Amon
Kim
Schoneboom
Bates Maloney
Scoville
Caohuy Nash Teel
Ekkens Newman
Tucker
Kazerouni Nielsen
Joseph J.
Amon
Department of Preventive Medicine and Biometrics
Doctor of
Philosophy
2002
Major Advisor: Dr. John H. Cross, Department of Preventive Medicine and Biometrics
Thesis Title: The Molecular Epidemiology of Malaria in Western Kenya
ABSTRACT
Malaria epidemiology reflects a complex web of inter-related factors: host, parasite, vector and environment. The nature, duration and severity of malaria infection depend upon these fixed and changing factors, and are complicated by varying levels of acquired immunity in individuals. The present research used molecular biology tools to examine three distinct topics related to malaria epidemiology: 1) the distribution of TNF-a, IFN-g, IL-6, TGF-b, and IL-10 gene polymorphisms; 2) the rate and pattern of mixed Plasmodium species infections; and 3) Plasmodium falciparum growth dynamics. The first two research topics were examined in a cohort of 248 males recruited from three highly endemic villages in western Kenya where severe malaria anemia is common. The third topic was investigated among 22 volunteers during the post-challenge phase of a malaria vaccine trial sponsored by Walter Reed Army Institute of Research. Individuals in the Kenya cohort were found to have a marked bias toward genotypes associated with low expression of IFN-g and IL-6, cytokines that, at high plasma levels, have been previously implicated in anemia and poor malaria outcomes. By contrast, the frequency of the TNFa -238A allele, which has been associated with severe malarial anemia, was found to be similar to frequencies reported in a number of diverse populations. Over the course of three malaria transmission seasons, non-falciparum malaria was consistently seen in a minority (5-10%) of volunteers, most commonly as a dual infection with P. falciparum. In contrast to previous reports of long-term stable parasite density among individuals with mixed species infections, in 80% of cases, multi-species infections were detected when individuals had their highest rate of parasite density. Among clinical trial volunteers, a highly sensitive real-time PCR assay found a ~48 hour periodicity in parasite density and a relatively wide range in parasite multiplication rate. Four individuals were identified as having some degree of resolution of infection, and an additional five volunteers were found to have a delayed pre-patent period. These results demonstrate the potential of molecular epidemiology and illustrate the subtle and complex relationship developed between humans and malaria parasites over millions of years of co-evolution.
Key words: malaria, epidemiology, molecular epidemiology, Kenya, cytokines, PCR, real-time PCR, molecular beacon probes, vaccine
Maj Mark Bates,
USAF
Department of Medical and Clinical Psychology
Doctor of
Philosophy
2002
Major Advisor: Wendy Law, Ph.D., Department of Medical and Clinical Psychology
Thesis Title: Risk Factor Model Predicting the Relationship between Stress and Performance in Explosive Ordnance Disposal Training
ABSTRACT
Stress is a common aspect of military operations. It therefore follows that
training to work effectively under conditions of stress is an essential component
of military training programs. The primary purpose of this proposed study
is to identify specific risk and protective factors that predict the effects
of stress responding on military operational performance. Because of the high
levels of stress and resultant program attrition, the military's Explosive
Ordnance Disposal (EOD) training program provides a unique opportunity to
study the interaction between stress and performance, and to identify specific
risk and protective factors.
The study recruited 500 students who were enrolled in EOD training. These
students were enlisted personnel in the US military who volunteered to participate
in the study at two points during the training program. Self-report assessments
of potential risk factors were collected at the beginning of the training
program and at one intermediate time point that is associated with the highest
level of student attrition. These risk factors included general cognitive
ability, inattention and impulsivity, problem solving, anxiety, personality
dimensions, social relations, and stressful events. In addition, the study
explored the role of the demographic, social desirability, and external stressor
control variables. The risk factors and control variables were used to predict
two measures of performance. The first measure of performance was the grade
on the first practical test of the training program, a continuous outcome
between 0-100. The second performance measure was program completion, which
was a dichotomous outcome of successful or unsuccessful completion.
