Objectives The objectives of our study were to: 1) summarize illness characteristics and functional ability difficulties among Children With Special Health Care Needs (CSHCN) in Virginia, 2) examine the distribution of socio-demographic, health insurance, and health-related factors among Virginia’s CSHCN, 3) quantify the influence of these socio-demographic, health insurance, and health-related factors on delayed and forgone care of CSHNC in Virginia, and 4) analyze reasons for delayed and forgone care among Virginia’s CSHCN. Methods Data from the 2005-2006 National Survey of Children with Special Health Care Needs (NS-CSHCN) were used for this study. Three categories of predictor variables were studied: socio-demographic (age, gender, race/ethnicity, household income, parental education, family structure and residence area), health insurance (type of insurance, insurance continuity, insurance adequacy, and whether a child has usual source of health care), and illness variables (severity of condition, impact on functional abilities). Outcome variable was whether respondents reported having delayed or forgone health care for their children. We explored the relationship between predictor variables and outcome variable using bivariate and multivariable logistic regression analyses. All analyses were adjusted for the complex survey design using SAS 9.1. Results Our data included 791 respondents, which represents 289,176 CSHCN from Virginia. Our results show that 6.1% of CSHCN in Virginia experienced delayed or forgone health care in the past 12 months. Bivariate analysis showed that lower income level, lower parental education, single parent household, lack of health insurance coverage, inadequate insurance, lack of continuous insurance coverage, lack of usual source of health care, severity of the condition, and impact on functional abilities were all significantly associated with delayed or forgone care. After adjusting for all potential confounders, multivariable analysis showed that family structure and insurance characteristics were significant predictors of delayed and forgone care. CSHCN who did not live in two-parent household (OR= 2.7; 1.05, 7.31), were ever uninsured during the past 12 months (OR = 17; 3.85, 75.58), had inadequate insurance (OR = 5.8; 2.06, 16.37), and who did not have a usual source of health care (OR = 22.6; 2.83, 180.55) had increased odds of delayed/forgone health care. Conclusion Lapse in health insurance coverage, lack of usual source of health care, and insurance that is inadequate, all which are modifiable, are strong predictors of delaying or forgoing health care among CSHCN. It is important to identify families that are experiencing these barriers and to place special emphasis on children who do not stem from two-parent households. Policies and programs that address health insurance coverage and continuity, that increase the number of children with medical homes and usual sources of health care, and that address the needs of families that are particularly vulnerable should be implemented to guarantee CSHCN receive timely and needed health care.

Biophysical entities are complex systems systems with strong environmental coupling, dominated by fluctuations on a hierarchy of timescales. These properties confound simulation of ligand binding and catalysis, inflating the scale of the problem to one tractable only with a considerable outlay of resources. In an attempt to ameliorate this restriction, several techniques are developed to accelerate biomolecular simulations while collaterally lending physical insight. The first segment of this dissertation is concerned with directed simulations of ligand binding in a model system. Using the serum retinol binding protein as a prototype, the potential of mean force associated with ligand binding is calculated and dissected. Desolvation is sufficient to drive formation of an intermediate binding state; however, a combination of electrostatic and van der Waals interactions pull the intermediate into a stable configuration. Association is accompanied by a change in the conformational flexibility of the portal domains of sRBP and subsequent "stiffening" of the holo sRBP, reflecting an "order-disorder" transition in the protein. The third and fourth chapters of this dissertation entail ab initio molecular dynamics (AIMD) and quantum Monte Carlo methods (QMC) for computational enzymology. An ideal system for the application of AIMD, are the cytochromes P450 (CYP450s). Most AIMD calculations are performed using plane-wave (PW) density functional theory as an electronic structure method; conversely, computational enzymology is generally performed using calculations with Gaussian basis sets. In this scenario, no benchmark exists to comparison of PW calculations with experimental data. To clarify this situation, benchmark PW calculations are performed on CYP450 Compound I, the iron-oxo species operant in these enzymes. Finally, lattice QMC methods are developed to characterize tunneling in mean-field backgrounds. Using AIMD simulations, a potential of mean force is constructed in the limit of classical nuclei. A framework for path integral Monte Carlo is introduced in which the Euclidean functional integral is discretized on a lattice, permitting calculations of correlation functions and ultimately the action of the system. As the action is quenched, instanton solutions and their contribution to degeneracy splitting are obtained. This technique is demonstrated for malonaldehyde, a system in which proton tunneling is critical.

