Francisella tularensis was a facultative intracellular pathogen and a gram-negative coccobacillus which has been categorized by the CDC as a potential class A select agent due to its highly infectious properties and high mortality rates. Francisella tularensis was also responsible for the zoonotic disease tularemia, which was usually transmitted by arthropod vectors or via contact with infected animals. Francisella tularensis subspecies novicida has been used by many researchers in genetic pathogenesis experiments to try to elucidate genes responsible for virulence factors. One of these virulence factors was a capsular material which has been thought to be involved in either increasing pathogenicity or infectivity of this organism upon engulfment by its principal host cell, the macrophage. There were many potential genetic loci which may be involved in this biosynthetic process of encapsulation. One such locus has excellent homology to the capsule biosynthesis operon of Bacillus anthracis, which, under certain conditions, creates a polyglutamic acid capsule (PGA). A transposon mutation in the amide ligase (capB) in LVS has a reduced virulence in murine infection models. I wished to investigate whether Francisella novicida was capable of producing such a capsule and under which environmental conditions this capsule was made. I have created a site-directed mutant of the capB gene in Francisella novicida U112 using targeted mutagenesis via PCR SOEing and have introduced this mutation via electroporation of a suicide vector. I have tested our mutant against preimmune serum treatments and have shown reduced viability as well as a reduced capacity for replication inside RAW 264.7 murine macrophages. I assayed for production of a PGA capsule via immunodot blot and electron microscopy as well as analysis by mass spectrophotometry of capsular extracts. I also tested various media constituents and different environmental conditions to determine which external stimuli may contribute to PGA capsule biosynthesis as well as regulatory changes in transcript levels of this operon.

The propensity for seizures in patients with epilepsy is due to underlying cortical hyperexcitability, the mechanisms of which are poorly understood. Particularly difficult to treat are patients with developmental malformations of cortex. Using the freeze-lesion rat model of one such malformation, polymicrogyria, we identified, in lesioned cortex, alterations in specific interneuron subpopulations that may promote hyperexcitability. Previous studies demonstrate increased excitatory input to the paramicrogyral region. An increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from pyramidal cells has also been shown. We report an increase in sEPSCs recorded from one subtype of interneuron, the low threshold-spiking interneuron (LTS), while sEPSCs in the fast-spiking (FS) interneuron remain unchanged. Distributed equally to pyramidal cells and interneurons, extra excitatory afferents should simply increase overall activity level but maintain the balance of excitation and inhibition. Selective changes in one or more interneuron subpopulations could allow inhibition to appear unchanged, while permitting problematic alterations in inhibitory circuitry. In what appears to be a morphological division of labor, interneurons with intralaminar orientations are typically characterized as FS, while intracolumnar orientations are associated with LTS cells. These cells are clearly distinguished by a combination of visual identification and electrophysiological and intrinsic properties. We report that these characteristics are unchanged in lesioned cortex, indicating that the malformation is not responsible for intrinsic alteration of the cell types. However, some firing properties demonstrate slight differences that may, in cooperation with the altered level of input, amplify the pro-epileptogenic changes in circuitry. Finally, we also report that there is anomalous expression of metabotropic glutamate receptors (mGluR) in malformed cortex. Our data show that the expression of mGluR5, normally causing no functional response in control cortex, contributes to the activation of interneurons in paramicrogyral (PMG) cortex. These findings provide new insight to the mechanisms of cortical hyperexcitability and identify a possible target for future pharmacological intervention.

