Robert McCullough Anthony
Department of Microbiology and Immunology
 
Doctor of Philosophy
2007
 
Major Advisor: William C. Gause, Ph.D., Department of Microbiology and Immunology
 
Dissertation Title: Characterization and Function of the Inflammatory
Response to Infection by a Gastrointestinal Nematode Parasite: New Insights into Protective Th2 Responses.
 
ABSTRACT
 
Effective immune responses to infectious diseases involve recognition of invading pathogens and result in appropriate primary and secondary reactions mediating host protection. Although these responses against many bacteria and viruses have been characterized extensively, Th2 effector mechanisms leading to host-protection remain elusive. Using an infectious model employing a natural murine gastrointestinal nematode parasite, Heligmosomoides polygyrus, we characterized the immune cell infiltrate surrounding invasive larval parasites in the small intestinal muscosa and submucosa (host:parasite interface) during early stages of a secondary infection. A primary is chronic, with established adult parasites detectable up to four months post infection, where as the parasites are naturally cleared from the intestinal lumen by 14 days follow challenge. This distinction between primary and secondary H. polygyrus infections allows a clear readout of protective immunity, making this infectious model useful for examining protective secondary Th2 responses.
 
A distinct and highly reproducible leukocyte architecture developed by the fourth day post challenge, which included Gr1+ neutrophils amassing adjacent to the parasite, and CD4+ T cells, CD11c+ dendritic cells, and MBP-1+ eosinophils surrounding the parasite in the lamina propria. Additionally, laser capture microdissected (LCM) samples from the host:parasite interface featured upregulated Th2 cytokine mRNAs relative to untreated intestinal tissue. This localized inflammatory response differed during primary infection, as CD4+ T cells did not infiltrate the host:parasite interface, and there were no increases in cytokine expression. These findings were extended to show that the peripheral inflammation during the memory Th2 response at the host:parasite interface is essential for host-protection leading to worm expulsion. Memory CD4+ T cells that express Th2 cytokines rapidly accumulate around the invading parasite in the intestinal submucosa, and induce the alternative activation of macrophages (IL-4Rhi, CD206+, arginase-1+, Fizz1+, Ym1+, iNOS-).
 
Alternatively activated macrophages metabolize the amino acid, arginine, by the enzyme arginase-1, which is essential for their differentiation and effector functions. Through intervention experiments, our findings demonstrate that macrophages and arginase contribute to the natural clearance of a secondary H. polygyrus infection. These observations provide new insights into mechanisms of host-protection mediated by Th2 responses, and establish a novel, protective role for alternatively activated macrophages.

Andrea S. Bertke
Emerging Infectious Diseases Program
 
Doctor of Philosophy
2007
 
Major Advisor: Dr. Philip R. Krause, M.D., Department of Microbiology and Immunology, Emerging Infectious Diseases and Food and Drug Administration, Center for Biological Research and Review
 
Thesis Title: Influence of Herpes Simplex Virus Latency Associated Transcript (LAT) on the Distribution of Latently Infected Neurons
 
ABSTRACT
 
Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are similar viruses with several notable differences. While both viruses establish latency in sensory ganglia and reactivate to cause recurrent disease, HSV-1 reactivates more efficiently from trigeminal ganglia to cause cold sores or keratitis and HSV-2 reactivates more efficiently from lumbosacral dorsal root ganglia (DRG) to cause genital herpes. Both viruses are capable of causing central nervous system (CNS) disease, but HSV-1 CNS infections typically manifest as severe necrotizing encephalitis while HSV-2 is more commonly associated with relatively benign meningitis. Potential mechanisms for type-specific differences between HSV-1 and HSV-2 have not been closely examined, although the latency-associated transcript (LAT) of HSV plays a critical role in the establishment of latency and site-specific reactivation and may have regulatory influence over viral replication and spread. In the work presented here, HSV-1 and HSV-2 were evaluated using the guinea pig model of infection to characterize differences in viral spread, replication, and the establishment of latency in the peripheral and central nervous systems. Chimeric viruses and a LAT promoter-deleted virus were also evaluated to determine the role of LAT in type-specific differences between HSV-1 and HSV-2, and also to determine the specific region of LAT that contributes the critical elements for HSV-2 reactivation. The major findings of this thesis are: 1) HSV-1 and HSV-2 spread to different regions of the central nervous system, preferentially utilizing different autonomic pathways in addition to sensory pathways; 2) type-specific LAT regions influence viral replication and gene expression differently in the DRG and the spinal cord mediated by host factors in specific types of neurons, contributing to differences in viral spread, neurotropism, severity of disease, and latent viral load; and 3) LAT exon 1 contributes the critical elements for type-specific reactivation of HSV-2. Taken together, the results provide insight into potential mechanisms for type-specific differences between HSV-1 and HSV-2, providing a basis for additional molecular analyses of the regulatory control of type-specific latency and reactivation as well as CNS involvement after peripheral HSV infections. In addition, the results suggest modifications to the current model of HSV pathogenesis.

