Dalgard
McCeney
Davis Mohyeldin
Elliott
Parrish
Ha Santos-Ciminera
Larsen
Simms
Lindsey Taylor
Clifton
Dalgard
Neuroscience Program
Doctor
of Philosophy
2005
Major Advisor: Ajay Verma, M.D., Ph.D. Department of Neurology
Dissertation Title: Hypoxia-Inducible Factor Hydroxylases Are Oxygen Sensors in the Brain
ABSTRACT:
In mammalian cells, molecular oxygen is a requirement for normal growth, metabolism,
and survival. Tissues in which oxygen demand surpasses oxygen supply become
hypoxic and cannot maintain normal function. Thus, the ability to sense oxygen
levels is necessary for an organism to survive and thrive in changing environmental
and physiological conditions. HIF-1 is a transcription factor, complex that
is essential and central to several cellular and systemic adaptations to hypoxia.
For example, vascular endothelial growth factor and erythropoietin are HIF-I
target genes that are important in angiogenesis and erythropoiesis, respectively.
HIF-I consists of two subunits, alpha and beta, and control of HIF-1 function
is accomplished through the hydroxylation of proline residues and an asparagine
residue on the a-subunit of HIF-l. Under normoxic conditions, hydroxylated
HIF-la is constantly and rapidly degraded, thus HIF-l is inactivated. Additionally,
undegraded HIF-la is hydroxylated at an asparagine residue in the c-terminal
region, which prevents it from binding to the co-transcriptional activator
p300. The post-translational modifications of HIF-la are performed by four
oxygen-dependent enzymes, the three HIF-la prolyl hydroxylase (HPH-l, HPH-2,
and HPH-3) and the asparaginyl hydroxylase FIH-l (Factor Inhibiting HIF).
Since these enzymes IV modify HIF-la in an oxygen-dependent manner, they have
been suggested to function as oxygen sensors in vivo. No studies of these
oxygen sensors have been conducted in the mammalian brain or brain derived
cells. This dissertation describes biochemistry, cellular and molecular biology,
and whole animal physiology of these oxygen sensors. Using human glioma cell
lines, we demonstrate that HPHs are themselves induced by hypoxia, thus suggesting
the presence of a negative feedback system to modulate hypoxic gene expression.
For the three HPHs, we found differential distribution of expression between
different brain cell types and different brain regions. The same HPH homologues
that are regulated in permanent cell lines are regulated in brain cells in
culture and in vivo. We found that different brain regions induce HPH expression
to different extents and hypoxic induction of the oxygen sensors was more
prominent in young animals than in old and was manifested by increases in
protein expression and enzymatic activity. We also found in addition to oxygen
availability, the HIF hydroxylases are also regulated by certain glycolytic
metabolites. We specifically identified pyruvate and oxaloacetate as the regulatory
metabolites and demonstrated that their mode of action involves a reversible
inactivation of HIF hydroxylation. Pyruvate and oxaloacetate induced HIF-l
in cells and also resulted in upregulation of HPH-l and HPH-2. These results
suggest HIF prolyl hydroxylases are sensor of oxygen tensions as well as glycolytic
metabolite accumulation. Moreover, both of these stimuli increase expression
of these hydroxylases which may serve as a negative feedback system for these
sensing mechanisms. Given that the brain is highly sensitive to low oxygen
tensions, these studies may provide valuable insight to develop novel tools
and therapies for oxygen-associated brain diseases like stroke, heart failure,
and brain cancer.
Doctor
of Philosophy
2005
Dr. Shiv Srivastava, Department of Surgery
DissertationTitle: Novel Insights into p63 Expression and Function in Prostate
ABSTRACT:
Introduction: The central hypothesis of this dissertation is that p53 homologues
exist and that identification of these genes will provide a better understanding
of the role p53 gene family plays in biology and cancer. At the beginning
of this research, no p53 related gene was known. Subsequently, p63, a p53
homolog was described by other investigators. p63 is necessary in the development
of epithelial structures. One of the isoforms of p63, DNp63, is highly expressed
in prostates, which led to the hypothesis that DNp63 has critical functions
in the prostate. The studies reported here provide new insights into the expression
and function of p63 in the prostate and prostate cancer.
Results: Initially, p53 homologs were sought in human prostate epithelial
derived cell cultures through degenerate RT-PCR and other techniques. When
the discovery of p63 using a similar approach was reported, our effort focused
on p63 evaluation in tissue microarrays of benign and malignant prostate tissues.
