Breast cancer (BC) is one of the most common diagnosed malignancies in females. Although 90% of early diagnosed women are expected to survive for at least 5 years, their quality of life is adversely affected by a cluster of symptoms which we collectively named “psychoneurological symptoms’’ (PN). Given that acquired telomere attrition has been speculated to be a causal factor in chronic diseases and the lack in the literature of mechanisms giving rise to PN symptoms, this study was performed to assess telomere length using a chromosome-specific telomere assay before receiving chemotherapy and at the first chemotherapy. We showed significant telomere attrition on the short arm of chromosome 9. In addition, we showed a negative correlation between telomere length and depression. Furthermore, we evaluated several variables as predictors of the change in telomere length and showed that chemotherapy was predictive of shortened telomere length. Taken together, one can speculate that shortened telomeres could result in epigenetic alterations in the genes juxtaposed to the telomeric region, giving rise to the development and persistence of PN symptoms. Knowledge gained from this study will offer hope for the development of therapeutic interventions that could prevent undesirable side effects and ensure a better quality of life for patients with BC.

Naegleria fowleri (N. fowleri) and Naegleria lovaniensis (N. lovaniensis) are closely related amebae found in the environment. N. fowleri causes Primary Amebic Meningoencephalitis (PAM), a fatal disease of the central nervous system, while N. lovaniensis is nonpathogenic. N. fowleri infection occurs when amebae enter the nasal passages, and migrate to the brain. The molecular mechanisms involved in the pathogenesis of PAM are not well-defined. Therefore, the purpose of this study was to define phenotypic characteristics that may be functionally linked to the pathogenicity associated with N. fowleri. Studies revealed that N. fowleri has a faster growth rate and is more resistant to complement-mediated lysis when compared to N. lovaniensis. Additionally, contact-independent cytotoxicity was observed only for N. fowleri. The ability to invade tissues can be a characteristic that distinguishes pathogens from nonpathogens. Therefore, adhesion to extracellular matrix components (ECM), laminin-1, fibronectin, and collagen I, was assessed. N. fowleri exhibited a higher level of adhesion to ECM components and was shown to invade tri-dimensional ECM scaffolds (matrigel and collagen I) to a greater extent than N. lovaniensis. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Attachment of N. fowleri to ECM components was decreased significantly when amebae were pretreated with trypsin, suggesting a role for a surface protein in this process. Pretreatment of N. fowleri amebae with periodate, a sugar oxidant, led to a decrease in attachment to laminin-1 and fibronectin suggesting that the surface component contained a sugar moiety. Western immunoblotting revealed two integrin-like proteins for both species. However, one with a molecular mass of approximately 70 kDa, was detected at a higher level for N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. An anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Zymographic analysis demonstrated differential expression of proteases occurs when N. fowleri and N. lovaniensis invade ECM components using an in vitro invasion assay. These results indicate a distinction in adhesion to, and invasion of, extracellular matrix proteins between N. fowleri and N. lovaniensis.

The chemokine receptor CCR5 has been implicated in the pathogenesis of cancers and AIDS. A series of novel piperidine derivatives were designed, synthesized, and evaluated as CCR5 antagonists. The ability of the new ligands to inhibit the increment of intracellular calcium level stimulated by endogenous ligand CCL5 was measured in the calcium mobilization assay as an indication of its CCR5 receptor antagonism. The anti-proliferation assay was performed to measure the ability of these new compounds to inhibit the proliferation of prostate cancer cell lines, PC-3 and M12. A new lead compound has been identified which showed micromolar level of inhibition to PC-3 cell line proliferation as well as calcium mobilization. These studies are the beginning of a thorough analysis of the CCR5 receptor antagonist binding pocket in the CCR5 receptor. Further examination may help identify next generation lead to develop highly selective CCR5 receptor antagonists and anti prostate cancer agents.