Heparin is a natural allosteric modulator, with numerous structural and conformational variations leading to many reports of bleeding complications and variations in anticoagulant effects. A flurry of research has been directed towards understanding this puzzle. This work entails the utilization of three unique strategies to further our understanding of this complex issue. Traditional synthetic, biosynthetic and biophysical approaches have failed to conquer the GAG-protein complexity. Computational analysis however could serve as a powerful approach to decipher this dilemma. A dual filter algorithm was incorporated to identify unique hexasaccharide sequences for HCII and AT. Our experimental studies exhibit a good correlation with our computational findings in addition, to the discovery of the first reported heparin based hexasaccharide sequence (HX1) as a potent activator of HCII and AT. In contrast to the enormity of GAG sequences, there appears to be a pattern where rare sequences have been identified to modulate characteristic functions in proteins. Our search led us to a biosynthetically rare GAG residue 2-O-sulfated glucuronic acid (GlcAp2S). Our computational studies indicated elements of selective recognition with coagulation enzymes propelling us towards synthesizing a polymer, HS2S2S enriched in GlcAp2S and GlcNp2S saccharides. Our biological studies indicate its potential in activating AT and HCII in addition to a previously unobserved inhibition of thrombin but not FXa, which is corroborated by our computational studies. These studies therefore showcase the importance of studying rare sequences to further our understanding of differential recognition of proteins of the coagulation cascade. An alternate anticoagulant strategy involves utilization of upstream enzymes like FXIa. Consequently, we devised a rational strategy, which targets the differential hydrophobic domain near the heparin binding sites of proteins through the design of molecules termed as sulfated allosteric modulators. Our endeavor led to the discovery of a library of quinazolin4-(3H)ones) dimers as selective inhibitors of FXIa. We recognized the linker length and geometry to be an important element affecting potency and selectivity. We therefore synthesized a library of 18 dimers using simple reaction schemes. Our inhibition studies do highlight a 9-fold improvement in potency.

Lyme disease is an emerging infection that is caused by the Borrelia burgdorferi sensu lato complex. These bacteria exist in nature through an enzootic life cycle involving Ixodes ticks and various reservoir hosts. One way that this bacterium adapts to the different hosts in the enzootic cycle is through the expression of outer surface protein C (OspC). OspC is a surface exposed lipoprotein encoded on circular plasmid 26 that forms homodimers on the bacterial surface and has distinct conserved and variable portions of sequence. When ospC is deleted, the spirochetes are unable to cause mammalian infection although the mechanism of this is unknown. Additionally, OspC is thought to be involved in reservoir host specificity/association and in tissue dissemination. In order to better understand the functional domains of OspC, the different conserved and variable portions of this protein were investigated. Three conserved portions of OspC were investigated: (1) the conserved cysteine residue at position 130 (C130), (2) the last ten C-terminal amino acids (C10), and (3) ligand binding domain 1 (LBD1). The C130 residue was mutated and this substitution disrupted OspC oligomerization in vitro and in vivo. A B. burgdorferi strain lacking the C10 retained full infectivity and plasminogen binding. The mutation of a single residue within LBD1 rendered B. burgdorferi noninfectious, indicating the importance of this domain in infection establishment. The variable portion of OspC was investigated by: (1) altering the surface charge of ligand binding domain 2 (LBD2), (2) inserting different OspC types into a constant genetic background, and (3) creating OspC hybrids. Alteration of the surface charge of LBD2 by site directed mutagenesis resulted in a lack of persistence in mice. By inserting an OspC type known to be noninfectious in mice into an infectious strain, infectivity was abolished. Strains expressing OspC hybrids indicated that multiple domains of OspC are involved in species specificity. Together these analyses demonstrated that OspC is as important protein that plays multiple roles in pathogenesis. The work presented here helps to increase the understanding of this crucial protein and the strains described can be used to decipher the full function of OspC.

Cytosine methylation of mitochondrial DNA (mtDNA) was first described several decades ago, but neither the mechanism generating this modification nor its functional significance was known. Because mitochondrial dysfunction is a hallmark characteristic of numerous human diseases, including neurological and cardiovascular disease, aging and cancer, this dissertation addressed whether epigenetic modification of mtDNA regulates mitochondrial function. We show that mtDNA contains not only 5-methylcytosine (5mC), but also 5-hydroxymethylcytosine (5hmC), suggesting that previous reports likely underestimated the degree of epigenetic modification within the mitochondrial genome. We questioned how these modifications were generated by looking for mitochondrial isoforms of the nuclear-encoded DNA methyltransferases. We found that an isoform of the most abundant mammalian methyltransferase, DNA methyltransferase 1 (DNMT1) translocates to mitochondria, driven by an in-frame mitochondrial targeting sequence (MTS) located upstream of the nuclear DNMT1 translational start site. This MTS is highly conserved across mammalian species, and directs a heterologous protein to the mitochondria. To investigate the function of mitochondrial DNMT1 (mtDNMT1), we created a cell line that carries a tandem-affinity purification (TAP) tag at the C-terminus of a single endogenous human DNMT1 allele. Using the DNMT1-TAP cell line, we showed that mtDNMT1 specifically binds mtDNA in a manner that is proportional to CpG density, proving its presence in the mitochondrial matrix. mtDNMT1 exhibits CpG-specific methyltransferase activity in vitro that is resistant to trypsin-treatment of intact mitochondria, but moderately susceptible to pharmacologic inhibition by the nucleoside analog 5-aza-2’-deoxycytidine (5-aza-dC). NRF1 and PGC1α, transcription factors that activate nuclear-encoded mitochondrial proteins in response to oxidative stress, were observed to up-regulate expression of mtDNMT1. Loss of p53, a tumor suppressor gene known to help control mitochondrial metabolism, also results in a striking increase in mtDNMT1 expression, and this up-regulation of mtDNMT1 appears to modify mitochondrial transcription in a gene-specific fashion. Our data suggests roles for mtDNMT1 in both the establishment and maintenance of cytosine methylation (from which 5hmC is presumably derived) and in the regulation of mitochondrial transcription. We propose that the enzymes responsible for epigenetic modification of mtDNA have potential as therapeutic targets, with relevance to a broad spectrum of human disorders.