The basal cell specific expression of DNp63 in normal prostate is very striking.
DNp63 is absent in other cell types in normal prostate and is not expressed
in prostate adenocarcinoma. Detectable p63 was found in immortalized and early
passage cell cultures derived from benign and malignant prostate tissue, but
not in senescent cultures. Experimental models using adenovirus p63 expression
vectors and prostate cell culture models have been developed. Evaluation of
proteins involved in cell growth and development regulation suggested that
p63 overexpression reduced serine 9 phosphorylation of GSK3 and AKT.
Conclusions: The selective presence of DNp63 in basal cells of the prostate
revealed it to be an excellent new bio-marker of prostate basal cells and
epithelial cell cultures. DNp63 has already found translational utility in
the diagnostic pathology setting for excluding prostate cancer in biopsies
due to its basal cell restricted expression in only benign glands. In the
prostate, DNp63 does not appear to stimulate growth, but rather functions
in the maintenance of basal cell phenotype. p63 does not appear to play a
role in prostate cancer. Further investigations of p63 mechanisms in basal
cell biology are warranted.
Doctor
of Philosophy
2005
Major Advisor:
Neil E. Grunberg, Ph.D., Department of Medical and Clinical Psychology
Dissertation Title: Environmental Enrichment, Performance, and Brain
Injury
in Male and Female Rats
ABSTRACT:
Environmental enrichment affects performance of intact organisms and improves
recovery from brain injury. The extent to which physical vs. social aspects
of enriched environments separately contribute to superior performance or
the extent to which males and females differ in their response to enrichment
has not been examined previously.
The goals of this doctoral research were to examine the separate and combined effects of social enrichment (SE) and physical enrichment (PE) on cognitive performance of neurologically-intact and brain-injured rats and to determine if there were gender differences in these effects. Measures of basic (i.e., locomotor habituation and ASR/PPI) and complex cognitive processing (i.e., passive avoidance, Morris water maze) were used to determine if enrichment affected performance on simple and complex cognitive measures. Experiment I examined the effects of enrichment on performance of 192 intact animals. Experiment II examined the effect of enrichment on performance of 96 injured animals.
The major findings from the current study were: 1) social enrichment has the greatest effect to improve performance for both intact and injured animals; 2) the effects of enrichment overall generally appear to be greater for males than for females; 3) overall enrichment has the greatest beneficial effect on tasks that require the active processing of information. These findings replicate and extend previous work on enrichment and may have important implications for educational programming and brain injury rehabilitation.
Doctor
of Philosophy
2005
Major Advisor: Gabriela Dveksler, Ph.D., Department of Pathology
Dissertation Title: Pregnancy Specific Glycoprotein 17 Binds to the Extracellular Loop 2 of its Receptor, CD9, and Induces the Secretion of IL-10, IL-6, PGE2 and TGFß1 in Murine Macrophages
ABSTRACT:
Pregnancy specific glycoproteins (PSGs) are a family of secreted proteins
produced by the placenta. In humans, the serum concentration of these glycoproteins
exceeds that of other pregnancy-related proteins, reaching up to 200-400 g/ml
at term. In species with hemochorial placentation, PSGs are believed to have
a critical role in the success of pregnancy. We previously reported that treatment
of human monocytes with PSGs induced IL-10, IL-6, and TGF-ß1 secretion.
Although the receptor for human PSGs remains unknown, we identified CD9 as
the receptor for both murine PSG17 and PSG19. To determine whether human and
murine PSG family members have similar functions and whether CD9 mediates
the response to PSG17, we treated macrophages from wild type and CD9-deficient
mice with recombinant murine PSG17N, which consists of the N1-domain of this
glycoprotein. Our data reveal that CD9 expression is required for PSG17N-induced
secretion of IL-10, IL-6, PGE2, and TGF-ß1 in macrophages. The ability
of PSG17N to induce IL-10 and IL-6 mRNA was significantly reduced in the presence
of cycloheximide, and secretion of these cytokines required induction of cyclooxygenase-2.
Further characterization of the response to PSG17 indicated that cAMP-dependent
protein kinase A is involved in the up-regulation of IL-10 and IL-6. The induction
of anti-inflammatory cytokines by various PSGs supports the hypothesis that
these placenta-secreted glycoproteins have an essential role in the regulation
of the maternal immune response during pregnancy.