The statistical procedures included multivariate analyses to identify unique predictors of performance and interactional analyses to clarify how pairs of variables might be interacting to affect performance. A substantial number of variables were found to predict performance on a univariate level. However, only select demographic variables, trait inattention, and situationally experienced cognitive anxiety emerged as key predictors of performance in multivariate comparisons of predictors. The findings also suggested that trait problem-solving skills might act as a protective factor against the negative performance effects associated with trait inattention. Finally, even though trait inattention was found to be a risk factor for poorer performance, the findings also suggested that inattention could be associated with enhanced performance if paired with either situational variables of high utilization of problem solving skill or low cognitive anxiety experienced during a test.
Hung Caohuy
Department of Anatomy, Physiology and Genetics
Doctor of
Philosophy
2002
Major Advisor: Harvey Pollard, M.D., Ph.D., Department of Anatomy, Physiology and Genetics
Thesis Title: Membrane Fusion Protein Annexin 7: A Common Site of Action for Calcium, Guanosine Triphosphate, Protein Kinase C and Botulinum Toxin Type C in Regulated Exocytosis
ABSTRACT
Numerous studies have shown that regulated exocytosis is activated simultaneously by calcium, guanosine triphosphate (GTP) and protein kinase C (PKC), and that this process is specifically inhibited by botulinum toxins (BoNTs). Although phenomenologically well known, the specific sites of action for these agents in the late stage of exocytosis, membrane fusion, remain unknown.
In this research project, we combined both in vitro and in vivo approaches to directly test the effects of these agents on annexin 7. Annexin 7 (ANX7) is a calcium-dependent GTP-activated membrane fusion protein. In a reconstituted membrane fusion system using artificial liposomes, ANX7 membrane fusion activity is substantially increased by the combination of individually optimal concentration of guanine nucleotide and PKC. This increasing ANX7 activity can be distinguished by a simple additive model when comparing activation by either guanine nucleotide or PKC alone. In other in vitro assays, the binding of GTP and its non-hydrolyzable analogues to ANX7 significantly enhance PKC phosphorylation, and conversely PKC phosphorylation markedly potentiates the binding and hydrolysis of GTP by ANX7. While certain other kinases label ANX7 efficiently, they do not substitute for PKC in potentiating GTP binding or membrane fusion.
To correlate the in vitro data with exocytotic events in cells, we examined the biochemical profile of endogenous ANX7 in secreting adrenal chromaffin cells. In vivo, both the ratio of ANX7-bound GDP/GTP as well as ANX7 phosphorylation by PKC change in proportion to the extent of catecholamine release from stimulated chromaffin cells. Thus, the stimulatory actions of calcium, GTP and PKC appear to specifically converge on ANX7 to drive membrane fusion activity occurring during exocytosis. To further support such an inference, we have found that BoNT type C efficiently cleaves ANX7 both in vitro and in permeabilized chromaffin cells. This proteolytic activity is concurrent with BoNT/C-dependent inhibition of ANX7 membrane fusion activity in vitro, and with inhibition of catecholamine secretion in vivo.
We therefore conclude that the exocytotic machinery includes ANX7 as a common site of action for calcium, GTP, PKC and Botulinum toxin in the exocytotic membrane fusion process.
Melinda Ekkens
Molecular and Cell Biology Program
Doctor of
Philosophy
2002
Major Advisor: William C. Gause, Ph.D., Department of Microbiology & Immunology
Thesis Title: The Role of Costimulatory Molecules in the Development of Memory and Effector T helper 2 Cells During an in vivo Immune Response to the Murine Gastrointestinal Parasite Heligmosomoides polygyrus
ABSTRACT
Previous studies have demonstrated the importance of costimulatory interactions for effector CD4+ T helper (Th) cell development during the primary immune response. However, the role of costimulatory molecules in memory CD4+ T cell differentiation is not well understood. One model used to study the Th immune response involves oral infection of mice with the gastrointestinal nematode parasite Heligmosomoides polygyrus. Although the primary immune response to H. polygyrus is a chronic infection, challenge immunization triggers a T-dependent memory response that impairs adult worm maturation. In the studies presented herein, the effects of costimulatory molecule blockade on T helper effector cell function during the memory response were examined.
Effector T cell development was inhibited during the primary response to H. polygyrus in B7-1/B7-2-/- mice; however, memory Th cells developed that produced IL-4 and mediated effective reductions in adult worm egg production, but did not provide effective Ag-specific B cell help or support increased germinal center (GC) formation. Parallel studies in H. polygyrus-challenged CD28-/- mice demonstrated similar IL-4 elevations and decreases in adult worm egg production. However, Ag-specific Ab responses and increased GC formation were significantly restored in H. polygyrus-inoculated CD28-/- mice.