Purpose: To evaluate the physical activity patterns of diabetic adults by the type of treatment they received Method: The study used secondary data collected by the National Health and Nutrition Examination Survey (NHANES) from 2001 to 2006. NHANES is a continuous study which measures the health and nutritional status of non-institutionalized citizens in the United States. To be eligible for the study, participants had to be an adult 18 years or older who responded during the interview phase of the survey that they had diabetes. Participants with any missing data pertaining to the variables were excluded. After exclusions, the final size of the study population was 957. The type of treatment was defined as: insulin only, oral antidiabetic medication only, or neither. Physical activity was defined according to the guidelines set forth by the American Diabetes Association. A logistic model was used to assess the association between the type of treatment and regular physical activity. All data analyses were performed using SAS 9.1. Results: Overall, only 28.2% of the study participants were involved in regular physical activity. With respect to the type of treatment they received, a majority of the participants (69.9%) took oral antidiabetic medication, while 23.1% used insulin. Only 7.1% didn’t take antidiabetic medication or insulin. Most of the study participants were either overweight or obese (86.1%). In relation to diabetes treatment type, the frequency of taking oral antidiabetic medication among those who were diagnosed with diabetes when they were 40 years of age or older was greater (76.9%) than the frequency of insulin use (64.9%) . Among this segment of the population, 81.6% didn’t use insulin or oral antidiabetic medication. Study participants who had diabetes for five years or less were more likely to take oral antidiabetic medication only, with 47.7% taking oral antidiabetic medication compared to 33.2% taking insulin. The crude odds ratio for insulin treatment and physical activity was 0.72 (CI, 0.32-1.61) while the crude odds ratio for treatment consisting of oral antidiabetic medication and physical activity was 0.61 (CI, 0.31-1.21). After adjusting for confounding, the odds of being physically active for patients on insulin treatment was 0.62 (CI, 0.28-1.39), and for those on oral antidiabetic medication the odds of being physically active was 0.53 (CI, 0.27-1.08), indicating that there was no statistical significance between either treatment group and physical activity participation. Conclusions: Although not statistically significant, the prevalence of regular physical activity was highest (37.3%) among diabetic individuals who used neither insulin nor oral antidiabetic medication to control their diabetes, while 26.7% of participants who used oral antidiabetic drugs and 30% of participants who used insulin exercised regularly. However, the failure to participate in physical activity is a common problem among all diabetics, irrespective of group distinctions. Thus, all diabetics should be encouraged to participate in physical activity to reduce future complications.

CD23 has long been appreciated to be a natural, negative regulator of IgE synthesis. This understanding is due in part to animal models in which CD23 deficient or CD23 transgenic animals display exacerbated or reduced IgE levels respectively. Interestingly, CD23 is susceptible to proteolytic cleavage from the cell surface. When this occurs, CD23 loses its regulatory capability and the solubilized form can lead to pro-inflammatory events through its cytokinergic activity on macrophages. Thus, targeting this specific cleavage would be beneficial to the control of allergic disease by stabilizing CD23 at the cell surface. Inhibitor studies performed by our group as well as others indicate that the enzyme responsible for CD23 ectodomain shedding is a hydroxamate-sensitive metalloproteinase. Through collaboration with the Blobel group, we analyzed various ADAM KO mouse embryonic fibroblasts (MEFs) and found no involvement of ADAMs 8,9,12,15,17,19, and 33 in CD23 shedding, however we did find a role for ADAM10. Using ADAM10 KO MEFs and ADAM10 specific inhibitors, we discovered that ADAM10 is indeed the CD23 cleaving enzyme or “sheddase”. Thus, developing strategies that would target ADAM10 could have an effect on sCD23 release and IgE production. In the CNS, signaling through the kainate receptor (KAR) by glutamate causes an increase in ADAM10 expression. Human B cells were found to express a GluK2 containing kainate receptor and its activation increased ADAM10 expression which is in agreement with KAR activation in the CNS. Although glutamate is considered a neurotransmitter, it signals in the periphery and elevated levels are associated with certain immune disorders. A significant corresponding increase in sCD23 release is observed as well. Remarkably, this activation induced a strong increase in B cell proliferation, IgG, and IgE production and these events can be reversed through the use of NS102, a specific KAR antagonist. Thus, we report for the first time the unique presence on B cells of a neurotransmitter receptor