Background: Women account for about a third of all new cases of HIV in India. Based on research examining trends of HIV infection in women, male perception and behaviors have emerged as strong potential risk factors. However, there has been limited research examining the relationship between male’s attitudes toward women and their sexual risk-taking behaviors. This study examined the degree to which men’s beliefs about power dynamics in heterosexual relationships are related to risky sexual behaviors in Indian men. Methods: Data on Indian men from the 2005-2006 National Family Health Survey (NFHS-3) was analyzed (N= 44,727). The outcome variable, risky sexual behavior, was created using a composite variables characterized by multiple sexual partners, pay for sex, sexual relationships with individuals other than wife or girlfriend, or a history of sexually transmitted diseases in the past 12 months. Men’s beliefs about power dynamics in heterosexual relationships was examined using the following predictor variables: a) women’s role in decision-making, b) acceptability of domestic violence, and c) acceptability of refusing sex. Age, marital status, standard of living, education, religion, region, knowledge of HIV, alcohol use, and family history of domestic violence were examined as covariates. Multiple logistic regression was used to examine the association. Results: Men who believed that women should rarely OR=1.73 [CI 1.36, 2.20] or sometimes be involved with decision-making OR=1.33 [CI 1.13, 1.56] were more likely to report risky sexual behavior as compared to those who believed that women should be involved most of the time. Additionally, men who had favorable attitude towards perpetrating domestic violence were 56% more likely to report risky sexual behavior OR=1.56 [CI 1.37, 1.79]. Similarly, men who believed that it was never acceptable for a woman to refuse sex were 43% less likely to engage in risky sex OR=1.43 [CI 1.27, 1.69]. Conclusion: Men who expressed a preference for male-dominant decision making, acceptance of violence towards women, or the belief that women should not have autonomy in sexual matters are more likely to engage in behaviors that put themselves and their female partners at risk for HIV infection. Comprehensive HIV prevention programs should address power dynamics as a component of HIV risk.

Little is known about the health of the indigenous peoples in Virginia. When compared to the total U.S. population, indigenous peoples nationwide disproportionately experience disparities in health status across multiple health indicators. Research shows that these disparities are largely due to the inequitable distribution of social and economic determinants present within indigenous communities. Because the indigenous peoples of Virginia are affected by inequitable social and economic conditions, there is reason to believe that health indicators may mirror that of the indigenous population at-large. Outlined in this paper is a framework for exploring the needs, strengths, and priorities of indigenous communities in Virginia. This paper proposes that the use of a culturally relevant methodology, such as Talking Circle, combined with an emergent and community-based participatory approach, will lead to a more authentic identification of the strengths, needs, and priorities of indigenous communities than traditional public health surveillance methodology, as well as build community capacity for on-going engagement.

Anaplasma phagocytophilum is an obligatory intracellular bacterium that infects neutrophils to cause human granulocytic anaplasmosis. Sialyl Lewis x (sLex)-modified P-selectin glycoprotein ligand-1 (PSGL-1) is the confirmed receptor utilized by A. phagocytophilum to bind and invade human neutrophils and myeloid cell lines. As an obligate intracellular pathogen, the binding of A. phagocytophilum to a host cell receptor is a prerequisite step for entry and replication, and thus its survival. However, the bacterial adhesins mediating this process have yet to be identified. In this study, we sought to identify surface proteins of A. phagocytophilum as putative adhesins. A. phagocytophilum undergoes a biphasic developmental cycle, transitioning between a smaller electron dense-cored cell (DC), which has a dense nucleoid, and a larger, pleomorphic electron lucent reticulate cell (RC), which has a dispersed nucleoid. We determined that the respective roles of the A. phagocytophilum DCs and RCs are adherence/infection and vacuolar replication, respectively, which is a finding that is consistent with the life cycles of other obligate intravacuolar pathogens that undergo biphasic development. Most importantly, we demonstrated the A. phagocytophilum DC is responsible for recognizing human PSGL-1. To identify surface proteins as putative adhesins we tested a variety of approaches. Three different computer prediction programs were compared, resulting in identification of 16 to 130 potential membrane proteins. As a more direct means to identify A. phagocytophilum surface proteins as PSGL-1 adhesins, several affinity capture approaches were tested. We used commercially available recombinant human PSGL-1 (rhPSGL-1) to try and capture adhesins by crosslinking and affinity purification. We were unsuccessful, but nevertheless gained insight into the binding properties of A. phagocytophilum. We next chose to take a broader approach to identify outer membrane proteins of the adherent DC by biotinylation. In the process we developed new density-gradient centrifugation approaches which successfully purified an RC-enriched population as well as a mixed population of RC and DC organisms. Results from this work demonstrate that A. phagocytophilum DC organisms are responsible for binding PSGL-1. Additionally, the results obtained thus far of gradient-purified bacteria will serve as a foundation for future experiments in identifying surface and developmental form specific proteins.