Although elevations in serum IgG1 and GC formation were intact in H. polygyrus-challenged OX40L-/- mice, elevations in IL-4 and serum IgE were partially inhibited, and associated with decreased worm expulsion and increased egg production. To further examine the role of OX40L in Ag-specific CD4+ T cell IL-4 production following priming, adoptively transferred OVA-specific DO11.10 T cells were analyzed in the context of the H. polygyrus response. Following immunization with OVA plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable in OX40L+/+ and OX40L-/- mice; however, Ag-specific T cell IL-4 production was reduced in OX40L-/- mice, suggesting a preferential role for OX40L costimulation in IL-4 production.
These studies extend our understanding of the role of costimulatory molecules in the development of memory Th2 cells during infectious disease. They also suggest that B7-1/B7-2 antagonists may be particularly effective in the treatment of chronic diseases where continuous renewal of effector populations from naïve precursor T cells mediates pathogenesis.
N. Neely Kazerouni
Department of Preventive Medicine and Biometrics
Doctor of
Public Health
2002
Major Advisor: Heidi Friedman, Ph.D., Department of Preventive Medicine and Biometrics
Thesis Title: Family History of Breast Cancer as a Determinant of the Risk of Developing Endometrial and Ovarian Cancers: A Nationwide Cohort Study
ABSTRACT
Statement of the problem: Although endometrial and ovarian cancers share some
of the same reproductive, hormonal, and genetic risk factors with breast cancer,
it is not well established if a family history of breast cancer is associated
with endometrial and ovarian cancer risk in a general population setting.
We examined these associations in a prospective cohort study.
Methods: The women in the endometrial (n=37,583) and ovarian (n=49,975) cancer studies were former participants in a national breast cancer screening who were selected for additional follow-up (1979-1998). During follow-up, 648 and 362 women with endometrial and ovarian cancers, respectively, were identified. We examined information on the breast cancer history of mothers, sisters, daughters, aunts, and grandmothers of the study participants as well as the number of relatives affected with breast cancer, their age at diagnosis, and breast cancer laterality. We used Poisson regression to estimate rate ratios and 95% confidence intervals to characterize the precision of these point estimates.
Results: The presence of breast cancer in a first-degree (RR=0.96, 95% CI= 0.78-1.2) or a second-degree (RR=1.0, 95% CI=0.81-1.2) relative did not influence the risk of developing endometrial cancer. In addition, the risk of endometrial cancer did not vary by age of the relative at breast cancer diagnosis or by the number of affected relatives with breast cancer. However, there was a non-significant increase in the risk of endometrial cancer among women with a 1st degree relative with bilateral breast cancer (RR=1.4, 95% CI= 0.84-2.4) but not among women with a 1st degree relative with unilateral breast cancer (RR=0.83, 95% CI=0.62-1.1). Women with a personal history of prior breast cancer were more likely to develop endometrial cancer during the course of follow-up (RR=1.3; 95% CI=1.1-1.7), but even in this subgroup, family history of breast cancer did not confer additional risk of endometrial cancer.
On the other hand, breast cancer in a first- or second-degree (RR=1.4, 95% CI=1.1-1.7), and any second-degree (RR=1.3, 95% CI=1.0-1.7) relative, increased the risk of ovarian cancer. Participants with two or more first-degree relatives with breast cancer also had a significantly increased risk (RR=1.8, 95% CI=1.1-2.8). Risk was particularly high among women with 2 or more first-degree affected relatives, at least one of whom had bilateral breast cancer (RR=4.2, 95% CI=1.7-10) or younger age (<50) at breast cancer diagnosis (RR=2.6, 95% CI=1.4-4.8), and among women with a personal history of breast cancer who also had a first-degree relative with younger age at breast cancer diagnosis (RR=3.5, 95% CI: 1.7-7.4).
Conclusions: These results provide support for the hypothesis that a family history of breast cancer is a strong predictor of the risk of developing ovarian cancer, but is not a predictor of endometrial cancer risk.