and that activation of this receptor could serve as a novel mechanism for enhancing B cell activation and Ig production. This enhancement and control thereof has implications for allergic and autoimmune diseases. Lastly, the CD23-ADAM10 axis was examined in a non-allergic disease state, B cell chronic lymphocytic leukemia (BCLL). BCLL is characterized by a large accumulation of CD23+ cells and high levels of soluble CD23 in the sera. After further analysis, we show that ADAM10 is indeed over-expressed in BCLL and could account for the high levels seen in this patient population. Furthermore, specifically targeting ADAM10 resulted in reduced soluble CD23 release, reduced proliferation, and enhanced apoptosis induction. Taken together the novel finding that ADAM10 is involved in CD23 shedding allows for targeted therapeutic intervention of both atopic and non-atopic disease states.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by beta-amyloid (Aβ) aggregation/oligomerization, biometal dyshomeostasis, oxidative stress, and neuroinflammation. The multifactorial nature of AD may indicate the therapeutic potential of multifunctional ligands that tackle various risk factors simultaneously as effective AD-modifying agents. This notion is further supported by the fact that while numerous AD-modifying agents targeting one single risk factor have been developed and a number of them entered clinical trials, none of them has been successfully approved by the FDA. Furthermore, neuronal cell membrane/lipid rafts (CM/LR) have been demonstrated to associate with all the indicated risk factors, indicating that this relationship can be exploited therapeutically to design strategically distinct multifunctional ligands by incorporating CM/LR anchorage into molecular design. With the long-term goal of developing multifunctional ligands to slow or stop the progression of AD, recently we have embarked on the development of bivalent multifunctional Aβ oligomerization inhibitors (BMOIs) as potential AD-modifying agents. These BMAOIs contain curcumin as the multifunctional moiety and cholesterol as the CM/LR anchorage moiety linked by a spacer to co-target AβOs, CM/LR, and oxidative stress. The hypothesis of the BMAOI strategy is that BMAOIs will anchor/target the multifunctional AβO inhibitor moiety inside, or in the vicinity of, CM/LR in which Aβ oligomerization, Aβ/biometal interaction and oxidative stress occur to efficiently interfere with these processes. In support of this hypothesis, proof-of-concept of the BMAOIs strategy has been reached through our preliminary studies. Our results demonstrated that: 1) BMAOIs containing curcumin as the multifunctional AβO inhibitor and cholesterol as CM/LR anchor primarily localize to CM/LR while curcumin does not; 2) BMAOIs with optimal spacer length efficiently inhibit the production of intracellular AβOs and protect MC65 cells from AβO-induced cell death (EC50~3 µM) while curcumin exhibits no significant activity; 3) these active BMAOIs retain curcumin’s antioxidant and metal complexation properties. Our preliminary studies also demonstrated the critical roles of spacer length and connectivity in the molecular design of BMAOIs and one lead compound was identified for further structural modification and optimization. Furthermore, this lead compound was shown to cross the blood-brain barrier (BBB) in a preliminary in vivo study as well as bind to Aβ plaques. Taken together, these results clearly reach the proof-of-concept of BMAOIs and confirm the rationale of designing BMAOIs to develop potential AD-modifying agents. In this thesis, we continued the exploration and validation of the BMAOI strategy by designing and biological characterizing a series of BMAOIs containing cholesterylamine as the CM/LR anchorage moiety and curcumin as the multifunctional moiety. Ten BMAOIs with the spacer length of 15, 17, 19, 21, and 23 atoms were designed and synthesized. Initially, these BMAOIs were tested for the neuroprotective activity against the AβO-induced cytotoxicity in human neuroblastoma MC65 cells. Then, Western blot analysis was performed for active BMAOIs to confirm the association of neuroprotection and suppression of AβOs. Furthermore, active BMAOIs were examined for antioxidant and metal complexation properties. Finally, Aβ plaque binding was examined using transgenic AD mice brain sections. Our results demonstrated that the same spacer length but different connectivity are preferred in this new series of BMAOIs for neuroprotective activity as that of the lead compound from cholesterol series. Moreover, the neuroprotection activity is closely associated with the inhibition of AβOs as demonstrated by Western blot analysis. In addition, the active BMAOIs retain the antioxidant and biometal binding properties of curcumin. More importantly, the binding affinity to the Aβ plaques was again confirmed for the new BMAOIs containing cholesterylamine. In summary, the design and characterization of the new series BMAOIs further confirmed the rationale of BMAOI strategy and their potential to lead to a new direction in development of effective AD-modifying and treatment agents.