As Pseudomonas aeruginosa adapts to the Cystic Fibrosis (CF) and chronic obstructive pulmonary disease (COPD) lung environments, mucoid strains often appear as a result of alginate overproduction. Such mucoid conversion is associated with the establishment of a chronic pulmonary infection. Alginate confers resistance to phagocytosis and has other pathogenic properties. The regulation of alginate production is complex and involves an alternate sigma factor, anti-sigmas and several DNA-binding transcriptional regulators. Here we examined the possibility that the catabolic repression control (Crc) protein repressor may affect alginate gene expression. A putative Crc binding site was observed adjacent to the ribosome binding site of algD, the first gene in the 12-gene alginate biosynthetic operon. We hypothesized that Crc binding here would act as a repressor of algD expression. Taking a genetic approach, Gateway technology was used to construct crc::GmR (nonpolar) mutants of P. aeruginosa strain PAO1 and its mucoid (mucA) mutant derivative, PDO300. The crc mutation had the expected phenotypes with respect to pyocyanin production, biofilm formation and diauxic growth. When a PalgD-lacZ (translational) fusion was tested, the crc mutant showed increased algD expression as predicted for a mRNA-binding repressor. Another Ptrc-algD-lacZ (translational) construct, but missing the upstream promoter (PalgD) elements, also showed increased activity in crc mutants as predicted if Crc was acting directly as a repressor of algD transcriptional / translational expression. However, this was not consistent with the production of alginate. The crc mutant of mucoid PDO300 showed lower levels of alginate production than its parent strain. Under conditions were the algD operon was induced by ammonium metavanadate in the growth medium to produce alginate, the crc mutation again resulted in a lower level of alginate production than wild-type, which was again inconsistent with the algD-lacZ expression data. This suggests that crc mutation, which has global effects on carbon flow in the cell, could be affecting metabolic pathways that feed precursors into the alginate biosynthetic pathway. Future directions for this research are discussed.

Treacher Collins Syndrome (TCS) is a rare autosomal dominant disorder characterized by severe craniofacial defects. The syndrome is associated with mutations in the TCOF1 gene, which encodes a nucleolar phosphoprotein called treacle. Model organisms have been generated to model the disease and have revealed knowledge about the etiology and pathogenesis of the disorder. The craniofacial abnormality observed in TCS patients is found to be caused by an increased level of apoptosis in the neuroepithelium and from this it has been suggested that treacle is important for proper formation and proliferation of neural crest cells that will ultimately contribute to the face. The best attempt to rescue the phenotype of TCS was made by inhibiting the expression of p53 in both zebrafish and mouse models. Although both rescues were successful, it resulted in tumor formation in mice which limits the potential of using this type of rescue in humans. Therefore, it is very important to identify p53-downstream genes that were not 5 related to cancer and use them to interrupt the apoptosis pathway and hence rescuing the phenotype.