Doctor
of Philosophy
2004
Major Advisor: Patricia Guerry, Ph.D., Department of Enteric Diseases, Naval Medical Research Center
Dissertation
Title: Characterization of a plasmid-encoded type IV secretion system in
Campylobacter jejuni 81-176
ABSTRACT:
The Gram-negative bacterium Campylobacter jejuni is major cause of diarrheal
illness in both the United States and the world abroad. Although well established
as an etiological agent, little is understood regarding the molecular mechanisms
underlying its pathogenesis. Additionally, inter-strain variations in clinical
manifestations, in vitro epithelial cell invasion levels, and virulence in
animal models suggest that genetic elements account for these phenotypic differences.
One strain of C. jejuni, 81-176, is a particularly virulent strain that invades epithelial cells at levels higher than most C. jejuni isolates. This strain possesses two plasmids, designated pVir and pTet, both of which contain genes with homology to type IV secretion systems (TFSS). TFSS are systems capable of translocating DNA, protein, or nucleoprotein complexes across bacterial membranes. Such systems are found in a number of human pathogens, where they facilitate various pathogenic processes. A mutational analysis in some of the pVir TFSS genes reduced levels of intestinal epithelial cell invasion and natural competence. Additionally, a mutation in one of the TFSS genes led to an attenuation of virulence in the ferret diarrhea model. These data led us to hypothesize that the pVir TFSS contributes to the pathogenesis of C. jejuni 81-176 and accounts for the phenotypic differences observed in virulence models.
This work represents a characterization of the pVir TFSS found in C. jejuni 81-176. Following purification and the generation of antisera, it was demonstrated that VirB10, a structural component of the pVir TFSS is glycosylated. Lectin affinity, enzymatic cleavage, and a reconstitution of glycosylation in Escherichia coli suggested that VirB10 was glycosylated by the general N-linked glycosylation pathway (pgl) of C. jejuni. Site-specific mutagenesis of VirB10 revealed two glycosylation sites at N32 and N97. Previously, mutation of virB10 resulted in a modest reduction in levels of natural competence. Mutation of N97 but not N32 resulted in levels of natural competence consistent with the virB10 mutant, suggesting glycosylation is required for the function of the pVir TFSS.
Secondly, an initial biochemical characterization of a putative type IV secretion ATPase was undertaken. A VirB11 homolog from the pVir plasmid was expressed and purified. This protein was shown in both a time and concentration dependent manner to possess ATPase activity and mutation of the nucleotide-binding site of VirB11 resulted in a form of the protein that was devoid of enzymatic activity. Yeast 2-hybrid analysis and chemical cross-linking experiments suggested that VirB11 formed hexameric structures, consistent with other VirB11 family members.
Lastly, characterization of a pVir-encoded protein linked with the TFSS was performed. This protein, Cjp29, bears homology to eukaryotic cell proteins and its expression is dependent on the TFSS genes. The regulation with the TFSS suggests that Cjp29 may be a protein substrate of the pVir TFSS.
Taken together, the characterization of the pVir TFSS represented here has provided a substantial foundation upon which to launch future research to elucidate the specific contributions of this system to clinical illness.
Doctor
of Philosophy
2005
Major Advisor: Kelly J. Rohan, Ph.D., Department of Medical and Clinical Psychology
Dissertation
Title: Surface Facial Electromyography Reactions to Light-Relevant
and Season-Relevant Stimuli in Seasonal Affective Disorder
Facial electromyography (EMG) activity was recorded from the zygomaticus major
and corrugator supercilii muscle regions to examine emotion-specific reactivity
in 24 currently depressed individuals meeting DSM-IV criteria for Major Depression,
Recurrent with Seasonal Pattern, and no other current Axis I diagnosis, and
24 controls with normal mood and no history of depression. Based on models
of seasonal affective disorder (SAD) and a proposed role for learned associations
between depressive behavior and environmental stimuli signaling low light
and winter season, participants were exposed to light- and season-relevant
environmental stimuli and were asked to imagine what they would be feeling
and thinking if they were actually in the picture. Skin conductance response
was also assessed to determine participants’ general sympathetic arousal
to the stimuli.