Objective: To determine if the prevalence of anti-diabetic and antilipidemic medication use among children treated with atypical antipsychotics is higher than those not treated with antipsychotics. Methods: Virginia Medicaid beneficiaries (2-17 years) continuously enrolled from August 1, 2010 to July 31, 2011 with at least two prescription claims for atypical antipsychotics were the exposed group. All other subjects during the study period were the non-exposed group. Prevalence of anti-diabetic and antilipidemic medication use in both groups were computed and compared using Chi-square test (α=0.05). Results: A total of 299,593 and 4,922 subjects were identified as the non-exposed and exposed groups, respectively. Prevalence of anti-diabetic medication use was 0.32% in the unexposed and 1.40% in the exposed group (p<0.0001). Prevalence of antilipidemic medication use was 0.09% in the unexposed and 0.35% in the exposed group (p<0.0001). Conclusion: Prevalence of anti-diabetic and antilipidemic medication use in the exposed group was significantly higher.

Proper T-cell activation and effector function are essential for effective immunity. T-cell antigen receptor (TCR) signals are known to regulate the outcome of differentiation, but the mechanisms remain unclear. Recent work from our lab demonstrates that the Src family protein tyrosine kinase, p56Lck, is able to specifically link TCR signals to activation of the Mitogen Activated Protein Kinase (MAPK) pathway through the function of its SH3 domain. The MAPK pathway is known to be involved in T-cell activation downstream of TCR ligation and has previously been implicated in T-helper type 2 (Th2) effector function. We have utilized an Lck SH3 mutant knock-in mouse line (Lck W97A) to investigate the potential role of this regulatory signaling mechanism in determining T-lymphocyte activation and effector function. Our results demonstrate that the Lck SH3 domain function is required for normal activation of T-lymphocytes following TCR stimulation as indicated by significantly reduced proliferation, IL-2 production, and CD69 induction in Lck W97A T-cells. Biochemical studies confirm that activation of the MAPK pathway is selectively altered in Lck W97A T-cells as P-ERK1/2 induction is significantly reduced but phospho-PLCg1 induction and calcium mobilization is unaffected. In vivo experiments demonstrate a specific and significantly impaired Th2 immunity in Lck W97A mice, with reduced serum levels of IgG1, IgE and IL-4 following immunization with DNP-KLH, or infection with the helminth Nippostrongylus brasiliensis. Th1 immunity does not appear differentially regulated in Lck W97A mice as serum levels of IgG3 and IgG2b are similar to WT following immunization with DNP-KLH, as well as serum levels of IFN-g1 following immunization with heat-killed Brucella abortus. In vitro differentiation studies confirm that Lck W97A T-lymphocytes are able to be directed to the Th2 phenotype, as indicated by intracellular staining for IL-4, with significantly increased levels of IFN-g under Th2 differentiating conditions compared to WT. These data indicate that the Lck SH3 domain regulates activation of T-lymphocytes by affecting MAPK pathway induction and demonstrate a novel and critical role for Lck in the regulation of Th2-type immunity. The Lck SH3 domain has also been implicated in the pathogenesis of Plasmodium, the causative agent of malaria. The role of the mosquito vector on malaria pathogenesis is not well understood. Initial studies examining the role of vector salivary gland proteins on cells of the innate immune system indicate that Anopheles stephensi saliva is able to enhance macrophage activation and phagocytosis as well as enhance macrophage Ag-presentation to T-lymphocytes in an in vitro model.