The main psychoactive and therapeutic effects of Δ9-tetrahydrocannabinol (THC) are mediated through cannabinoid type 1 receptors (CB1Rs). The therapeutic uses of THC are mitigated by the development of tolerance to these therapeutic effects, whereas tolerance does not readily develop to some of the side-effects of THC, like motor impairment and reward. The development of tolerance occurs through adaptations at CB1Rs, which include desensitization (G-protein uncoupling) and downregulation (receptor degradation). Brain region-dependent differences in THC-mediated adaptations are proposed to explain the differences in tolerance to various THC-mediated effects. These studies focused on whether ΔFosB, a stable transcription factor, could regulate CB1R adaptations since regions resistant to CB1R adaptations, like the basal ganglia, exhibit THC-mediated ΔFosB induction. The studies in this dissertation tested the hypothesis that THC-mediated induction of ΔFosB is regulated through interactions between cannabinoid and dopamine systems and that brain region-dependent differences in ΔFosB transcriptional regulation could explain some aspects of long-term CB1R signaling and CB1R adaptations. Results determined that THC induced ΔFosB primarily in forebrain areas, like striatum, that are innervated by midbrain dopamine neurons. An inverse, brain region-dependent correlation was found between CB1R desensitization and ΔFosB induction. Studies utilizing bitransgenic mice with overexpression of ΔFosB, or its dominant negative ∆cJun, determined that ΔFosB regulates CB1R signaling and reduces CB1R desensitization. Based on this regional profile, studies determined the role of dopamine signaling in THC-mediated ∆FosB induction. Results showed that THC-mediated induction of ΔFosB required dopamine type 1 receptors, but not the dopamine-and cAMP-dependent phosphoprotein of Mr 32kDA. Finally, the functional consequences of THC-mediated ΔFosB induction were assessed by measuring expression of known targets of ΔFosB following both acute and repeated THC administration. Results found that, in prefrontal cortex, known targets of ΔFosB exhibited functionally different signaling expression patterns when comparing acute THC with THC-challenge in THC-experienced mice, which enhanced ΔFosB induction. These studies establish a role for ΔFosB in regulating long-term CB1R signaling/adaptation following repeated THC administration and could have implications for changes in the effects of THC during repeated administration, including the development of differential tolerance to motor-impairing and rewarding effects of THC versus other pharmacological effects.

Introduction: Among women, breast cancer is the most prevalent cancer and the second leading cause of cancer death. Although technology advances have improved survival rates for breast cancer overall, improvements have not been universally experienced by all socioeconomic and racial groups. Known determinants of breast cancer care disparities include socioeconomic status, race, age, and social support. As a part of the Breast and Cervical Cancer Mortality Prevention Act of 1990 and with the help of CDC funding, the Virginia Breast and Cervical Cancer Early Detection Program (BCCEDP) or Every Woman’s Life (EWL) was created. EWL provides breast cancer screening to female VA residents between the ages of 18 and 64 who lack health insurance and fall at or below 200% of the Federal Poverty Level. Objective: The purpose of this study is to determine if delays in the diagnosis and treatment of breast cancer, within the VDH program EWL, differs based on sociodemographic characteristics and/ or regional location. Methods: From its inception to July 2008, 705 women received a breast cancer diagnosis through the EWL program. For these 705 cases prevalence and crude odds ratios were calculated for both diagnosis and treatment delays for all of the demographic variables along with 95% confidence intervals. Adjusted odds ratios were calculated for sociodemographic variables against screening to diagnosis delays and diagnosis to treatment disparities along with 95% confidence intervals. Results: According to the crude odds ratios more women who fall into the other category of race experienced diagnosis delays (OR=2.28 [1.11, 4.67]), but they were more likely to receive treatment in a timely manner (OR=0.29 [0.11, 0.79]). Women living alone were also more likely to experience diagnosis delays (OR=1.49 [1.10, 3.02]). Hispanic women were more likely to receive treatment in a more timely manner than non-Hispanic women (OR=0.21 [0.05, 0.81]). Also, women being treated in any other region than northern VA were more likely to experience treatment delays. However, according to the adjusted odds ratios, the only significant timing delay was the one experienced more often by women in the other race category. Conclusion: The research indicates known indicators of disparities within cancer care as socioeconomic status, race, ethnicity, age, and social support. The findings of this study indicate that the only significant indicator of disparity within the Every Women’s Life program is race. Although, African-American women were just as likely to receive timely diagnosis and treatment as white women in the program, it was the combined groups of Asian, American Indian, and other women that were more likely to experience diagnosis, but not treatment, delays. The fact that no other significant indicators of disparities were found within EWL indicates a success of the program, as EWL is targeting those women that would have otherwise been missed by the system.

