern of gorging (eating fewer, larger meals later in the day) is ill defined in the literature and inconsistently linked to metabolic rate suppression and body composition. The purpose of this study was to further understand the relationship between gorging, metabolic rate, and body composition with a consideration of daily physical activity and purposeful exercise. Gorging was defined as two or fewer meals per day with at least seven hours between waking and the first meal for at least three days per week. It was hypothesized that gorgers would have 1) lower metabolic rates, 2) more body fat, 3) lower energy and higher fat intakes, and 4) more pathological eating attitudes than non-gorgers. Participants were 12 obese gorging (OG), 11 obese non-gorging (ON), 14 normal weight gorging (NG), and 14 normal weight non-gorging (NN; age-matched), non-smoking, otherwise healthy women. Metabolic testing included assessment of resting metabolic rate (RMR), active metabolic rate (AMR) while riding a stationary bicycle at a rate of 50 rpm and workload of 1kg, and dietary induced thermogenesis (DIT) where postprandial metabolic rate was assessed. Results were partially supported. Contrary to the first hypothesis, the eating pattern groups did not differ by RMR [F(1,47) = 3.96, p = 0.05], AMR [F(1, 47) = 2.03, p = 0.16], or DIT [F(1, 47) = 0.40, p = 0.53] after covarying lean body mass. Lean body mass was the best predictor of metabolic rate accounting for 72% of the variance. These findings are limited by the small effect sizes for these analyses. Future investigations should increase statistical power and consider increasing the both the exercise and meal challenges so they may have a greater affect on metabolic rate. The eating pattern groups did not differ by body composition [F(1,47) = 1.02, p = 0.32], contrary to the second hypothesis. However, the gorging group reported less energy intake than the non-gorging group [F(1,47) = 14.50, p = 0.001] supporting the third hypothesis. Estimated energy intake for the participants was calculated using measured RMR multiplied by an activity factor of 1.3. Based on these results the gorgers underreported food intake to a greater degree than the non-gorgers. There was an interaction effect for fat consumption. The ON consumed the greatest amount of fat followed by the obese and normal weight gorgers, and then the NN group. Contrary to the fourth hypothesis, there were no significant differences between the eating pattern groups’ eating pathology. This was one of the first studies to thoroughly operationalize gorging and examine existing eating patterns. While several of the major hypotheses were not supported, the results of this study do support the importance of the relationship between weight regulating behaviors, body composition, and metabolic rate. The findings are clearly worth further study and it is clear that a biopsychosocial framework is needed when examining the interrelationship of eating patterns and metabolism.

Kimberly L. Kalupa
Department of Medical and Clinical Psychology

Doctor of Philosophy
2003

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

Title of Dissertation:The impact of a low-fat diet and the use of fat substitutes on fat preferences among overweight women seeking weight loss treatment

ABSTRACT

Overweight and obesity form the basis of the second leading cause of preventable death in the United States and are on the rise ENRfu(NIH, 1998). Traditionally, recommendations for weight loss include a reduction in dietary fat (Drewnowski, 1990). However, adherence to low-fat diets remains a challenge. Hedonic responses to food may be important to understand the adherence problem. When compared to bland-tasting foods, nutritionally-equivalent foods that are flavorful are more satiating (Rolls, 1995; Warwick et al., 1993). Two previous studies that examined the sensory impact of dietary fat modification reported conflicting results (Mattes, 1993; Guinard et al., 1999).

The current study evaluated the impact of dietary fat modification on fat preference in three weight loss groups. The impact of changes in fat preference on short-term adherence to a 6-week weight loss program was assessed. Sixty-one otherwise, healthy overweight women (BMI 25-37 kg/m2) between the ages of 18-60 were randomly assigned to one of three brief, 6-week weight loss programs. All three groups were asked to reduce dietary intake to 1800 kilocalories per day, but one group was asked to maintain fat at 36%, and two additional groups were asked to consume 20-25% of kilocalories in fat. One low-fat group was instructed to use fat substitutes and the other was instructed to avoid substitutes. It was hypothesized that decreased fat intake and sensory exposure to fat would cause a decrease in preference for high fat foods.

Excellent adherence was observed with over 91% of participants completing treatment. In addition, guidelines regarding kilocalories, respective fat intake, fat substitute use were followed. Taste changes that occurred were subtle and did not affect on short-term dietary adherence. More time on the prescribed fat levels might be required to induce a change in fat preference. Differential changes in fat preference following dietary fat modification were expected, but not observed in the current investigation. However, results suggest that a modified 6-week Behavior Choice program might be a viable treatment option for women in this age and weight category (BMI < 37 kg/m2).