Steven Kenneth
Kim
Department of Anatomy, Physiology & Genetics
Doctor of
Philosophy
2002
Major Advisor: Meera Srivastava, Ph.D., Department of Surgery
Thesis Title: the Regulation of Nucleolin Expression in Prostate Epithelial Cells; Possible Involvement of Myc
ABSTRACT
Note: both abstracts for the two papers that comprise this thesis manuscript are located with the papers; that is, abstract 1 is included with paper 1, and abstract 2 is included with paper 2.
Elizabeth
M. Maloney
Department of Preventive Medicine and Biometrics
Doctor in
Public Health
2002
Major Advisor: Terry L. Thomas, Ph.D., National Cancer Institute
Thesis Title: A Cohort Study of Health Effects of HTLV-I Infection in Jamaican Children and their Associations with Viral, Immunologic and Host Genetic Markers
Abstract
Statement of the problem: Human T-lymphotropic virus type I (HTLV-I) infection is associated with infective dermatitis of childhood. Early childhood infection is also thought to play a role in development of a rare malignancy associated with HTLV-I, adult T-cell leukemia/lymphoma (ATL). ATL develops in <5.0% of persons infected with HTLV-I in childhood. Identifying markers associated with increased risk of ATL among infected persons could be used to target persons for early clinical intervention. Several case reports have documented ATL patients with childhood histories of infective dermatitis. Infective dermatitis may be a cutaneous marker of risk for ATL. There may be additional health effects of HTLV-I infection in children that could be potential markers of risk for development of ATL, however this age group has not been well studied.
Methods: A cohort study of 28 HTLV-I infected and 280 uninfected children born to women who attended one of two antenatal clinics in Kingston, Jamaica between January, 1989 and August, 1990 were enrolled in this study. Children received physical examinations and phlebotomy at clinic visits scheduled from six weeks to ten years of age. The primary analysis compared incidence rates for targeted health outcomes between HTLV-I infected and uninfected children. Based on the results of the primary analysis, the secondary analysis examined associations of HTLV-I-associated health outcomes with pre-diagnostic levels of viral and immunologic markers, as well as host genetic markers among HTLV-I infected children. Additionally, levels of immunologic markers at the time of diagnosis were described in infected children with a specific health outcome.
Results: HTLV-I infected children had significantly increased incidence rates of seborrheic dermatitis, eczema and hyperreflexia of the lower limbs compared to HTLV-I uninfected children. Additionally, compared to uninfected children, HTLV-I infected children had elevated rates of lymphadenopathy, severe anemia, abnormal lymphocytes and a decreased rate of eosinophilia that were of borderline statistical. Among infected children, seborrheic dermatitis and severe anemia were associated with elevated HTLV-I proviral loads in pre-diagnostic specimens. Children with seborrheic dermatitis had elevated pro-inflammatory cytokines at the time of diagnosis, but not 12 months post-infection. Health outcomes among infected children were not associated with the HLA Class II alleles studied.
Conclusions: HTLV-I infection in children may be associated with seborrheic dermatitis and eczema. Further study is needed to confirm these associations and clinically define these HTLV-I associated diseases. Other abnormalities associated with HTLV-I have previously been reported in association with HTLV-I in adults. HTLV-I infected children with seborrheic dermatitis, eczema or severe anemia had elevated levels of proviral load 12 months post-infection that were of borderline statistical significance. At the time of diagnosis, infected children with seborrheic dermatitis had elevated levels of pro-inflammatory cytokines. HTLV-I associated seborrheic dermatitis may be an obvious marker of immune system dysregulation in children.