Results indicated that SAD participants: 1) responded to bright light stimuli with decreased corrugator mean EMG activity relative to low light stimuli; 2) demonstrated no increases in zygomatic mean EMG activity to bright light stimuli; 3) reported an exacerbation of baseline depressed mood following low light and winter stimuli and an improvement in depressed mood following bright light stimuli; and 4) evidenced increased SCR magnitude to bright light stimuli as compared to low light stimuli. Notably, corrugator and self-report mood ratings support previous findings of heightened psychophysiological reactivity and exacerbated depressed mood after exposure to light-relevant stimuli in SAD and suggest that light intensity may be more salient than seasonal cues in determining affective reactivity. Further research is needed to understand how these associations develop, and to establish the clinical implications for psychophysiological measures in SAD assessment and treatment monitoring.
Doctor
of Philosophy
2004
Major Advisor:
David S. Krantz, Ph.D., Department of Medical and Clinical Psychology
DissertationTitle: Biobehavioral Triggers of Cardiac Arrhythmia during
Daily Life: The Role of Emotion, Physical Activity, and Heart Rate Variability
ABSTRACT:
Biobehavioral factors, such as physical activity and emotions, have been associated
with adverse cardiac outcomes, including myocardial ischemia and infarction,
in individuals with coronary artery disease. However, cardiac arrhythmia has
largely been neglected with regard to psychosocial influences. The present
study examined the role of physical activity and acute emotions in triggering
arrhythmia in susceptible individuals during everyday activities. In addition,
it addressed the impact of chronic factors, including trait anxiety, hostility,
depression, and usual levels of physical activity on arrhythmia. Finally,
this research explored short-term and long-term heart rate variability (HRV)
as a potential mechanism for the impact of biobehavioral factors on cardiac
arrhythmia.
Participants included 40 men and 10 women with documented coronary artery
disease who have received implantable cardioverter defibrillators (ICDs).
As part of the Triggers of Arrhythmia in Defibrillator Patients (TRIAD) study,
these individuals were monitored for 48 hours using ambulatory electrocardiogram
(ECG) recorders. During the monitoring period, participants completed two
structured diaries, recording their activities and moods. Participants also
completed standardized measures of depression, hostility, and trait anxiety.
Ambulatory ECG tapes were analyzed to identify arrhythmic events, and these
events were correlated with changes in physical activity and/or emotions to
identify acute triggers of arrhythmia. Acute changes in HRV immediately coincident
and following these triggers were evaluated. Long-term HRV and chronic factors
were be correlated with arrhythmia.
As predicted, higher levels of trait anxiety and chronic depressive symptoms
were associated with lower measures of heart rate variability. Although all
associations between hostility and HRV were negative (i.e., higher hostility
was associated with lower HRV), the associations were small and did not reach
statistical significance.
Participants appeared to have been more likely to be exposed to acute negative
emotions prior to more severe cardiac arrhythmia, such as bigeminy, triplets,
and ventricular runs, compared to control periods when no arrhythmia occurred.
This association was not present when couplets were included in the analyses.
In the present sample, HRV did not change significantly prior to arrhythmic
events. However, nonsignificant changes were consistently in the predicted
direction (decreasing prior to arrhythmias). The changes in HRV prior to more
severe arrhythmias, particularly ventricular runs, were considerably larger
than those prior to more benign arrhythmias (such as couplets), but the small
number of more severe arrhythmias experienced by the present sample prevented
those changes from reaching statistical significance.
Surprisingly, participants were less likely to have been exposed to acute
physical activity prior to arrhythmia than during control periods when no
arrhythmia occurred. This finding is likely due to the fact that patients
didn’t feel well prior to arrhythmias and therefore chose not to be
physically active at that time. Although they did not report increased chest
pain or shortness of breath prior to arrhythmias, there was no measure of
general malaise, and patients were significantly more likely to report feeling
tired prior to arrhythmias compared to control periods.
Further, higher depressive symptoms were unexpectedly associated with fewer
arrhythmias. This disparate finding may be due to a type of selection bias
due to the length of time that had passed since participants had their first
infarct and the length of time that had passed since their ICDs were implanted.
Given the low levels of depressive symptoms in this sample, this finding may
also reflect a maladaptive level of unrealistic optimism. Further study is
necessary to determine the role of unrealistic optimism in cardiovascular
outcomes. Neither hostility nor trait anxiety was associated with cardiac
arrhythmia.
Self-reported usual levels of light, vigorous, heavy, and extreme physical
activity were not significantly associated with any type of arrhythmia. However,
nonsignificant correlations were all in the predicted direction; that is,
more frequent heavy and extreme activity were associated with fewer arrhythmias.