Matthew J. LeBaron
Department of Pathology

Doctor of Philosophy
2003

Major Advisor: Hallgeir Rui, M.D., Ph.D., Associate Professor,
Department of Pathology

Thesis Title: Technologies for Genome-Wide Identification of Stat5 Regulated Genes

ABSTRACT

Each year more than one million new cases of breast cancer are diagnosed worldwide and an estimated 370,000 women die from breast cancer. Although the vast majority of fatal breast cancer cases involve metastatic spread of the primary tumor, the formation of metastases is still a poorly understood, complex process. Identifying the early molecular changes that facilitate metastasis of breast cancer will lead to new molecular targets for prevention of metastases and improved therapies.

Intriguing data from the mentor's laboratory show loss of activation of the transcription factor Stat5 during breast cancer progression and that tumors without active Stat5 have higher histological grade, increased mitotic rate, and unfavorable prognosis. Furthermore, data developed in the preparation of this dissertation indicate a substantial growth-inhibitory and pro-differentiation role for Stat5 in mammary epithelial cells. Based on these and other recent observations, we postulate that loss of Stat5 activation in breast cancer represents a progression event that leads to dedifferentiation and increased risk of metastatic invasion. Therefore, a critical analysis of the role of Stat5 in human breast cancer is warranted, including systematic efforts to identify genes directly controlled by Stat5.

The recent completion of the human genome sequence presents new opportunities for global identification of Stat5 target genes. Work performed in the preparation of this dissertation has established new methodology to capture, clone, sequence, and validate physiological Stat5 DNA-binding sites in a genome-wide manner. The method can also be used to determine whether Stat5 interacts with a known Stat5-responsive promoter within a given experimental context when coupled with PCR amplification of the target DNA. Using this methodology, we have demonstrated that glucocorticoids markedly alter the pattern of chromatin access for Stat5 binding in breast cancer cells. Furthermore, we also report novel methodology to specifically identify transcripts directly regulated by Stat5, based on dominant-negative, differential suppression of Stat5 regulated transcripts and large-scale gene chip analysis. As a result of this work, rapid progress in genome-wide identification of Stat5 target genes is now possible, as well as molecular mapping of the regulatory role of Stat5 in breast cancer.

John F. Quigley
Department of Medical and Clinical Psychology

Doctor of Philosophy
2003

Major Advisor: David S. Krantz, Ph.D.

Title of Dissertation: Autonomic and Hemodynamic Correlates of Daily Life
Activity and Ambulatory Myocardial Ischemia in Patients with Stable Coronary Artery Disease

ABSTRACT

Impaired autonomic-cardiac regulation, as defined by reduced levels of heart rate variability (HRV), is an independent predictor of death in patients with coronary artery disease (CAD) and a history of myocardial infarction. Moreover, transient shifts in HRV have been observed before the acute onset of ambulatory myocardial ischemia in CAD patients. The present study investigated whether or not changes in autonomic-cardiac regulation: (1) were associated with altered hemodynamic regulation in CAD patients; (2) could identify CAD patients at-risk for ambulatory myocardial ischemia; or (3) were related to changes in physical exertion and heart rate levels before the onset of ambulatory myocardial ischemia. Fifty-three patients with documented CAD (44 men; mean age 65 years) participated in 48-hours of ambulatory electrocardiographic (Holter), physical activity, and blood pressure monitoring. Holter tapes were analyzed for evidence of myocardial ischemia and were used to generate several indices of HRV, including markers for sympathetic and parasympathetic tone, and their ratio, sympathovagal tone.

Results indicated that CAD patients with lower levels of HRV (n = 26) had significantly higher levels of heart rate than did patients with preserved levels of HRV (n = 27; p < .05), whereas HRV was found to be unrelated to patients’ blood pressure levels (p’s > .05). Contrary to predictions, ischemic CAD patients (n = 9) were found to have significantly higher levels of HRV and significantly lower levels of heart rate when compared to non-ischemic patients (n = 44; p’s < .05). These differences were not related to patients’ medication regimens, ejection fraction, or history of reperfusion procedures (p’s > 05). Ischemic and non-ischemic CAD patients did not differ in terms of their HRV, heart rate, or blood pressure levels during prescribed non-ischemic periods of matched “low” and “high” physical activity (p’s > .05). Regarding ambulatory ischemia (18 episodes), results indicated that there was a steady, significant increase in physical activity (p < .05) and heart rate levels (p < .05) over the half-hour preceding the onset of ischemia. During the peak in this activity (i.e., for the 10 minutes preceding ischemia), there was also a corresponding increase in sympathovagal balance (p < .05). The duration of ischemia was significantly longer in CAD patients with lower levels of 24-hour vagal tone (p < .05). Collectively, these findings suggest that activity-related changes in autonomic tone and heart rate are complicit in the induction of exertional ischemia.