Holly H. Nash
Neuroscience Program
Doctor of
Philosophy
2002
Major Advisor: Juanita J. Anders, Ph.D., Department of Anatomy, Physiology, and Genetics
Thesis Title: Regeneration of the Adult Rat Spinal Cord in Response to Ensheathing Cells and Methylprednisolone
ABSTRACT
Axons fail to regenerate after spinal cord injury (SCI) in adult mammals, leading to permanent loss of function. Following SCI, ensheathing cells promote recovery in animal models, whereas methylprednisolone study, a new method of purifying ensheathing cells was developed, resulting in a final population of ensheathing cells that were 93% pure. In the second study, the ability of a modified directed forepaw reaching (DFR) apparatus to accurately assess function of the corticospinal tract (CST) was examined. The data demonstrated that the modified apparatus prevented extinguishing of DFR behavior after SCI. In addition, the modified apparatus allowed for the collection of quantitative data to accurately assess CST function after bilateral, cervical spinal cord lesions. In the third study, the effectiveness of combining ensheathing cells and methylprednisolone after SCI was investigated. After lesioning the CST in adult rats, a purified population of ensheathing cells was transplanted into the lesion, and methylprednisolone was administered for 24 hours. At six weeks post injury, functional recovery was assessed by measuring successful DFR performance. Axonal regeneration was analyzed by counting the number of anterogradely labeled CST axons caudal to the lesion. Lesioned control rats, receiving either no treatment or vehicle, had abortive axonal regrowth (1 mm) and poor DFR success (38% and 42%, respectively). Compared to controls, rats treated with methylprednisolone for 24 hours had significantly more axons at 7 mm caudal to the lesion, and DFR performance was significantly improved (57%). Rats that received ensheathing cells with methylprednisolone had significantly more regrowing axons than all other lesioned rats up to 13 mm caudal to the lesion. Successful DFR performance was significantly higher in rats with ensheathing cell transplants, both without (72%) and with (78%) methylprednisolone, compared to other lesioned rats. These data confirm previous reports that ensheathing cells promote axonal regeneration and functional recovery after spinal cord lesions in a rat model. In addition, this research provides new evidence that, when used in combination, methylprednisolone and ensheathing cells improve axonal regrowth up to 13 mm caudal to the lesion.promotes neurological recovery in humans. The aim of this research was to explore the effectiveness of ensheathing cells and methylprednisolone after acute SCI in the adult rat.
Sara B. Newman
Department of Preventive Medicine and Biometrics
Doctor of
Public Health
2002
Major Advisor: Heidi Friedman, Ph.D., Department of Preventive Medicine & Biometrics
An Epidemiologic Analysis of Chlamydia trachomatis and Neisseria gonorrhoeae Infections in Female Federal Prisoners
ABSTRACT
Statement of the problem: Infections caused by Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are the most commonly reported bacterial sexually transmitted infections in the U.S. No studies have been conducted to determine the prevalence of these two infections in women federal prisoners. Information about the prevalence of and risk factors for the infections may assist the Federal Bureau of Prisons to implement a rational screening approach for CT and GC in female inmates.
Methods: Two study phases were implemented as part of this protocol. The first study phase relied on qualitative techniques: focus groups and group-based cognitive interviews were implemented to assist in the design of the study instrument and study procedures to be used in the second phase of the study. For the second, quantitative phase, urine specimens and self-collected swabs were taken from 983 women incarcerated in two federal prison facilities in the U.S. Participants completed a self-administered questionnaire on sociodemographic characteristics, and sexual and clinical history. Another questionnaire was administered to participants after specimen collection on preferences for self-collected swab, urine collection and pelvic examination. Specimens were analyzed at the Johns Hopkins University Chlamydia laboratory using strand displacement amplification technology to detect the presence of CT and/or GC DNA. Prevalence of CT and GC was calculated for each of the prison sites. Potential risk markers associated with infection were assessed. Participants' preferences of self-collected swab and urine were also compared between the two sites.
Results: CT infection was found in 1.3% of the participants at one site where women are screened when they enter the prison, and in 2.3% at the other site, where women are not screened. One case of gonococcal infection was detected at the site where women are screened and no cases in the other site. Among women age 18-22, prevalence of CT infection was 8.5% in the prison with the highest prevalence of infection. Prevalence of CT infection among women age 30 and younger exceeded 3.5%. Screening women age 30 and younger would identify more than 60% of cases at an estimated cost of less than $60,000 per year. Approximately 83% of infections could be detected if women age 35 and younger were screened, but the cost for screening would approach $90,000.
More
than half of the participants (57%) found no difference between giving urine
or swab samples in terms of ease of collection. Approximately 30% of participants
said they would prefer to give a swab specimen in the future as compared to
urine (21%), but nearly half of women expressed no preference for one method
over the other. Most participants (60%) expressed a preference for doing a
self-collected swab rather than having a pelvic exam (23%) to test for the
infections in the future, but nearly 17% had no preference for one over the
other.
Conclusions: While prevalence among the study population was low, targeted
screening in women age 30 and younger is recommended to detect more than half
of the cases cost-effectively. This study provided evidence that to inmates
noninvasive screening techniques are acceptable alternatives to pelvic examination.