Kathleen Daddario-DiCaprio
Department of Pathology
 
Doctor of Philosophy
2006
 
Major Advisor: Dr. Elliott Kagan, M.D., F.R.C. Path, Uniformed Services University of the Health Sciences, Professor of Pathology, Professor of Emerging Infectious Diseases, and Professor of Preventative Medicine & Biometrics and Dr. Thomas Geisbert, Ph.D, Department of Virology, United States Army Research Institute of Infectious Diseases, USAMRIID, Adjunct Professor, Uniformed Services University of the Health Sciences, Department of Pathology
 
Thesis Title: Evaluation of the protective efficacy of recombinant vesicular stomatitis virus vectors against Marburg hemorrhagic fever in nonhuman primate models
 
ABSTRACT
 
Marburg virus (MARV) causes a severe and often fatal hemorrhagic disease in both humans and nonhuman primates (NHPs) for which there are no proven therapies or vaccines. The need for efficacious interventions is underscored both by the recent MARV outbreak in Angola and by the risks of potential MARV laboratory exposures. Unfortunately, the majority of attempts at developing effective vaccines and therapeutics against MARV HF have been unsuccessful. While several vaccine platforms have demonstrated utility in rodent models, these platforms have been inconsistent in transitioning protective responses into NHP models. In this study, we investigated whether live, attenuated, replication-competent recombinant vesicular stomatitis virus vectors (rVSVs), which express the transmembrane glycoprotein of MARV strain Musoke (rVSV-MARV), could protect NHPs against MARV challenge. This study evaluated the utility of rVSV-MARV as both preventative and postexposure treatment strategies against homologous and heterolgous MARV challenge in NHPs. Results showed that rVSV-MARV provided complete protective responses when used as a preventative vaccine as well as a postexposure treatment and that protection associated with both strategies were composed primarily of MARV-specific antibody responses that constituted only low to moderate induction of neutralizing antibodies. Additional evaluations illustrated the absence of detectable T-lymphocyte responses, thus highlighting the protective role of humoral immunity. Although the protective mechanism of the rVSV-MARV vaccine in NHPs remains to be determined, the study design for evaluating postexposure protection against MARV in NHPs provides a paradigm to outline correlates of immunity for which other interventions could apply. These studies suggest that survival is weighted heavily on antibody responses; however, the possibility that other mechanisms of immunity are contributing to viral clearance cannot be excluded. Possible mechanisms include direct viral interference, induction of a robust innate response, or a "jump-start" of the adaptive immune response. Accordingly, these results provide critical insight into the nature of effective anti-MARV immunity.

Gabriel Nji Defang
Emerging Infectious Diseases Graduate Program
 
Doctor of Philosophy
2007
 
Major Advisor: CAPT Gerald V. Quinnan, Jr., M.D. Chair, Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences
 
Thesis Title: Toward Viral Vaccine Development: A modified Venezuelan Equine Encephalitis Replicon As A Strategy For Optimizing Immunogenicity.
 
ABSTRACT
 
Alphaviral vectors have been used as vaccines for immunization against several viral infections. However, these vectors typically induce rapid cytopathic effects in mammalian cells. The cytopathicity of these vectors allow for only transient expression of transgenes. In viral models where long-term expression may be needed to induce adequately differentiated immune responses, the current wild-type alphaviral vectors may not be sufficiently immunogenic. This thesis describes development of attenuated, prolonged-expression, Venezuelan equine encephalitis virus (VEE) vectors by modifying the non-structural protein-2 gene (NsP2) through the introduction of single or double point mutations.We studied the potential of wild-type and modified VEE replicon particles (VRPs) for optimizing and enhancing the immunogenicity of HIV-1 envelope glycoprotein and to the fusion (F) and attachment (G) glycoproteins of two zoonotic pathogens, Hendra and Nipah viruses, in a murine and rabbit model. We demonstrate in this study that modified VRPs are efficient in optimizing neutralizing antibody responses. Three-fold lower doses and only two vaccinations of modified VRPs were sufficient to induce maximal HIV-1 neutralizing antibody responses in mice compared to three vaccinations of wild-type VRPs.We also report here for the first time that, high-level cross-reactive neutralizing antibodies against henipaviruses were achieved using VRPs. Overall, these data suggest that the use of modified alphavirus-based vaccine platforms should be given consideration for the development of viable antiviral vaccines.

David A. Florin
Department of Preventive Medicine and Biometrics
 
Doctor of Philosophy
2006
 
Major Advisor: Phillip Lawyer, Ph.D., Department of Preventive Medicine and Biometrics
 
Thesis Title: Morphometric and molecular analyses of the sand fly species Lutzomyia shannoni (Dyar 1929) (Diptera: Psychodidae: Phlebotiminae) collected from seven different geographical areas in the southeastern United States
 
ABSTRACT
 
Morphometric and molecular analyses were used to elucidate the variation among the sand fly Lutzomyia shannoni collected from seven widely separated locations in the southeastern United States: Baton Rouge, LA; Fort Bragg, NC; Fort Campbell, KY; Fort Rucker, AL; Ossabaw Island, GA; Patuxent NWRR, MD; and Suwannee NWR, FL. Lu. shannoni is a wide-ranging phlebotomine sand fly has been implicated in the transmission of a number of parasitic and viral pathogens of medical/veterinary importance. In light of this, it is imperative to answer the question of whether or not significant variation exists among the purported biogeographical populations so as to make a determination on the possibility of a cryptic species complex. A balanced approach to answering this question was emphasized by using the two-prong method of morphological and molecular data. The morphometric analysis entailed using univariate and multivariate techniques on a sample size of 40 males and 40 females from each collection site (with the exceptions, due to inadequate number of collected specimens, of Baton Rouge where morphometrics were not conducted for the specimens of either gender and of Suwannee NWR where morphometrics were not conducted on the male specimens) A total of 54 characters from the male specimens and 49 characters from the female specimens were measured by an inserted micrometer in the ocular eyepiece of a compound microscope. Results indicate that while there is a certain amount of variation, it is not sufficient to discriminate among the collection sites. Two molecular markers, the mitochondrial DNA CO I and the nuclear DNA ITS2, were PCR-amplified and the resulting sequences compared. For both markers, the small amount of variation observed in the sequences did not have a diagnostic distribution and were not informative in distinguishing the specimens based upon collection site. There may exist one population of Lu. shannoni throughout the United States. In a corollary study, the population dynamics of Lu. shannoni were examined at the Patuxent NWRR, MD from June 23, 2005 to June 15, 2006 by conducting weekly light trap collections. The abundance pattern appears to be unimodal although only multi-year data can provide a definitive determination.