In conclusion, the findings of this study revealed that global markers of HRV do not discriminate CAD patients at-risk for ischemia, but are nonetheless important variables to consider whenever investigating the “triggering” of ambulatory myocardial ischemia by periods of heightened physical activity and cardiac demand levels. Study limitations include the low incidence of ambulatory ischemia – a possible consequence of keeping patients on their medications during their participation in this study. Future research should consider analyzing HRV in a larger sample of CAD patients at risk for myocardial ischemia.

Keith E. Steele
Department of Pathology

Doctor of Philosophy
2003

Major Advisor: Radha K. Maheshwari, Ph.D.

Title of Dissertation: Tunicamycin Enhances Neuroinvasion and Pathogenicity in Mice with Venezuelan Equine Encephalitis Virus

ABSTRACT

Venezuelan equine encephalitis (VEE) virus infects horses and humans and is a potential biothreat pathogen. Agents that alter the blood-brain barrier (BBB) may enhance viral encephalitides. The current studies explored whether tunicamycin (TM) and other agents affect the pathogenesis of VEE.

Following infection with the molecularly-cloned virulent V3000 virus TM-treated mice had a significantly (p < .05) decreased mean survival time (MST) of 7.3 days versus 9.9 days in controls. Using plaque assay, V3000 reached nearly 107 pfu/gram in the brains of TM-treated mice at 48 hours post inoculation (PI) versus 103 pfu/gram in controls. The attenuated V3034 strain invaded the brains of TM-treated mice by 48 hours PI versus 72 hours for controls and TM-treated mice had 100-fold or more virus at all times. TM-treated and control mice had similar viremia profiles with both viruses and similar V3000 brain replication curves. Immunohistochemistry showed that V3000 invaded the brains of TM-treated mice by 24 hours, versus 48 hours for the controls. V3000 appeared to enter the brain via the olfactory tract, not via the BBB, in both groups. TM-treated mice with V3000 exhibited earlier clinical signs and greater weight loss than controls. The brains of TM-treated mice had earlier and more severe inflammation, neuronal damage, and extravascular fibrinogen and upregulation of several cytokines. Electron microscopy of the brains of uninfected, TM-treated mice revealed perivascular edema and swollen astrocyte endfeet.

Additional studies failed to show that pyridostigmine, LPS or TNF-a significantly altered VEE. Pyridostigmine and LPS-treated mice with V3034 showed similar neuroinvasion patterns as controls and those with V3000 showed similar MSTs as controls. Serum TNF-a was increased in V3000-infected mice (peak - 52.9 pg/ml at 24 hours). However, mice given antibodies to TNF-a had identical neuroinvasion patterns as controls.

These findings indicate that TM enhances neuroinvasion and pathogenicity in mice infected with VEE viruses but fail to indicate that pyridostigmine, LPS and TNF-a affect VEE similarly, suggesting TM is unique in its effect on VEE virus. Tunicamycin could be a useful tool in further studies of VEE virus neuroinvasion.

LTC Kelly G. Vest
Department of Preventive Medicine and Biometrics

Doctor of Public Health
2003

Major Advisor: CAPT David Trump

Title of Dissertation: Risk of Peripheral Nerve Disease in Military Working Dogs Deployed in Operations Desert Shield/Storm

ABSTRACT

A population-based, cohort study was conducted to determine the importance of Gulf War deployment to Southwest Asia, from 1 August 1990 to 31 December 1991, in explaining neurologic mortality and peripheral nerve disease among United States military working dogs. The study cohort consisted of 2,123 military working dogs that were eligible to deploy to the Gulf War and died between 4 September 1990 and 30 June 2001 with complete medical records maintained at the Department of Defense Military Working Dog Training Center and Veterinary Services, Lackland AFB, San Antonio, TX. Within this Gulf War cohort was a prospectively followed cohort of 651 dogs; 347 of these dogs had complete peripheral nerve histopathologic diagnostic records.