The primary aims of this study were to examine acquired genetic and epigenetic changes that occur in individuals with increasing age and to determine how these changes are influenced by genetic/environmental factors. Acquired genetic changes were assessed by determining the frequency and chromosomal contents of spontaneously occurring micronuclei in identical and fraternal twins. A total of 115 individuals (48 twin pairs and 19 singletons) were evaluated, ranging in age from 7 to 85 years. As expected, micronuclei frequencies, which are indicative of genomic damage, significantly increased with age (p<0.0001, r=0.446). The majority of micronuclei (32%) contained sex chromosomes and the frequency of sex chromosome-bearing micronuclei significantly increased with age (p<0.0001). The frequency of autosome-containing micronuclei was not significantly influenced by age or gender. However, some autosomes were seen more (chromosomes 4, 8, and 9) or less (chromosomes 17 and 22) frequently than expected by chance (p<0.05). An evaluation of the numerical contents of the sex chromosome-containing micronuclei and their corresponding binucleates showed that the majority of the binucleates had an abnormal chromosomal complement (either hypodiploid or hyperdiploid), with the subset of binucleates having a normal chromosomal complement decreasing with age for both the Y chromosome in males and the X chromosome in females. Model fitting, implemented in Mx, showed the variation in the frequency of micronuclei to be best explained by either additive genetic and unique environmental components, or common and unique environmental factors. Specific environmental exposures and health conditions that were shown to influence micronuclei frequencies, included: multivitamins, leafy green vegetables, fruit, vitamin E supplements, arthritis, heart disease, allergies, and alcohol. To assess acquired epigenetic changes, global methylation profiles of two identical twin pairs were compared and found to differ, indicating that individuals do develop alterations in their methylation profiles with age. Furthermore, the twin pair having a significant difference in their micronuclei frequencies and environmental exposures had more differences in their methylation pattern compared to the twin pair whose micronuclei frequencies and environmental factors did not differ. Overall, genetic and epigenetic changes were shown to occur with age and to be influenced by genetic and lifestyle factors.

Methyl-CpG Binding Proteins (MCBPs) are thought to function as the interpreters of epigenetic information encoded in cytosine methylation. Their ability to translate DNA methylation into local transcriptional repression has sparked interest in the role of Methyl-Binding Domain Proteins (MBDs) in cancer, where repatterning of CpG methylation is common. In this dissertation I summarize and discuss observations made in the Ginder Lab linking MCBPs to the progression of neoplastic disease. It is clear from our work that the Methyl Binding Domain Protein 2 (MBD2) is necessary for the persistent repression of critical tumor suppressor genes in breast cancer. We show that stable knockdown of MBD2 also leads to growth suppression in cultured human mammary epithelial cancer lines (MCF-7, 49% suppression; MDA-MB-231, 77%; MDA-MB-435, 94%; SK-BR-3, 92%) with the peak cytotoxicity and anti-proliferative effect occurring as late as 2-3 weeks after knockdown. MBD2 knockdown also led to a decrease in viable tumor cells at equivalent doses of the histone deacetylase inhibitor, SAHA (Vorinostat™), and chemotherapeutic agents Doxorubicin, and Paclitaxel. Stable MBD2 knockdown in MCF7 cells led to an increased proportion of normal epithelial structures in 3D culture (70%, [CI=0.55-0.83]) when compared to untransfected (46%, [CI=0.39-0.53], p≤0.038) or scrambled shRNA transfected (37%, [CI=0.29-0.45], p≤0.012) controls. In vivo xenograft studies show tumor growth in BALB/C nu/nu mice was significantly impaired when mice were implanted with human breast cancer cells harboring MBD2 targeted shRNA. Following MBD2 knockdown, tumor suppressor promoter methylation remained unchanged despite sustained increases in gene expression, arguing against the convention that passive demethylation occurs with increased transcription. Our data suggest that uncoupling CpG methylation from histone modifications or other repressor functions by removing MBD2 is sufficient to initiate and maintain anti-tumor gene transcription in the absence of secondary changes in DNA methylation. In this dissertation I present evidence for the pathologic role of MBD2 in breast cancer and provide mechanistic support for the prospect of targeting MBDs in neoplastic disease..