Ardath W. Grills
Department of Medicine
 
Doctor of Philosophy
2006
 
Major Advisor: COL Christian F. Ockenhouse, MD Assistant Professor, Department of Medicine
 
Thesis Title: The complexity of Plasmodium falciparum infections in children in western Kenya
 
ABSTRACT
 
To investigate the allelic complexity of infection (COI) of Plasmodium falciparum infections in children living in Kisumu, western Kenya, samples from three studies conducted from June, 2003 through May, 2006 were analyzed: a longitudinal cohort, a phase 1 field trial and a phase 2 field trial. Samples from the studies were analyzed using nested PCR of the highly polymorphic msp1 block 2 to observe potential selective effects of the vaccine, a MSP1 formulation.
 
The longitudinal cohort, used to determine the baseline COI, followed 60 children 1-4 years old for 13 months who donated scheduled samples monthly and additional samples when ill with clinical malaria. Results revealed a COI that was dependent on age, parasite density, illness, village location and bed net use. Nearly all infections were with multiple genotypes. Fluctuations of the three examined alleles of msp1, K1, MAD20 and RO33, were rapid and random within individual children, as well as the entire study group, indicating a highly diverse parasite population. Parasite density was found to be directly correlated with COI in those children with clinical illness. As the density increased, the contribution of the K1 allele proportionately increased while the contribution of the RO33 allele decreased. Presence of the invariable RO33 allele was also found to be mildly protective against clinical illness. For the first time, bed net use was found to decrease COI in 1-2 year old children who were both asymptomatic carriers of parasites and ill with clinical malaria; the RO33 allele was again most associated with the decrease in COI. In the phase 1 dose-escalation trial with 135 children, those participants who received the full vaccine dose had a decrease in COI following vaccine administration. In that group of children, the RO33 allele was identified in much greater prevalence following vaccine administration. Samples that were RO33 positive were also predominately chloroquine sensitive. The phase 2 vaccine trial with 400 participants is still currently blinded; initial analysis showed an increased COI in ill patients, a finding that contrasts with previous reports. Combined, the three studies provided evidence of the rapidly evolving immunity to malaria, even within the limited 1-4 year age range of the study participants.

Nader Halim
Molecular & Cell Biology Program
 
Doctor of Philosophy
2006
 
Major Advisor: Ajay Verma M.D., Ph.D. Professor, Department of Neurology
 
Thesis Title: Regulation of Brain Glucose Metabolic Patterns by Protein Phosphorylation and Drug Therapy
 
ABSTRACT
 
Glucose metabolism is the main energy-producing pathway of the central nervous system under normal conditions. Thus the regulation of brain glucose metabolism is essential to maintaining homeostasis. This study aims to determine whether the phosphorylation status of the pyruvate dehydrogenase complex differentiates the metabolic phenotype of astrocytes and neurons in vitro; and to determine whether antipsychotic drug administration affects glucose metabolites in vivo. The results of this study demonstrate that the phosphorylation status of the pyruvate dehydrogenase complex underlies the glycolytic phenotype of astrocytes and the oxidative phenotype of neurons in culture. In cultured astrocytes this phenotype can be altered to resemble that of neurons by treatment with the pyruvate dehydrogenase activating compound dichloroacetate. These data also suggest a possible molecular mechanism for the metabolic exchange of monocarboxylates between astrocytes and neurons in vivo. We also demonstrate in rats that chronic antipsychotic administration results in an increase in postmortem lactate levels. We also show an increase in the postmortem lactate levels in the cerebellum of patients with schizophrenia. These results suggest that postmortem metabolic alterations in the brains of patients with schizophrenia may be the result of antipsychotic treatment rather than a primary feature of the disease.

Jeremy R. Hershfield
Anatomy, Physiology and Genetics
 
Doctor of Philosophy
2006
 
Major Advisor: Aryan Namboodiri, Ph.D. Assistant Professor, Department of Medicine
 
Thesis Title: Mutational Analysis of Aspartoacylase: Implications for Canavan Disease
 
ABSTRACT
 
Mutations in aspartoacylase, which catalyzes the deacetylation of N acetyl Laspartate in the CNS, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. Although several mutations result in undetectable aspartoacylase activity, the underlying biochemical mechanisms are poorly understood. Therefore, we developed and tested a three dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc binding residues (H21E/E24H and E24H/H116E) yielded wild type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss of function aspartoacylase mutations associated with Canavan Disease.

Xiaolong Jiang
Neuroscience Program
 
Doctor of Philosophy
2007
 
Major Advisor: He Li MD., Ph.D. Associate Professor, Department of Psychiatry
 
Thesis Title: Effect of Traumatic Stress on Multiple Aminergic Systems in Amygdala and Hypothalamus: Specific Impairment of 5-ht2a Receptor Signaling and its Pathophysiological Role in an Animal Model of Post-Traumatic Stress Disorder
 
ABSTRACT
 
The amygdala and hypothalamus are central brain regions participating in stress response. This response also requires participation of multiple aminergic systems, which extensively interconnect with the amygdala and hypothalamus. Thus, dysregulation of aminergic systems, particularly the serotonergic and noradrenergic systems, is closely linked with multiple anxiety and stress disorders. The present study, utilizing a learned helplessness stress model, determined if alterations of aminergic systems in the amygdala and hypothalamus were involved in stress-induced behavioral and physiological abnormalities associated with anxiety and stress disorders.
 