Background: Forty percent of all head and neck cancers occur in the oral cavity. According to ICD-O (International classification of diseases for oncology) C00-C14 includes cancers of the lip, oral cavity and pharynx. Studies have indicated that increased population density or Metro living have increased oral cancer incidence. The objectives of this study are to look at the distribution of Oral and Oro pharyngeal Cancers in Virginia from 2001-2005 The study aims to determine if there is an association between metro living (beale code 3) and advanced Oral Cancers. This study is also being done to determine if Metro living is a predictor of Oral Cancer after adjusting for gender. Methods: The data for this study was obtained from the Virginia Cancer Registry. Cancer counts were obtained based on gender, beale code distribution and stage at diagnosis. The counts were collected for the years 2001-2005 based on the ICD-O codes C00-C14. Analysis of this secondary data was done using SAS 9.1. Descriptive statistics presents the distribution of oral cancer according to the stage, gender and urbanity level of the patient. A log-linear model was done to look for association between metro living and Oral Cancers in Virginia after adjusting for gender and stage. This model was fit using a Poisson’s regression to observe if the cancer counts are influenced by the urban beale code 3. Results: During the five year period of 2001- 2005 the Virginia Cancer registry received a total of 3,390 reported cases of oral and pharyngeal cancers. Out of the 3,390 cases 67.35% (2283) of the cases were diagnosed in males and the rest 32.65% were females (1107). Based on the stage at diagnosis, 34.45% (1168) of Oro-pharyngeal cancers were diagnosed to have localized staging as compared to 50.18% (1701) regional and 11.03% (374) distant. 4.34% of the cancers were unstaged (N=147). 82% of all Oro-pharyngeal cancers were seen amongst whites. Majority of oral cancers were seen amongst age groups 35-74 years (78.41%). While looking at the distribution of oral cancers reported from the urban populations; 82.3% (2790) were reported from beale code1. Only 9.73% (330) cases were reported from beale code 2 and 7.96% (270) cases were reported from beale code 3 (population fewer than 250,000 people). More than 50% of cancers were diagnosed at an advanced stage in the urban populations; we did not see a significant relation between advanced oral cancers and metro living. (p =0.2878). After performing a scaled deviance Poisson regression model indicated that there was a stable trend in counts of advanced oral cancer after adjusting for race, age and gender.(θ=-0.04 p-value =0.617). Conclusions: With a linear trend between increased population density and advanced oral cancers, our study observed a stable trend within the metro populations. Due to lack of a clear understanding of all the possible contributing factors further research is recommended to observe the various etiological differences within the urban populations and advanced oral cancers.

Purpose: The purpose of this study was to determine the relationship of the CYP2B6 genotype to the clinical response to meperidine in pediatric dental patients. Methods: Forty-nine patients, ASA I/ II, 41–101 months old, received an oral sedative regimen containing meperidine for dental treatment. The North Carolina Behavior Rating Scale (NCBRS) and Overall Effectiveness of Sedation Scale (OESS) were used to assess their behavior and sedation outcome. Saliva DNA samples were genotyped by PCR-RFLP. Results: We found the following genotype distributions: homozygous wild-type 1*1 (n = 19, 39%), heterozygous 1*6 (n = 25, 51%), and homozygous variant 6*6 (n = 5, 10%). The genotypes showed a significant difference in the North Carolina Behavior Rating Scores and a trend towards significance of the Overall Effectiveness of Sedation Scale during meperidine oral sedations. Conclusion: This research concludes that variations of the CYP2B6 enzyme can be used in the prediction of successful behaviors for oral sedations that include meperidine in the drug regimen. Future research regarding the enzyme kinetics of meperidine is needed to determine the exact enzymatic function of CYP2B6 and its variants.

BACKGROUND: Increasing rates of hospital acquired Clostridium difficile (CDI), as well as mortality owing to CDI, warrants further exploration of risk factors. Clostridium difficile bacteria typically produce toxins which inactivate the Rho proteins found in the colonic epithelium. Statins also disrupt Rho protein cell signaling functions. The objective of this study was to estimate the extent to which use of statins increases the risk of healthcare-acquired CDI. METHODS: Patients over 18 years of age admitted to hospitals contributing data to the University HealthSystem Consortium between 2002 and 2009 were eligible. We identified 25, 111 incident cases of CDI and matched up to 5 controls, matched on hospital, year of admission date, and age +/- 10 years (N=68,259). Patients with ICD-9-CM code of 008.45 who initiated a minimum three day course of either metronidazole or oral vancomycin on/after day 5 of admission were considered incident cases of CDI. RESULTS: Compared to non-users, users of any drug within the statin class as monotherapy were 0.63 times less likely to be classified as a case (95% CI = 0.60-0.66) adjusting for potential confounders. CONCLUSIONS: Our data did not support the Rho protein hypothesis. Rather it was consistent with a growing body of literature demonstrating reduced risk of infections with statin use. Statins’ pleiotropic properties may provide protection against CDI.