Unexpectedly, higher levels of moderate activity were significantly associated
with more frequent arrhythmias, which may indicate that the acute risk of
physical activity outweighs the benefit of consistent activity. It may also
be that participants in the present sample did not exercise frequently enough
for physical activity to be beneficial. It is unclear whether self-reported
usual levels of physical activity adequately reflect physical fitness levels.
However, correlations between reported activity levels, resting heart rate,
and body mass index suggest that they do reflect physical fitness, at least
to some extent. Additional research using more objective measures of physical
fitness, such as aerobic capacity and cardiovascular endurance, may help to
clarify these findings. Usual levels of physical activity were not associated
with any measure of heart rate variability.
Contrary to predictions, heart rate variability did not change following negative
emotions, perhaps due to low baseline levels of HRV that did not allow for
much fluctuation. This finding may help to explain why exposure to acute negative
emotions did not increase the risk of arrhythmia. Measures of 24-hour HRV
were not associated with the frequency of arrhythmias in the present sample,
which may reflect the lack of an association between HRV and arrhythmia in
ICD patients, but may also be the result of lingering effects of beta-blockers.
Doctor
of Philosophy
2005
Major Advisor: Ajay Verma, M.D., PhD, Department of Neurology
Dissertation Title: The Role of Erythropoietin Signaling in Human Cancer
ABSTRACT:
Hypoxia in solid tumors emanates from a structural and functionally disturbed
vascular supply. Intratumoral oxygen levels are associated with poor prognosis,
treatment resistance and cancer metastases, yet mechanisms for such phenomenon
remain poorly understood. The major objective of this dissertation was to
test whether or not erythropoietin (Epo), a hypoxia inducible cytokine, plays
a role in astrocytoma treatment resistance and progression. The specific aims
of this dissertation were to: 1) Determine whether or not hypoxia regulates
the expression of Epo and EpoR (erythropoietin receptor) in astrocytomas.
2) Examine if Epo treatment results in treatment resistance in astrocytomas
against chemotherapy. 3) Evaluate if Epo signaling promotes invasiveness in
human astrocytomas. We examined the expression of erythropoietin and its receptor
using immunohistochemistry in human glioma and head and neck tumor biopsies.
We also established several in vitro cell death and cell invasion assays to
examine the effects of Epo signaling on human malignant astrocytoma and head
and neck cancer cell lines. In addition, we developed primers to measure baseline
and hypoxia-inducible Epo and EpoR mRNA expression in cancer cells with quantitative
RT-PCR.
Collectively, this work answers key questions that provide insight into how
hypoxia promotes cancer malignancy. Human cancers express Epo as well as functional
EpoR. Expression of these proteins is most pronounced in hypoxic tumor regions
and in invasive tumor margins. This work demonstrates that recombinant human
Epoetin-a can directly stimulate the invasiveness of human cancer cells through
Matrigel®. Epo also promotes tyrosine phosphorylation in human glioma
cell lines. Hypoxia upregulates the expression of both Epo and EpoR in cancer
cell lines and also promotes invasiveness. Moreover, hypoxia-induced invasiveness
is blunted in stably transfected cells expressing a truncated form of the
Epo receptor and diminished by Epo neutralizing antibodies. Together these
findings suggest that autocrine or paracrine Epo signaling may play a significant
role in cancer cell invasiveness. Furthermore, the use of Epo to treat anemia
in cancer patients may have the deleterious side effect of promoting local
cancer spread. Our work may also have profound implications for the treatment
and management of cancer patients since Epo is used to treat anemia associated
with cancer therapy.
Doctor
of Philosophy
2005
Major Advisor: CDR Gary L. Hook, Department of Preventive Medicine and Biometrics
Dissertation Title: Application of Solid Phase Microextraction with Gas Chromatography-Mass Spectrometry as a Rapid, Reliable, and Safe Method for Field Sampling and Analysis of Chemical Warfare Agent Precursors
ABSTRACT:
Solid phase microextraction was combined with gas chromatography-mass spectrometry
(SPME-GC-MS) for detection of hydrogen cyanide (HCN) in the headspace above
deionized (DI) water samples, with linear results that were sensitive to below
Department of Defense short-term drinking water standards. HCN and several
common volatile organic contaminants were also detected in 3 water types in
a laboratory and field setting. The method provides an advantage over the
standard drinking water detection methods for HCN as it can also simultaneously
detect common low molecular weight hydrocarbons.