The first part of the study investigated the electrophysiological function of serotonergic, histaminergic and noradrenergic systems in the basolateral amygdala (BLA) in control rats and associated these functions with identification of the primary aminergic receptors. The principal electrophysiological function of serotonin in the BLA was to facilitate GABA release and the 5-HT2A receptor was the primary serotonin receptor involved in this effect. The principal electrophysiological function of histamine in the BLA was to suppress excitatory synaptic transmission through presynaptic histamine H3 receptors. In certain BLA neurons, histamine also potentiated excitatory synaptic transmission via a currently unknown mechanism. The primary function of norepinephrine in the BLA was also to suppress amygdala excitability, and the ?2 adrenoceptor was confirmed to be one of the mechanisms mediating this inhibitory effect.
 
In rats exposed to three-day restraint/tail shock, 5-HT2A receptor-mediated serotonergic facilitation of GABA release was severely impaired, while ?2 adrenoceptor-mediated and H3 receptor mediated suppression of BLA excitatory synaptic transmission were not significantly changed. Quantitative RT-PCR and western blot analysis further demonstrated that stress specifically decreased BLA 5-HT2A receptors, while other aminergic receptors were not significantly altered. In addition, treatment with the selective 5-HT2A antagonist, MDL 11,939 during stress, which is assumed to prevent amygdala 5-HT2A receptor from being impaired, prevented the occurrence of enhanced acoustic startle response (ASR), a stress-induced behavioral manifestation that depends on the amygdala.
 
Since serotonin in the hypothalamus, by mechanisms of 5-HT2A,5-HT2C, 5-T1A, 5-HT1B receptors, provides an important mechanism mediating feeding and body weight, the present study also examined whether sustained body weight loss in stressed animals is associated with dysregulation of hypothalamic serotonergic system. Stress decreased the mRNA levels of hypothalamic 5-HT2A receptor and increased 5-HT1B receptor mRNA levels, while 5-HT2C and 5-HT1A receptor mRNA levels remained unchanged. Pretreatment with the selective 5-HT2A antagonist, MDL 11,939, which is assumed to prevent hypothalamic 5-HT2A receptors from being decreased by stress, dose-dependently reversed sustained body weight loss in stressed animals.
 
These findings indicate that BLA and hypothalamic 5-HT2A receptors, but not H3 receptor, ?2 adrenoceptor and other serotonin receptors, play a critical role in pathophysiological response to traumatic stress, and alteration of this receptor in the BLA and hypothalamus may represent an essential mechanism underlying the emergence of behavioral and physiological abnormalities resulting from stress, such as enhanced ASR and sustained body weight loss. Hence, 5-HT2A receptor ligands may be potential preventive or therapeutic agents for stress-associated psychiatric disorders, especially post-traumatic stress disorder.

Johnson
Neuroscience Program
 
Doctor of Philosophy
2007
 
Major Advisor: He Li MD., Ph.D. Associate Professor, Department of Psychiatry
 
Thesis Title: Effect of Traumatic Stress on Multiple Aminergic Systems in Amygdala and Hypothalamus: Specific Impairment of 5-ht2a Receptor Signaling and its Pathophysiological Role in an Animal Model of Post-Traumatic Stress Disorder
 
ABSTRACT
 
Since serotonin in the hypothalamus, by mechanisms of 5-HT2A,5-HT2C, 5-T1A, 5-HT1B receptors, provides an important mechanism mediating feeding and body weight, the present study also examined whether sustained body weight loss in stressed animals is associated with dysregulation of hypothalamic serotonergic system. Stress decreased the mRNA levels of hypothalamic 5-HT2A receptor and increased 5-HT1B receptor mRNA levels, while 5-HT2C and 5-HT1A receptor mRNA levels remained unchanged. Pretreatment with the selective 5-HT2A antagonist, MDL 11,939, which is assumed to prevent hypothalamic 5-HT2A receptors from being decreased by stress, dose-dependently reversed sustained body weight loss in stressed animals.
 
These findings indicate that BLA and hypothalamic 5-HT2A receptors, but not H3 receptor, ?2 adrenoceptor and other serotonin receptors, play a critical role in pathophysiological response to traumatic stress, and alteration of this receptor in the BLA and hypothalamus may represent an essential mechanism underlying the emergence of behavioral and physiological abnormalities resulting from stress, such as enhanced ASR and sustained body weight loss. Hence, 5-HT2A receptor ligands may be potential preventive or therapeutic agents for stress-associated psychiatric disorders, especially post-traumatic stress disorder.

Katherine Shi-Hui Lee
Pathology
 
Doctor of Philosophy
2006
 
Major Advisor: Clifford M. Snapper, M.D., Professor, Department of Pathology
 
Thesis Title: The host immune response to Streptococcus pneumoniae: bridging innate and adaptive immunity
 
ABSTRACT
 
Streptococcus pneumoniae (Pn) remains the primary cause of community-acquired pneumonia throughout the world, leading to high morbidity and mortality rates in the young and elderly. A better understanding of the host response to the organism would aid in the development of more effective antibiotics and vaccines. Toll-like receptors (TLRs) play an important role in the initial recognition of pathogens by binding conserved moieties known as pathogen associated molecular patterns (PAMPs). This interaction is translated through the induction of signaling cascades that ultimately results in the production of various chemical mediators necessary for the activation of the adaptive arm of the immune response. Presentation of processed antigen in the context of major histocompatibility complex (MHC) by antigen presenting cells occurs, leading to effective help by primed T cells (Teff) to naïve B cells in a process known as linked recognition. The activation and proliferation of B cells into mature plasma cells results in the development of appropriate antibody responses that are critically important in the clearance of extracellular bacteria such as Pn. These responses, however, need to be modulated so that inappropriate immune activation does not lead to anergy or overresponsiveness. The naturally occurring thymic population of CD4+CD25+ regulatory T cells (Tregs) survey and monitor the actions of Teff to prevent such inappropriate responses.
 