Joseph Allen
Nielsen
Molecular and Cell Biology
Doctor of
Philosophy
2002
Major Advisor:
Regina Armstrong, Ph.D., Department of Anatomy, Physiology
and Genetics
Thesis Title:Nuclear Organization and Myt1 Interaction in Transcriptional Control of Neural Cell Differentiation
Abstract
Neural
cell differentiation is a complex set of events beginning with cells responding
to both soluble and cell contact-dependent external signals. These signals
activate pathways that lead to changes in gene expression patterns, which
ultimately give rise to the differentiated cell phenotype. In these studies,
potential mechanisms regulating different aspects of oligodendrocyte differentiation
were explored. Specifically, these studies examined the contribution of gene
and protein localization to the establishment and/or maintenance of terminally
differentiated oligodendrocyte gene expression patterns and the role of myelin
transcription factor 1 (Myt1) in the regulation of oligodendrocyte proliferation
and differentiation.
Myt1 is a zinc-finger DNA-binding protein that is expressed in neural progenitors
and is localized to discrete domains within the nucleus of oligodendrocyte
progenitors. Primary oligodendrocyte lineage cells were examined during cell
differentiation in order to study the localization of the highly expressed
tissue-specific proteolipid protein gene relative to nuclear proteins such
as Myt1 and splicing factors within interphase nuclei. These data support
a nuclear organization model in which nuclear proteins and genes exhibit specific
patterns of distribution within nuclei, and activation of tissue-specific
genes is associated with changes in protein distribution rather than changes
in gene localization.
Myt1 contains six zinc-finger DNA-binding domains with sets of two N-terminal and of four C-terminal zinc-fingers. A retroviral expression system was used to overexpress the four zinc-finger DNA-binding domain of Myt1 (4FMyt1) which lacks the putative domains for protein-protein interaction and transcriptional activation. In a dominant negative study, expression of 4FMyt1 inhibited both proliferation and differentiation of oligodendrocyte progenitors. These data indicate that Myt1 contributes to the regulation of oligodendrocyte lineage development in the transitional period between proliferating progenitor cells and terminally differentiated oligodendrocytes.
These studies demonstrate the importance of Myt1 and nuclear organization to the regulation of oligodendrocyte progenitor differentiation and the establishment of tissue-specific gene expression patterns.
Major Bruce
A. Schoneboom
Neuroscience Program
Doctor of
Philosophy
2002
Major Advisor: Franziska B. Grieder, DVM, Ph.D., Microbiology and Immunology
Thesis Title: Neuro-Immune Mechanisms in Response to Venezuelan Equine Encephalitis Virus Infection
ABSTRACT
Venezuelan equine encephalitis virus (VEE) is an emerging pathogen with epizootics
and epidemics occurring in the Western Hemisphere. Recent outbreaks in South
America have caused significant morbidity and mortality among domesticated
livestock and surrounding human communities. VEE pathogenesis is characterized
by infection of the central nervous system (CNS) where the virus targets neurons,
resulting in significant neurodegeneration. VEE encephalitis can result in
profound neurological deficits or even death. Because of the devastating nature
of this disease and the lack of interventional therapies, it is important
to understand the intricate details of VEE neuropathogenesis in order to identify
targets for treatment to effect a cure.
Inflammation has recently been implicated as a component of neurodegeneration. Inflammation in the CNS in response to acute infections is a protective mechanism that attempts to contain and clear neuro-invasive pathogens, however this upregulation of pro- inflammatory genes may be deleterious to surrounding neurons. The combined effects of direct infection and inflammation may be additive or synergistic in the amount of injury sustained in the CNS.
Glial cells are of particular importance in the CNS immune response. These resident cells of the CNS have intimate associations with neurons and regulate the CNS milieu. One type of glial cell is the astrocyte. Astrocytes are found in vast numbers in the CNS and have essential functional roles in maintaining a healthy environment for neurons. Further, astrocytes playa role in the pro-inflammatory innate immune response.
To identify the role of astrocytes in VEE infection, I characterized astrocyte susceptibility to VEE infection using an in vitro culture system and have further described their pro-inflammatory responses following VEE infection. Specifically, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-6 are upregulated in response to VEE infection in primary astrocyte cultures as shown by reverse transcriptase-polymerase chain reaction and analyses of protein synthesis. I have also demonstrated that there were quantitative differences in the upregulation of these responses between virulent and attenuated strains of VEE.