Linear results were achieved for the detection of diisopropylamine (DIPA) by SPME-GC-MS in soil from 0.72 - 3584.5 g DIPA/g soil and in DI water from 0.018 - 17.9 g/mL. The methods were successfully field tested with common hydrocarbon contaminants in 3 common agricultural soil types and 3 water types. In particular, this methodology would be useful for investigation of a suspected VX nerve agent production facility.
Solid phase dynamic extraction (SPDE) was compared to passive SPME for vapor sampling of DIPA and dimethyl methylphosphonate (DMMP) with analysis by fast GC-MS. Equilibrium sampling by SPDE for DIPA and DMMP vapor provided linear results at lower concentrations and gave larger extracted ion peak areas than comparable SPME sampling. This unique application has shown great potential for further laboratory and field use, both for health risk assessment and initial chemical detection employment.
A fast GC capillary column was integrated with a novel low pressure, quadrupole ion trap, time-of-flight photoionization mass spectrometer (Qit Tof PI-MS). The addition of a GC injection port and column allowed the use of SPME to provide headspace extraction of several chemical warfare agent (CWA) precursors for introduction to this GC/PI-MS. This prototype instrumentation was shown to be able to detect CWA precursor vapors in air individually and in mixtures.
The central research goal was to develop the ability to rapidly detect CWA precursors in a field setting. The greatest benefit to using these SPME-GC-MS methods is they allow unambiguous detection and identification of CWA precursors as well as common environmental contaminants. These detection methods are applicable to the military environmental scientist as well as homeland defense and hazardous material detection personnel. Identification of environmental chemicals is the first step in assessing military deployment exposures and health risks.
Doctor
of Philosophy
2005
Major Advisor: Gerald V. Quinnan, Jr., M.D., Department of Preventive Medicine and Biometrics
Dissertation Title: Molecular Epidemiology of Epidemic Severe Malaria Caused by Plasmodium vivax in the State of Amazonas, Brazil.
ABSTRACT:
Malaria in South America is a major public health problem. In Brazil, most
of the cases occur in the Amazon Region, particularly in the State of Amazonas.
In Manaus, the capital of Amazonas, atypical cases of P. vivax infections,
including patients presenting with severe thrombocytopenia and bleeding, led
to the formulation of the hypothesis that severe disease could be related
to a particular, emergent and more pathogenic genotype of P. vivax. We described
the epidemiology of malaria for the Amazonas State and city of Manaus by comparing
patients admitted in the hospital to those treated as outpatients in the Fundação
de Medicina Tropical do Amazonas (FMT-AM). Admissions due to vivax malaria
increased significantly from 1997 through 2003 suggesting a change in clinical
presentation. The admitted group presented higher mean parasite counts, lower
platelet counts, and higher levels of liver enzymes, higher total and indirect
bilirubin, and higher blood urea nitrogen when compared to the outpatient
group. Clinical symptoms of severe disease, including hematuria, hemolytic
anemia, and thrombocytopenia were only noted in the admitted group. Furthermore,
the presence of a palpable liver was more frequent in admitted patients. Nucleic
acid sequences of three genes from P. vivax, the 18S SSUrRNA Type A gene,
CSP gene and MSP-1 gene, were determined. Strains from test samples were compared
to each other, to the reference strains Salvador I and Belém and to
sequences retrieved from the Gene Bank. There were two main polymorphisms
in the 18S SSUrRNA Type A gene, a cytosine/thymidine polymorphism at residue
100 of the alignment and a thymidine (T)/adenine (A) polymorphism at residue
117. Ten of eleven (10/11) admitted patients were 117:T compared to 13/21
outpatients. This frequency difference was statistically significant (P<0.05).
The Salvador I strain was T at this position and the Belém strain was
A. In the CSP gene, we identified 15 unique sequences of the VK210 strain;
one sample had a mixed infection with P. vivax-like. The sources of variation
in the CSP gene included the numbers of repeat segments, alanine/aspartic
acid polymorphism at position five of a common repeat, and sporadic mutations.