Objectives: This study estimates the proportion of intravenous drug use (IDU) partners and HIV positive sexual partners among black and white and examine the association between repeat HIV testing and sex with high risk partners. Methods: A cross-sectional study of MSM was conducted by using pooled data from counseling, Testing, Referral services in Virginia, USA 2002-2007. Results: We obtained a sample of 19679 MSM out of which 10924(56%) and 6739(34%) were white and black MSM respectively. The proportion of IDU partners and HIV positive partners were among white MSM (5% and 11%) and black MSM (3% and 9%) respectively. Age rather than race was significantly associated with having more IDU and HIV positive partners. MSM in 30 - 39 age groups were thrice likely to have sex with HIV partners compared to young MSM. 79% and 74% of the white MSM and black MSM had undergone previous HIV testing. Previously tested MSM were twice likely to report to have sex with HIV positive partners compared to first time testers. Discussion: These findings suggest that fewer black MSM compared with white MSM report having HIV positive sex partners. HIV test repeaters continue to engage in sex with high risk individuals. Further study is needed to compare high risk behaviors between HIV positive repeaters and HIV negative repeaters. Identifying the epidemiological dynamics driving HIV infection among black MSM that go beyond individual level risk behaviors may be warranted.

The purpose of this study was to compare shear bond strengths of brackets bonded to stainless steel crowns using various orthodontic adhesives and surface conditioning techniques. One hundred and twenty mandibular first molar stainless steel crowns were randomly divided into groups: (1): Aluminablasting + Metal Primer + Assure; (2): Aluminablasting + Silane Coupling Agent + Transbond; (3): Diamond Bur Abrasion + Metal Primer + Assure; (4): Diamond Bur Abrasion + Silane Coupling Agent + Transbond; (5: control): Acid Etching + Metal Primer + Assure; (6: control): Acid Etching + Silane Coupling Agent + Transbond. Bond strength was tested using a universal testing machine. Both aluminablasting and diamond bur abrasion surface preparation techniques, when used in conjunction with metal primer and Assure bonding resin, reached clinically acceptable bond strength values (9.05 and 9.30 MPa, respectively). These techniques seem to offer viable options to bond orthodontic brackets to stainless steel crowns.

The oxygen uptake (VO2) kinetic response to exercise provides insight into aerobic performance and the efficiency of the body to maintain oxygen demand during the initiation of exercise. Previous research in normal weight children reports insignificant differences in gender VO2 on-kinetic responses to moderate exercise. No study has evaluated the impact obesity may have on gender VO2 on-kinetics. PURPOSE: To determine if sex differences exist in the VO2 kinetic response to moderate exercise in obese adolescents. METHODS: Male (n=16) and female (n=39) adolescents completed a graded exercise test to exhaustion on a treadmill. Data from initial 4-min treadmill walking was used to determine a time constant. RESULTS: The time constant was significantly different (P=0.009) between obese male and female adolescents (15.42±7.31 s vs. 22.03±8.56 s, respectively). CONCLUSION: Sex differences exist in VO2 on-kinetics during moderate exercise in obese adolescents indicating an enhanced potential for males to deliver and/or utilize oxygen.