To characterize the pro-inflammatory response in vivo, I measured cytokine gene expression in the CNS using a murine model of VEE infection. The cytokine responses to virulent VEE resulted in the upregulation of multiple genes important in inflammation and apoptosis. In contrast, cytokine responses in the CNS were delayed or absent following infection with attenuated VEE, depending on the specific mutant VEE strain.
Finally, CNS tissue from mice infected with VEE was double-labeled for astrocytosis and apoptosis, and stained for VEE antigen in adjacent tissue sections. Apoptosis occurs not only in areas of the brain where VEE antigen could be detected, but also in areas of acute astrogliosis, where no VEE antigen could be demonstrated. This association of apoptosis and astrogliosis suggests that inflammation may be contributing to neuronal degeneration in response to VEE infection.
Stephanie
L. Scoville
Department of Preventive Medicine and Biometrics
Doctor of
Public Health
2002
Major Advisor: COL John Gardner, MC, USA, Department of Preventive Medicine and Biometrics
Thesis Title:
"Mortality During U.S. Armed Forces Basic Training:
A 25-year Review (1977-2001)"
ABSTRACT
Background: Efforts to understand and prevent the rare, but tragic, occurrence
of death among healthy, young military recruits trying to serve their country
depend upon medical surveillance data and accurate determination of mortality
rates by specific cause. The purpose of this study was to create a Recruit
Mortality Registry that includes deaths that have occurred during Air Force,
Navy, Marine Corps, and Army basic military training from 1977 through 2001,
and to describe the epidemiology of recruit mortality due to traumatic and
nontraumatic deaths.
Methods: Recruit deaths were identified and confirmed through redundant sources. Attempts were made to obtain a complete file on each death including all available medical and personnel records. Demographic, circumstantial, and medical information was recorded on an abstraction form developed for this study. Mortality rates per 100,000 recruit-years were calculated using recruit accession data from the Defense Manpower Data Center.
Results: There were 276 recruit deaths from 1977 through 2001 identified through the Recruit Mortality Registry. Age-specific recruit mortality rates were less than half of same-age U.S. civilian mortality rates. Between Services, the age-adjusted mortality rates were highest in the Army and lowest in the Air Force and Navy, with the Marine Corps in between. The majority (71%) of recruit deaths were classified as nontraumatic and 69% (136/196) of these were exercise-related. Of the exercise-related deaths, 42% (57/136) were cardiac deaths and at least one-third (45/136) were related to heat stress. Infectious agents accounted for 24% (48/196) of the nontraumatic deaths. Only 29% (80/276) of recruit deaths were classified as traumatic. Of these, the majority (58%) were due to suicide, followed by unintentional injuries (37%), and homicide (5%).
Conclusions: Comparison of military recruit mortality rates with the same-age U. S. civilian population establishes the safety of the basic military training environment. This can be attributed to selection factors in inducting healthy recruits, a well-supervised training environment, the tremendous focus on safety during recruit training, and lack of access to alcohol and motor vehicles. Preventive measures focused on reducing heat stress during exercise may be effective in reducing high rates of exercise-related death.
Louise D.
Teel
Department of Microbiology and Immunology
Doctor of
Philosophy
2002
Major Advisor: Alison D. O'Brien, Ph.D., Department of Microbiology and Immunology
Title of Dissertation: The Regulation of Expression of the Stx2d Toxins in Shiga Toxin-producing Escherichia coli O91:H21 Strain B2F1
ABSTRACT
Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx2e from one human STEC isolate has been reported to be encoded within a toxin-converting phage. In this study, I examined O91:H21 STEC isolate B2F1 that carries two functional alleles (stx2d1 and stx2d2) for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages and other potential regulators of toxin expression. Mutants of B2F1 that produced one or the other Stx2d toxin were made. The Stx2d1-producing mutant (stx2d2::cat) was less cytotoxic for Vero cells than the Stx2d2-producing mutant (stx2d1::cat). Consistent with those results, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection, while the Stx2d2-producing mutant was nearly as virulent as wild-type B2F1. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. An stx2d1-containing lysogen was isolated from plaques on strain DH5a that had been exposed to lysates of the mutant that produced Stx2d1 only. However, that RecA- lysogen could not be induced for phage nor were culture lysates from it cytotoxic for Vero cells. By contrast, when the lysogen was transformed with a plasmid encoding RecA and induced with mitomycin C, culture extracts were cytotoxic for Vero cells. Furthermore, electron microscopic examination of extracts from the fB2F1-lysogen showed hexagonal particles that resembled the prototypic Stx2-converting phage 933W. These observations provide strong evidence that expression of Stx2d1 is bacteriophage-associated.