In the following dissertation, I have used a murine model to study the host response to Pn. Specifically, I have focused on previous findings in the lab that suggested a possible role for Tregs as well as TLRs in our model system. I found that Tregs do not appear to play a part in modulating acute humoral responses to a bacterial pathogen such as Pn. The length of antigen stimulation may determine the need for regulation by Tregs, but since Pn is rapidly cleared by the immune system, Treg modulation does not appear to be necessary in such an infection model. However, I was able to show that cooperation between TLR2 and TLR4, as well as TLR2 and TLR9, is important for the induction of MyD88-dependent innate immune responses to Pn.

Sean T. Manion
Psychiatry and Neuroscience
 
Doctor of Philosophy
2006
 
Major Advisor: He Li, M.D., Ph.D., Associate Professor Department of Psychiatry and Neuroscience
 
Thesis Title: Amygdala, Anxiety & á1 Adrenoceptors: Investigations Utilizing a Rodent Model of Traumatic Stress
 
ABSTRACT
 
Exposure to traumatic stress can result in post-traumatic stress disorder (PTSD) and other pathophysiological conditions. PTSD is characterized by a number of persistently heightened physiological and behavioral indicators, including exaggerated acoustic startle response (ASR) and alterations in processing of emotional memory. Similar effects can be seen in an animal model of traumatic stress in which stress results from restraint and inescapable tailshocks to rats. The basolateral amygdala (BLA) is an area known to be involved in the processing of emotional memory and startle modulation. Synaptic plasticity in the BLA is thought to play a key part in this memory formation, and therefore can be involved in subsequent stress related pathologies seen in PTSD.
 
The first part of this project used this model of traumatic stress to investigate the effects of prazosin, an á1 adrenergic receptor (AR) antagonist, on stress induced elevation of ASR. Recent studies have shown the effectiveness of prazosin in treating PTSD. This investigation sought to determine its effectiveness in reducing the effects of traumatic stress when given prior to stress. Male Sprague-Dawley rats were injected with 0.5 mg/kg of prazosin 30 minutes before inescapable tail shock on three consecutive days. ASR testing was performed 1, 4, 7 and 10 days post-stress and compared to baseline and control values. Results show a significant reduction of ASR hyperarousal in the pre-stress injection group. Pre-stress treatment with lower levels of prazosin (0.25, 0.1 and 0.05 mg/kg) showed similar reduction in ASR hyperarousal due to stress. The second part of this project sought to investigate á1A adrenoceptor involvement in long-term potentiation (LTP) in the BLA and to determine the effects of traumatic stress on this type of plasticity. In the BLA of control animals, the á1A AR specific agonist A61603 (1 ìM) completely abolished theta-burst stimulation-induced LTP. In animals previously exposed to a repeated restraint and tailshock stress protocol, only a partial reduction of LTP was detected in the presence of A61603. This blocking effect of A61603 on LTP in control animals was occluded in the presence of á1A AR specific antagonist WB4101 (1 ìM), while the antagonist did not eliminate the partial reduction of LTP in stressed animals. These findings suggest a possible mechanism contributing to the emotional memory in PTSD and support that the á1A ARs can be a specific pharmacological target in PTSD.
 
Taken together, these findings offer both the possibility of preventative treatment for certain physiological symptoms of PTSD by administering prazosin prior to stress, as well as the potential involvement of á1A ARs in the BLA in the emotional memory aspects of PTSD.

Beth Ann McNichol
Microbiology and Immunology Program
 
Doctor of Philosophy
2007
 
Major Advisor: Alison D. O'Brien, Ph.D., Professor and Chair, Department of Microbiology and Immunology
 
Thesis Title: Identification of the Regions of Cytotoxic Necrotizing Factor Type 1 Responsible for Receptor Binding and Enzymatic Activity
 
ABSTRACT
 
Cytotoxic necrotizing factor type 1 (CNF1) and dermonecrotic toxin (DNT) share homology within their catalytic domains and possess deamidase and transglutaminase activities. Although each toxin has a preferred enzymatic activity (i.e., deamidation for CNF1 and transglutamination for DNT) as well as target substrates, both modify a specific glutamine residue in RhoA, Rac1, and Cdc42, which renders these GTPases constitutively active. Here we show that despite their similar mechanisms of action CNF1 and DNT induced unique phenotypes on HEp-2 and Swiss 3T3 cells. CNF1 induced multinucleation of HEp-2 cells and was cytotoxic for Swiss 3T3 cells (with binucleation of the few surviving cells) while DNT showed no morphological effects on HEp-2 cells but did induce binucleation of Swiss 3T3 cells. To determine if the enzymatic domain of each toxin dictated the induced phenotype, we constructed enzymatically active chimeric toxins and mutant toxins that contained single amino acid substitutions within the catalytic site and tested these molecules in tissue culture and enzymatic assays. Moreover, both site-directed mutant toxins showed reduced time to maximum transglutamination of RhoA compared to the parent toxins. Nevertheless, the substitution of threonine for Lys1310 in the DNT-based mutant, while affecting transglutamination efficiency of the toxin, did not abrogate that enzymatic activity.

Randall K. Merling
Molecular and Cell Biology Program
 
Doctor of Philosophy
2007
 
Major Advisor: Chou-Zen Giam, Ph.D. , Professor, Department of Microbiology and Immunology
 
Thesis Title: The Effects of HTLV-1 Tax on Mitotic Regulation
 
ABSTRACT
 
The human T-cell leukemia virus 1 (HTLV-1) encodes the tax gene (transactivator encoded by the pX region) which is a potent activator of viral transcription and Nuclear Factor kappa B (NF-.B). Recent data indicate that Tax protein activates the anaphase promoting complex/cyclosome (APC) ahead of schedule, thereby causing the premature degradation of cyclin A, cyclin B1, securin, and Skp2. The premature loss of these mitotic regulators is accompanied by mitotic aberrations and leads to rapid senescence and cell cycle arrest in HeLa and S. cerevisiae cells. This tax-induced rapid senescence (tax-IRS) of HeLa cells is mediated primarily through a dramatic stabilization of p27KIP, as deficiencies in p27KIP prevent Tax-IRS. Since p27KIP stabilization is accompanied by a surge in the level of p21CIP1/WAF1, this cyclin-dependent kinase inhibitor may also play a role in tax-IRS.
 