Descriptive analysis of the study variables defined neurologic mortality incidence for the Gulf War cohort at 1.90 cases per 1,000 dog-months. Rates among dogs assigned to the United States, overseas, and Southwest Asia were 1.83, 1.91, and 2.44 cases per 1,000 dog-months, respectively. Analysis of other exposures showed highest neurologic mortality among dogs assigned to Southwest Asia countries other than Saudi Arabia, dogs arriving before or departing after the war, dogs that arrived before and departed after the war, and dogs that spent more than 176 days in Southwest Asia. Peripheral nerve disease incidence was 3.69 cases per 1,000 dog-months for the prospective cohort. The rate for dogs assigned to the United States was higher than for those overseas or in Southwest Asia (3.86, 3.53, and 3.21 cases per 1,000 dog-months, respectively). Using survival analysis, adjusted neurologic mortality and peripheral nerve disease rates were similar between the United States, overseas, and Southwest Asia assignment locations. An increasing trend of neurologic mortality was evident with increased time spent in Southwest Asia.

Results from this study suggest that there was not a relationship between Southwest Asia deployment during the Gulf War and neurologic mortality or peripheral nerve disease. This study is consistent with previous human Gulf War neurologic studies. The military working dog remains a viable sentinel model for measuring outcomes of unique military exposures. This study and available data sets can form the basis for future studies.

Anthony Williams
Neuroscience Program

Doctor of Philosophy
2003

Major Advisor: LTC Geoffrey Ling, MC, USA

Title of Dissertation: Neuroprotection Profile of the High Affinity
NMDA Receptor Antagonist Conantokin-G

ABSTRACT

Conantokin-G (Con-G or CGX-1007), a potent NR2B subunit selective NMDA receptor antagonist, was evaluated for its neuroprotective properties in experimental models of neuronal injury. In primary neuronal cultures Con-G was shown to decrease excitotoxic calcium responses to NMDA and provide 100% neuroprotection against hypoxia/hypoglycemia (34 µM[13-91]), NMDA (77 µM[42-141]), glutamate (819 µM[346-1937]) or veratradine (2136 µM[1508-3026]) induced injury (numbers in parentheses indicate EC50 and 95% confidence limits). Con-G (0.1-1 µM) also provided up to 80% protection against staurosporine-induced apoptotic injury (P<0.01, n = 12/group), which was linked to the NR2B subunit. For in vivo brain injury studies, middle cerebral artery occlusion (MCAo) in the rat was used as a model of transient focal brain ischemia. In this model Con-G (0.01-2.0 nmoles, i.c.v.) reduced brain infarction and improved both neurological and electroencephalographic (EEG) recovery as evaluated both 24 and 72 h post-injury. The maximal neuroprotective effect was measured with the highest dose of Con-G tested (2.0 nmol, i.c.v) with an 89% reduction of core infarct volume (P<0.05, n = 6-10/group). Post-injury time course experiments demonstrated a therapeutic window out to at least 4 h from the start of the injury. These neuroprotective effects were also associated with a 50% reduction in the early expression (i.e. 1-4 h) of the c-fos gene (P<0.05, n = 3-4/group), a preservation of Bcl-2 immunoreactivity at 24 h (P<0.05, n = 4), and with a reduction in DNA strand breaks in the ischemic hemisphere as evaluated 24 h post-injury (P<0.05, n = 6/group). Clinically relevant routes of administration were evaluated by administering intrathecal (i.t.) injections of Con-G (20-160 nmol), which provided up to 62% reduction in brain infarction (P<0.05, n = 8-9/group) along with significant neurological recovery and a therapeutic window of up to 8 h post-injury. Con-G (i.t.) treatment was also associated with fewer ischemia-induced EEG seizures with a positive albeit non-significant trend (P>0.05, n = 6-7/group) between the number of brain seizures and infarct volume. Intrathecal Con-G was not associated with changes in c-fos gene expression although, similar to i.c.v. administration, Bcl-2 immunoreactivity was preserved in cortical tissues (P<0.05, n = 3/group) and presence of TUNEL positive cells decreased at 24 h (P<0.05, n = 6/group). These data provide evidence for the potent and highly efficacious effect of Con-G as a neuroprotective agent with an excellent therapeutic window for the potential intervention against ischemic/excitotoxic brain injury in humans.