Breast cancer is the second leading cause of cancer death in women according to the American Cancer Society. The standard treatment regimen for breast cancer involves ionizing radiation combined with surgery and chemotherapy. Ionizing radiation induces a complex signaling response in cells resulting in either growth arrest, senescence or cell death, and the cell killing after exposure to radiation results largely from DNA double-strand breaks (DSBs). There are two main mechanisms in mammalian cells responsible for repairing the DSBs; the primary mechanism is non-homologous end joining (NHEJ) and the secondary mechanism is homologous recombination (HRR). Previous studies showed that breast tumor cells depend mainly on NHEJ for repairing induced DNA damage. XRCC4 and XLF are two essential proteins in the NHEJ process. The interaction between XRCC4 and XLF (also called Cernunnos) is responsible for stimulating ligase IV for rejoining DNA ends. A single mutation on the XLF-binding interface of XRCC4 at M61, F106, M59 or D58 has been shown to disrupt its interaction with XLF and thus inhibiting NHEJ. Therefore, it is proposed that small natural cyclic peptides that bind to the XLF interface of XRCC4 near M61 and F106 can be identified through an mRNA display in vitro selection, and these peptides will inhibit NHEJ and thereby radiosensitize breast tumor cells. We have synthesized five DNA libraries that produced mRNA-peptide fusions containing a trillion unique peptide sequences that will be used for the selection of peptide inhibitors of the XRCC4/XLF interaction, and we have verified their randomness. Tagged wild-type and mutant versions of the head domain of XRCC4 protein, containing the XLF binding site, were successfully purified, and the wild-type version was applied to initial stages of selection of inhibitory peptides by mRNA display. The percentage of the mRNA-peptide fusions that bound to the XRCC4157 after the first round was 2.1%. The recovery after the second and third rounds was 1.14% and 2%, respectively. Results obtained thus far, although preliminary, suggest that the mRNA display method can be successfully applied to the XLF/XRCC4 interaction.

Trends in childhood obesity have lead to a generation that has a shortened lifespan, where excessive weight may decrease life expectancy by as much as five years. Physical inactivity is a major risk factor for childhood obesity as well as cardiovascular disease, osteoporosis, and cancer. Addressing inactivity in children could help keep the obesity epidemic from continuing to grow as well as help address other health concerns. The specific aims of this study are 1) To examine the effect of parental role-modeling of exercise on children’s activity level by specifically looking at the role of the mother and the father, 2) To determine what variables affect the relationship between parental role-modeling of physical activity and child’s physical activity, and 3) To assess what other variables are associated with children’s physical activity. This cross-sectional study uses data from the National Survey of Children’s Health (NSCH) 2003. The results of this survey are weighted to represent the population of non-institutionalized children ages 0-17 nationally and in each state. This study focused on Virginia as approximately 13.8% of children ages 0-17 in Virginia are overweight and only 23.7% exercise vigorously everyday (26% nationally). The study sample was n=949 children. The sample was predominantly White (76.2%) with approximately equal numbers of males and females. The exposure of interest was parent’s regular exercise (yes/no) and the outcome of interest was child’s regular exercise (0-3 days a week/ 4+ days a week). Potential confounding variables included age, gender, race, BMI, the mother’s and father’s health, and smoking. The crude results of the study show that children with mothers who did not exercise regularly were more likely to exercise than children with mothers who did exercise regularly (OR= 1.19, 95% CI [0.83, 1.72]). Children with fathers who did not exercise regularly were less likely to exercise than children with fathers that did exercise regularly (OR=0.86, 95% CI [0.57, 1.28]). However, after adjustment, children with mothers who did not exercise regularly were less likely to exercise than children with mothers who did exercise regularly (OR=0.99, 95%CI [0.71, 1.32]). The same was true for fathers (OR=0.77, 95%CI [0.49, 1.22]). The results of this study could have possible policy implications. Interventions and programs that are implemented in schools and in communities need to have a family component as this study, as well as others, have shown that parents can influence their child’s exercise behaviors purely by being active themselves. Encouraging and facilitating exercise by parents could be a cost-effective way of implementing obesity prevention efforts with children as well as helping to improve the health of the adult population. Further research needs to be undertaken where all current known risk factors for childhood obesity are included so that the relative importance of these factors can be investigated.