The finding that synthesis of Stx2d1 but not Stx2d2 was associated with phage induction led me to investigate regulation of Stx2d2 production. Transposon mutagenesis of DH5a revealed genes associated with reduced expression of an stx2d2 promoter::lacZ fusion in a reporter plasmid, observations that suggested the inactivation of potential activators of transcription of stx2d2. The mutant genes were isolated and sequenced. Two mutations identified were in caiD and the "div" gene of DH5a; these mutants showed reduced cytotoxicity and virulence for mice when transformed with an Stx2d2 toxin gene clone as compared to wild-type DH5a similarly transformed. However, introduction of the same mutations into strain B2F1 did not result in reduced cytotoxicity or virulence. The influence of the caiD and"div" gene mutations on toxin expression in DH5a may be polar and not correspond to the arrangement of these genes in B2F1. Alternatively, the effects of these mutations may only be evident when multiple copies of the toxin gene are present. In sum, the regulation of expression of the two Stx2d alleles in B2F1 differs such that Stx2d1expression is tightly repressed except under conditions that induce the toxin-converting phage in which it is encoded. In contrast, Stx2d2 is expressed at higher levels, independent of phage induction, under the direction of some other yet-to-be-defined host factor. The DNA sequences that flank stx2d2 share extensive homology to those flanking stx2d1 and other phage-borne Stx genes, a finding that suggests that stx2d1 and stx2d2 share a common phage origin but the phage sequences associated with stx2d2 are defective.
Dana L. Tucker
Department of Medical and Clinical Psychology
Doctor of
Philosophy
2002
Major Advisor: David S. Krantz, Ph.D., Department of Medical and Clinical Psychology
Thesis Title: Hostility and Anger Expression: Behavioral and Cardiovascular Responses to Mental Stress among Cardiovascular Disease Patients
ABSTRACT
Hostility and anger expression have been postulated as contributing factors to cardiovascular disease and have been associated with cardiovascular reactivity and stress-induced myocardial ischemia. The present research investigates relationships between hostility, anger expression, and defensiveness and cardiovascular measures of reactivity and ischemia. The study is an analysis of previously collected data from the Triggers of Myocardial Ischemia Study, in which participants underwent mental stress tasks (math, anger recall speech, and Stroop Color-Word) with concurrent assessments of cardiovascular reactivity and ischemia as well as brief emotional state assessments in the laboratory. Outside of the laboratory, a subset of these participants (n= 59) completed a set of questionnaires including the Marlowe-Crowne Scale of Social Desirability and the Cook-Medley Hostility Scale.
Results indicated that higher Cook-Medley Hostility scores were positively correlated with anger expression changes following the math task (r=.308, p< .05), and Composite Hostility predicted anger expression changes to the same task (r=.334, p<.05). The Composite Hostility component Hostile Affect was a significant predictor of anger expression changes during all three tasks. Although hostility measures did not appear to be consistently predictive factors in stress-induced cardiovascular reactivity, changes in anger expression predicted systolic and diastolic blood pressure changes during the anger recall task, and marginally predicted heart rate changes to anger recall. Higher Total Hostility was marginally related to presence of ischemia during Anger Recall and Stroop tasks, but anger expression changes were not related to presence of ischemia. Hypothesized interaction effects between hostility and anger expression in eliciting cardiovascular reactivity and ischemia were not observed. Defensiveness, contrary to predictions, was not significantly negatively associated with anger expression changes, nor was it a significant predictor of cardiovascular reactivity or ischemia.
The study suggests the importance of assessment of acute emotional states when assessing personality characteristics and their impact on measures of cardiovascular reactivity and ischemia. Study limitations include low power in the evaluation of factors related to myocardial ischemia, and the study's use of a data set not specifically designed to test the present study hypotheses. Further investigation into anger expression in addition to personality traits may help in understanding factors contributing to cardiovascular reactivity and adverse coronary events.