Frequent synonymous substitutions of the common repeat occurred in codons
1, 2 and 7, while the mutations at codon 5 were always non-synonymous. Among
MSP-1 gene sequences, four recombination sites were distinguished between
the interspecies conserved regions 5 and 6. Recombination among progenitor
strains, closely related to the Salvador I and Belém strains, was the
main source of diversity among the strains. The second most significant form
of variation was in the polyglutamine region of strains with Belém-like
sequences in the central part of this gene segment. There was no clustering
of MSP-1 sequences in patients with severe disease. It was not possible to
demonstrate the evolutionary relationship among our test samples by tests
of phylogeny that incorporated sequence data for all three genes tested. The
factors that may have limited the power of a combined analysis included small
sample size and differences in the mechanisms and extent of variation among
the genes. The retrospective study was unable to demonstrate that a particular
strain of P. vivax was responsible for severe disease requiring hospitalization.
This is the first detailed description of the genetic diversity among the
P. vivax population in the Amazonas State of Brazil.
Doctor
of Philosophy
2005
Major Advisor: Ann E. Jerse, Ph.D. Department of Microbiology and Immunology
Dissertation Title: Examination of Neisseria gonorrhoeae Opacity Protein Expression During Experimental Murine Genital Tract Infection
The opacity (Opa) proteins of Neisseria gonorrhoeae are a family of phase- variable outer membrane proteins that bind to host cells. Phase variable expression occurs via a reversible frameshift mechanism within each opa gene. Opa protein expression is selected for, or induced during experimental genital tract infection of female mice, similar to that which was reported in male volunteers. Using a genetically marked strain of FAI090 to follow recovery of a specific population of Opa variants during murine infection, here we showed that selection of a pre-existing population of Opa-positive gonococci present in the inoculum was responsible for the reisolation of mainly Opa-positive variants early during infection. We conclude that the preferential recovery of Opa-positive gonococci observed early during murine infection is due to selection of a pre-existing population of Opa-positive variants caused by factors other than binding to human CEACAM receptors. In long-term infection of mice, a cyclical pattern of Opa protein expression was observed in which a decreased recovery of Opa- positive variants followed early selection for Opa protein expression; reemergence of Opa-positive gonococci occurred later in infection. To examine rates of phase variation in a N gonorrhoeae background under physiologically relevant conditions, we engineered a translational opaB::phoA fusion and introduced it into the chromosome of N. gonorrhoeae strain FAIO90 to generate strain ANSIOO. No change was seen in the frequency or rate of opaB::phoA phase variation in all in vitro conditions tested. We next examined whether increased phase variation of the opaB::phoA fusion occurred during murine infection. No significant change occurred in the frequency of "on" variants among vaginal isolates when mice were inoculated with predominantly "off' variants; however, a marked increase in the recovery of "off' variants was observed following inoculation with predominantly "on" variants. The inability to show differences in opa gene phase variation under different conditions in vitro leads us to conclude at this time that induction ofopa gene phase variation may be spontaneous and random. However, the in vivo studies suggest that increased opa gene phase variation may occur under conditions that we have not yet been able to mimic in vitro.
Doctor
of Public Health
2005
Major Advisor: COL Robert Lipnick, USA, Department of Preventive Medicine & Biometrics
Dissertation Title: Birth Weight and Acute Leukemia: A Meta-Analysis of Observational Studies
ABSTRACT
The major objective of this study was to determine whether high birth weight
is associated with ALL and AML among children and to quantify the strength
of the relationships. We conducted a meta-analysis of nine case-control studies
(published between 1991 and 2004) encompassing over 6,200 children with ALL
and over 12,000 controls. We found that children weighing 4,000 g or more
at birth had 24% (OR: 1.24; 95% CI: 1.12, 1.37) higher odds of developing
ALL than children weighing less (without consideration to reference weight).
Regardless of peer-review status, response rates among cases and controls,
or choice of threshold for high birth weight, studies consistently demonstrated
a similar overall odds ratio ranging from 1.23 to 1.29. In addition, our data
analysis identified possible reasons for inconsistent findings among previous
studies that examined high birth weight as a risk factor for ALL. Possible
explanations include: use of different reference birth weights, different
data source for birth weight (i.e., birth certificate vs. interview), and
different ethnic makeup of the study population. Our data supports the growing
evidence for the link between high birth weight and childhood ALL. Women who
are pregnant or considering pregnancy should know that unbounded weight gain
may increase the odds of childhood ALL in their baby. Whether a positive association
with high birth weight applies to AML is less clear from our results. Based
on a meta-analysis of only three case-control studies (published between 1997
and 2004) involving over 700 children with AML and over 1,900 controls, high
birth weight (> 4,000 g) appeared to increase the odds of developing AML
by 14% (OR: 1.14; 95% CI: 0.84, 1.54).