As part of the research presented in this dissertation, a collection of HTLV-1 tax point mutants were isolated that permit normal growth of S. cerevisiae. Similar to wild-type tax, many mutants (C23W, A108T, L159F, and L235F) transactivated both the HTLV long terminal repeat (LTR) and the NF-.B reporters. The V19M mutant preferentially activated NF-.B, but was attenuated in LTR activation. None of the mutants significantly elevated the levels of p21CIP1/WAF1 and p27KIP1, indicating that dramatic Tax-induced surges in p21CIP1/WAF1 and p27KIP1 occurs by mechanisms distinct from NF-.B and LTR activation. Importantly, the ability of these mutants to activate APC was attenuated or abrogated. These data show that Tax-induced rapid senescence is causally associated with APC activation and suggest that the mitotic abnormalities that are associated with premature APC activation might play an important role in cell transformation by Tax.
 
In a parallel study, we demonstrate that Tax interacts with both centrosomal Nek2associated protein 1 (C-Nap1), a large coiled-coil protein that maintains centrosome cohesion, and protein phosphatase 1 (PP1) and that these interactions localize to the centrosome. Furthermore, in cells expressing tax, C-Nap1 phosphorylation is greatly diminished. C-Nap1 phosphorylation status plays an important role in centrosome cycle progression; PP1-mediated dephosphorylation of C-Nap1 maintains centriolar cohesion during interphase, and never in mitosis A (NIMA)-related kinase 2 (Nek2A)-mediated phosphorylation of C-Nap1 during G2/M triggers C-Nap1 dissociation from duplicated centrosomes permitting normal centrosome disjunction. Our data suggest that Tax, through recruitment of PP1 to C-Nap1, may permit prolonged C-Nap1 dephosphorylation thereby disrupting normal centrosome disjunction leading to observed Tax-induced centrosome abnormalities and distinct microtubule organizing center (MTOC) loss.
 
Our studies have revealed two distinct pathways through which Tax could contribute to oncogenesis. First, our Tax mutants that retained LTR and NF-.B activation properties but were impaired in APC activation were also defective in the ability to transform Rat1 fibroblasts. Therefore, premature APC activation by Tax appears to correlate with the ability of Tax to induce cellular transformation. Second, our demonstration of Tax-mediated alterations in C-Nap1 phosphorylation cycle offers an explanation for the centrosome abnormalities and mitotic apparatus defects observed in response to Tax expression. Chromosomal abnormalities resulting from such mitotic apparatus defects may be expected to contribute to the oncogenic potential of Tax.

LTC Joseph C. O'Sullivan
Anatomy, Physiology and Genetics
 
Doctor of Philosophy
2006
 
Major Advisor: Joseph T. McCabe, Ph.D., Professor and Vice-Chair of the Department of Anatomy, Physiology and Genetics; and Professor of Neuroscience and Molecular and Cell Biology
 
Thesis Title: The Effect of Diazoxide Upon Heat Shock Protein Expression and Physiological Response to Hemorrhagic Shock and Cerebral Stroke
 
ABSTRACT
 
Our team tested the hypothesis that pharmacological induction of ischemic preconditioning (IPC) can offer cytoprotection and preserve vital tissues after cerebral stroke with hemorrhagic shock. The compound, diazoxide (DZ), is known to mimic IPC through its effects as a mitochondrial KATP channel opener and succinate dehydrogenase inhibitor. The effect of DZ was examined under two IPC protocols: delayed preconditioning and postconditioning in an anesthetized rat model of hemorrhagic shock, combined in some experimental groups with cerebral vascular occlusion (stroke).
 
When DZ was administered 24 hours prior to shock (delayed preconditioning), it significantly reduced hyperglycemia, which in vehicle-treated animals persisted after resuscitation. DZ also attenuated hyperlactatemia during the 1 hour shock period. With more severe trauma from combined stroke and shock, DZ also decreased hyperglycemia, but the apparent reduction of hyperlactatemia did not reach statistical significance. DZ also appeared to increase the survival rate of animals that sustained combined stroke and shock, but not significantly (p=.058). The expression levels of heat shock proteins 25 (HSP25) and 70 (HSP70) were used as biomarkers for assessing the response of the kidney, liver, and right cerebral cortex and hippocampus to combined stroke and shock. Compared to vehicle-treated animals, DZ- pretreated rats subjected to stroke and shock exhibited increased HSP25 and HSP70 expression in kidney, liver and brain tissue.
 
When administered in a postconditioning Stroke + Shock model, where DZ was administered at two time points after stroke and shock, DZ again caused a significant increase in HSP25 and HSP70 expression in the ipsilateral cerebral cortex and hippocampus. As a postconditioning trigger, given after stroke and shock, DZ was effective when it was administered 60 minutes after shock immediately prior to reperfusion but not when given 10 minutes after Stroke and Shock (without immediate reperfusion). Taken together, these results suggest DZ attenuates physiological indicators of metabolic stress following shock or combined shock and stroke, that it may increase survivability, and that it enhances the upregulation of cytoprotective heat shock protein expression in key organs.