Acanthamoeba culbertsoni is an opportunistic free-living amoeba that is causative of granulomatous amoebic encephalitis (GAE), a chronic and often fatal central nervous system (CNS) disease that is most prevalent in immune compromised individuals. One hallmark of this disease is the formation of granulomas within the CNS, which are commonly absent in immune compromised individuals. Granulomas are usually composed of amoebae, microglia (CNS macrophages), macrophages, T cells, B cells, and neutrophils. Previous studies have demonstrated that microglia respond to Acanthamoeba by producing pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF)-α, interleukin (IL)-1α, and IL-1β. In addition, activated microglia and macrophages have been demonstrated to be cytolytic (i.e., amoebicidal) to Acanthamoeba. Furthermore, previous studies also indicated that Acanthamoeba secrete a myriad of factors including proteases. The role of these proteases during GAE has not been fully elucidated; however, it is thought that these factors may aid in the chronic persistence of Acanthamoeba within the CNS by modulating the host immune response. Using two-dimensional (iso-dalt) gel electrophoresis, we demonstrated that A. culbertsoni secrete factors that degrade culture medium proteins. Initial gelatin zymography studies demonstrated that propagation of A. culbertsoni in medium with high iron content leads to augmentation of protease activity. Gelatin zymography in concert with protease inhibitors demonstrated that A. culbertsoni secrete proteases predominantly of the serine protease class. Using an in vitro co-culture model, we demonstrated that co-culture of A. culbertsoni with mouse microglial-like cells (BV-2 cells) results in the augmentation of A. culbertsoni serine protease activity and stimulation of pro-inflammatory cytokine and chemokine protein expression by microglial-like cells. However, the A. culbertsoni-elicited proteases were shown to degrade microglial-like cell elicited cytokines and chemokines. Collectively, our results suggest that A. culbertsoni- secreted serine proteases may play a role in A. culbertsoni CNS immune evasion by increasing A. culbertsoni CNS dissemination via the diminution of granuloma formation and by dampening microglial-dependent cytokine response.

Current anticoagulants carry a serious risk of bleeding complications. In addition, narrow therapeutic index, drug interactions, immunological reactions, toxicity and high cost to benefit ratio limits the effective use of these drugs in patients with thrombotic conditions.Heparin is the most widely used anticoagulant. We hypothesized that one of the major drawback of heparins, its non-specific interaction with the plasma proteins arises as a result of negative charges. To reduce these non-specific interactions, our laboratory designed sulfated low molecular weight lignin (LMWL) like biomacromolecules, which were found to be direct inhibitors of thrombin and factor Xa, acting through a unique exosite-2 mediated process. To elucidate the structural basis of this mechanism, we studied unsulfated and size fractionated LMWLs. Detailed enzyme inhibition studies with sulfated and unsulfated LMWLs of ferulic and caffeic acid oligomers revealed that sulfation was not absolutely critical for dual inhibition property and smaller oligomers can yield a potent anticoagulant. Mechanistically, unsulfated LMWLs retained exosite-2 mediated inhibition mechanism. A major advantage expected of the unsulfated LMWLs is the possibility that orally bioavailable anticoagulants may become possible.To identify target specific structures within the heterogeneous population of sulfated LMWLs, we prepared sulfated β-O-4-linked oligomer using chemical synthesis. Enzyme inhibition studies revealed that the sulfated β-O-4 LMWL were highly selective direct inhibitors of thrombin. These results show for the first time that specific structural features on LMWL scaffold dictate inhibition specificity. Studies in plasma and blood display highly promising anticoagulant profile for further studies in animals. To further study the LMWL scaffold as macromolecular mimetic of heparin; we investigated their effect in preventing cellular infection by herpes simplex virus-1 (HSV-1). Based on previous findings on sulfated lignins a size-dependent study on unsulfated LMWLs was done. The unsulfated lignins were found to not only inhibit HSV-1 entry into mammalian cells, but were more potent than sulfated lignins. Interestingly, shorter chains were found to be as active as the longer ones, suggesting that structural features, in addition to carboxylate groups, may be important. It can be expected that unsulfated lignins also antagonize the entry of other enveloped viruses, like HIV-1 and HCV that utilize heparan sulfate to gain entry into cells. The results further present major opportunities for developing lignin-based antiviral formulations for topical use.

CB1 cannabinoid receptors are G-protein-coupled receptors that mediate the central nervous system (CNS) effects of marijuana and endocannabinoids. Recently, cannabinoid receptor interacting protein 1a (CRIP1a) was discovered as a novel protein that binds to the CB1 receptor C-terminus and inhibits CB1 receptor activity without affecting CB1 expression. This thesis investigated the functions of CRIP1a by characterizing the first CRIP1a knockout (KO) mouse line. The absence of CRIP1a was confirmed in KO mice using quantitative PCR and immunoblotting. I hypothesized that CRIP1a KO mice would exhibit enhanced CB1 receptor-mediated G-protein activity in the CNS, as well as cannabimemetic phenotypes and enhanced sensitivity to cannabinoid agonists in vivo. Results showed increased CB1 agonist-stimulated G-protein activity in the amygdala of CRIP1a KO relative to WT mice, but not in cerebellum, hippocampus or spinal cord. CB1 receptor levels did not differ between genotypes in in any region examined. Interestingly, CRIP1a KO mice exhibited an anxiolytic-like phenotype and decreased nociceptive sensitivity in vivo, but did not differ from WT mice in tests of motor activity or coordination. Surprisingly, sensitivity to agonist-induced antinociception, hypothermia, catalepsy or motor incoordination did not differ between genotypes. Our findings suggest that CRIP1a could play a selective role in modulation of anxiety by endocannabinoids, and this action could be mediated through the amygdala. Thus, CRIP1a might serve as a future pharmacological target for studying and treating anxiety disorders.

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.