Jared Renford Patch
Emerging Infectious Diseases Program
 
Doctor of Philosophy
2007
 
Major Advisor: Dr. Christopher C. Broder, Professor and Director, Emerging Infectious Diseases Graduate Program
 
Thesis Title: The Central Role of the Matrix Protein in Nipah Virus Assembly and Morphogenesis
 
ABSTRACT
 
Nipah virus (NiV) is an emerging paramyxovirus distinguished by its ability to cause fatal disease in both animal and human hosts. Together with Hendra virus (HeV), they comprise the genus Henipavirus in the Paramyxoviridae family. NiV and HeV are restricted to Biosafety Level-4 (BSL-4) containment and this has hampered progress towards examining the details of their replication and morphogenesis mechanisms. Here, recombinant gene expression systems to study NiV particle assembly and budding through the formation of virus-like particles (VLPs) have been established to circumvent these obstacles. When expressed by recombinant Modified Vaccinia virus Ankara (rMVA) or by plasmid vector transfection, individual NiV matrix (M), fusion (F) and attachment (G) proteins were released into cell culture supernatants in a membrane-associated state as determined by sucrose density gradient flotation and immunoprecipitation analysis. However, co-expression of F and G along with M revealed a shift in their distribution across the gradient, indicating association with M in VLPs. Protein release was also altered depending on the context of viral proteins being expressed, with F, G and nucleocapsid (N) protein reducing the overall release of M, and N release completely dependent on the co-expression of M. Immunoelectron microscopy and density analysis revealed VLPs that were structurally similar to authentic virus. Differences in the budding dynamics of NiV proteins were noted between rMVA and plasmid-based strategies. Mutational analysis of M revealed a sequence (YPLGVG) that was required for budding. Replacement of the known Ebola VP40 late budding domain (L-domain) with the NiV M sequence resulted in restoration of VP40 budding, and immunofluorescent microscopy revealed cells morphologically similar to those expressing wild-type VP40. Taken together, these results indicate that NiV M, F, and G each possess some ability to bud from expressing cells, and that co-expression of these viral proteins results in a more organized budding process with M playing a central role. Additionally, NiV M contains a sequence that is important for budding and appears to serve as an L-domain. These findings further our understanding of paramyxovirus particle assembly in general and could help facilitate the development of a novel vaccine approach for henipaviruses.

Schilpi Paul
Molecular and Cell Biology Graduate Program
 
Doctor of Philosophy
2007
 
Major Advisor: Teresa M. Dunn, Ph.D., Professor and Interim Chair, Department of Biochemistry and Molecular Biology
 
Thesis Title: Characterization of Enzymes Involved in Fatty Acid Elongation
 
ABSTRACT
 
The very long chain fatty acids, synthesized by a microsomal chain-elongating enzyme system known as the elongase, are essential components of many cellular lipids. Each cycle of elongation involves four successive enzymatic reactions: condensation, reduction, dehydration, and a second reduction, and lengthens the fatty acid by 2 carbon units. Several enzymes that mediate condensation, including the soluble fatty acid synthases and the FAE1-like 3-ketoacyl-CoA synthases (FAE-KCSs) possess a catalytic triad of Cys, His, and His/Asn. In contrast, the Elop proteins, which are implicated in the condensation reaction lack any homology to the well-characterized condensing enzymes. There are three Elop proteins (Elo1p, Elo2p and Elo3p) in yeast and our in vitro assays with microsomes from wild type and the single elo mutants (elo1Ä, elo2Ä and elo3Ä) demonstrate that the Elops are essential for condensation. Heterologous expression of several Arabidopsis FAE-KCSs can substitute for the Elops in yeast. Similar to the Elop proteins, genetic and coimmunoprecipitation experiments provide evidence that the FAEKCSs work in conjunction and are physically associated, with the reductases of the elongase complex. These studies indicate that FAE-KCSs and Elops might have evolved independently, but are still able to utilize similar reductases.
 
Since the elongase proteins localize to the ER, behave as integral membrane proteins, and are predicted to have several membrane spanning domains, an important step toward elucidating the organization of the elongase complex is to determine the membrane topology of the elongase proteins. Several biochemical approaches including protease protection, glycosylation, factor Xa protease cleavage and the split-ubiquitin yeast two hybrid assays were utilized to resolve the topology of the 3-ketoreductase and enoyl-CoA reductase of the elongase complex. A six membrane-spanning topology model with both termini facing the cytosol for both yeast and Arabidopsis Tsc13p, the enoyl-CoA reductases is proposed. By alanine substitution two conserved functionally critical residues of yeast Tsc13p were identified. Based on this model, these residues (K140 and R141) lie towards the cytosolic side of TMD2. These studies also revealed that Ybr159p, the 3-ketoreductase of the elongase system, has an N-terminal membraneassociated domain with the active site lying in the cytosol.

Alisa W. Schaefer
Neuroscience Program
 
Doctor of Philosophy
2007
 
Major Advisor: Sharon Juliano, Ph.D., Professor, Department of Anatomy, Physiology and Genetics
 
Thesis Title: Repair of Neocortex in a Model of Cortical Dysplasia
 
ABSTRACT
 
Our lab developed an animal model to elucidate factors associated with abnormal neocortical development and to attempt repair of cortical dysgenesis. We disrupt corticogenesis using an anti-mitotic methylazoxy methanol (MAM), which inhibits mitosis for several hours. The effects of MAM on neocortical development are assessed during early (embryonic day 24; E24) and late (E33) corticogenesis in ferrets. These animals have protracted cortical development with neurogenesis and migration continuing postnatally. MAM treatment on E24 leads to disorganized cortical laminae, abnormal radial morphology, precocious differentiation of radial glia, and dispersal of Cajal Retzius cells. MAM treatment on E33 leads to less severe effects including diminished layer 4, widespread termination of thalamocortical afferents, and abnormal distribution of GABAA? receptors.
 
Reelin is a protein that plays a role in cortical layering and may also be a key factor in radial alignment. To assess the role of reelin in migration and radial morphology in our model, organotypic cultures were paired with wild type, heterozygous, or reeler mouse cortex. We observed that although reelin is necessary for cortical migration, other factors in neocortex rescue radial morphology and reelin is not required for proper elongation of radial glia.
 
In attempts to repair E33 MAM treated cortex, we transplanted E27, E33 ferret and E14 mouse neural progenitor (NPs) cells into organotypic slices. Using a different paradigm, E27 and E33 fNPs were injected into the brains of E33 MAM treated and normal ferret kits. All donor cell types survive well in culture and differentiate into multiple cell phenotypes of neural origin. When transplanted into organotypic cultures, all donor cells survive and migrate into the cortical plate, although injections into the ventricular zone were significantly more likely to reach the cortical plate than transplants into the intermediate zone. In vivo transplants of fNPs into ferret kits also migrate into the cortex, differentiate, and become neurons, but not glia. The in vivo studies further revealed that the migration pattern of transplanted cells into MAM cortex varies from their distribution in normal cortex and that donor cells of different ages migrated into distinct layers in normal versus MAM cortex.

Adam Vana
Anatomy, Physiology and Genetics
 
Doctor of Philosophy
2006
 
Major Advisor: Dr. Armstrong
 
Thesis Title: The Oligodendrocyte Progenitor Response to Demyelination
 
ABSTRACT
 
In multiple sclerosis (MS), demyelination results in impaired axon conduction and functional deficits. Remyelination is often observed early in the MS disease course, but over time becomes limited. Factors that may influence remyelination are important, as denuded axons have impaired neurotransmission and increased vulnerability to transection. The general belief is that remyelination requires robust oligodendrocyte progenitor (OP) amplification prior to remyelination. Myelin transcription factor 1 (Myt1) influences OP proliferation, differentiation, and myelin gene transcription in vitro. The potential of Myt1 to influence OP responses leading to remyelination was examined using murine hepatitis virus (MHV) induced demyelination. Myt1 expression was dramatically increased in lesioned white matter. Cells expressing Myt1 proliferated extensively during active demyelination and early remyelination, and Myt1 was observed predominantly in OPs. Increased expression of Myt1 was found within MHV lesions and in MS tissue adjacent to and within lesions. These results suggest a potential role for Myt1 in oligodendrocyte lineage cell regeneration in response to acute demyelination.
 
With MS being a chronic disease we were interested in the responses that occurred following chronic demyelination. Studies using the chronic cuprizone model of demyelination display limited remyelination, a depleted pool of OPs, and decreased oligodendrocytes. We now show that after chronic demyelination apoptosis continues even after cessation of cuprizone to evaluate means to promote remyelination. Overexpression of platelet-derived growth factor-A (PDGF-A) was tested with chronic cuprizone demyelination in hPDGF-A transgenic (tg) mice. Remyelination was improved in hPDGF-A tg mice during recovery after chronic demyelination. OP density and proliferation increased only transiently in hPDGF-A tg mice during acute demyelination but not during chronic demyelination or recovery. Importantly, hPDGF-A tg mice had increased oligodendrocyte regeneration associated with reduced apoptosis during recovery. The effect of increased PDGF-A is likely as a survival factor during the regeneration of oligodendrocytes and remyelination, as preventing apoptosis of oligodendrocytes may be important not only during acute demyelination but also during chronic demyelination. Overall, we found that following demyelination Myt1 may have a potential role in the regeneration of oligodendrocyte lineage cells, whereas the overexpression of PDGF-A appears to enhance survival of newly differentiated myelinating oligodendrocytes.

Ali A. Weinstein
Department of Medical and Clinical Psychology
 
Doctor of Philosophy
2007
 
Major Advisor: Willem J. Kop, Ph.D., Associate Professor, Department of Medical and Clinical Psychology
 
Thesis Title: Biobehavioral Correlates of Depression in Reaction to Mental and Physical Challenge
 
ABSTRACT
 
Depression is the most common mental disorder in the United States. Individuals with depression are at an increased risk of cardiovascular morbidity and mortality. However, little is known regarding the possible mechanisms to explain this relationship. One of the possible pathways includes exaggerated responsiveness to challenge in depressed individuals. The overarching hypothesis of this investigation is that depressed individuals display higher reactivity to mental and physical challenge than non-depressed controls. Specifically, it was examined whether elevated neurohormonal and negative mood responses to challenge tasks would result in elevated cardiovascular and inflammatory responsiveness.
 
Reactivity to mental (mental arithmetic and anger recall) and physical challenge (exercise bout on treadmill) was assessed in 14 depressed and 16 non-depressed control participants. Neurohormonal (adrenocorticotropic hormone, cortisol, norepinephrine, and epinephrine), negative mood, cardiovascular (systolic blood pressure, diastolic blood pressure, and heart rate), and inflammatory (IL-6, TNF-?, and CRP) responses to mental challenge (anger recall and mental arithmetic) and physical challenge (treadmill exercise) tasks were assessed.
 
Results indicated that depressed participants: 1) displayed higher reactivity of neurohormonal and negative mood measures, as well as increased cardiovascular and inflammatory responses during the challenge tasks; 2) neurohormonal and negative mood responsiveness were associated with cardiovascular and inflammatory reactivity; and 3) these relationships displayed variability across measures and challenge tasks. This study demonstrates that hyper-reactivity to challenge tasks can be documented among depressed individuals. Future research is needed to determine the consequences of hyper-reactivity to the development of adverse cardiovascular health outcomes. These findings may also lead to novel interventions aimed at reducing hyper-reactivity to challenge with potential positive effects on quality of life for individuals with depression.

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