Background: Ischemic heart disease remains the number one killer in the developed world. A protein transduction peptide specific for the heart capable of efficiently delivering agents of therapeutic potential at the time of injury, would be of immense public health significance. This work was undertaken to identify peptide(s) able to transduce heart tissue in vivo in a tissue-specific manner. Biopanning was performed in cell culture followed by in vivo with an M13 phage peptide display library. Using the heart-specific peptide, we delivered nemo-binding domain peptide in a murine infarct model to test if NF-kB inhibition can reduce infarct size. Methods and Results: A cardiomyoblast cell line, H9C2, was incubated with M13 twelve amino acid phage peptide display library. Internalized phage was recovered, amplified and subjected to a total of three rounds of in vivo biopanning where infectious, internalized phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). This peptide was synthesized either fluorescently labeled, biotinylated, or in combination with a NEMO-binding peptide (NBD), an inhibitor of the inducible NF-kappa B Kinase (IKK). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was not observed with a cationic transduction domain. CTP-NBD was able to inhibit NF-kB activation in cell culture in a dose-dependent fashion. When administered to mice in a murine infarct model, CTP-NBD showed a trend towards smaller infarct size, which did not reach statistical significance. Conclusions: Biopanning using a peptide phage display library identified a peptide, termed CTP, able to transduce cardiomyoblast cell line in vitro, and heart tissue in vivo, efficiently and specifically. Administration of CTP-NBD to post-infarct mice showed a trend towards reduction of infarct size. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.

ERCC1-XPF is a mammalian structure specific endonuclease that incises at the junction of double and 3 single-stranded DNA. ERCC1-XPF and its homologs are implicated in multiple DNA repair pathways including, nucleotide excision repair, interstrand crosslink repair, double strand break repair, repair of AP sites, and telomere maintenance, making it essential for viability. The pleiotropic phenotypes of ERCC1-XPF deficiency in humans, range from the cancer-predisposition syndrome xeroderma pigmentosum to the XPF-ERCC1 progeroid syndrome, a disease of accelerated aging. This document concentrates on dissecting the role of ERCC1-XPF in crosslink repair, elucidating the mechanisms underlying the dramatic phenotypic differences in patients with inherited XPF mutations, and exploring the utility of ERCC1 and XPF as biomarkers of tumor prognosis. Ercc1 and Xpf knockout mice are phenocopies, illustrating that the two proteins function exclusively as a heterodimer. These models present a progeroid phenotype, reflected in some patients with XPF or ERCC1 mutations. We hypothesize that this accelerated aging is consequent on a failure to repair crosslinks, which are highly cytotoxic DNA lesions. The crosslink repair mechanism in animals remains poorly elucidated and the exact relationship of ERCC1-XPF to other players in this pathway, largely unexplored. Herein we show that ERCC1-XPF functions in the same pathway of crosslink repair as the Fanconi anemia proteins and that crosslink unhooking by ERCC1-XPF is required for the efficient binding of FANCD2 to the chromatin. The pleiotropy of phenotypes associated with human ERCC1-XPF deficiency is puzzling. Often, the severity of phenotype does not correlate with the DNA incising ability of the mutant proteins. We show here that the phenotypic heterogeneity is at least partially explained by the mislocalization of mutant proteins to the cytoplasm. The public health importance of this work comes from the increasing interest in the regulation of ERCC1-XPF expression and activity, due to its involvement in DNA repair pathways implicated in the resistance of tumors to platinum-based chemotherapy. A panel of antibodies was tested extensively and optimized for use in clinical measurement of ERCC1-XPF. This will facilitate improved measurement of DNA repair proteins in clinical specimens and greater understanding of the mechanisms of tumor resistance to genotoxic therapy.

E47, an alternative splice product of the transcription factor 3 (TCF3) gene, has been mechanistically linked with multiple leukemias and lymphomas, and thus it is of great public health importance to study the mechanisms by which E47 influences the development of the hematopoietic system. Throughout life, all mature blood cells are constantly replenished from rare, self-renewing bone marrow hematopoietic stem cells (HSCs) and downstream non-renewing multipotent progenitors (MPPs). Little is known about the gene regulatory network that controls the integrity of these essential bone marrow subsets. Previous evidence has suggested a crucial role for the transcription factor E47 in lymphocyte lineage commitment. However, the specific stages of hematopoiesis that require E47 and the underlying mechanisms through which it acts on remain unclear. Our study aims to elucidate the role of the transcription factor E47 in the earliest, multipotent stages of hematopoiesis. Using E47 deficient mice, we found that E47 is required for the development and functional integrity of uncommitted hematopoietic progenitors. Our results showed that E47 deficient mice had a 5070% reduction in non-renewing MPPs, and the residual MPPs failed to initiate V(D)J recombination, a hallmark of lymphoid lineage progression. The long-term lineage repopulation and self-renewal activities of the primitive HSCs are also compromised in the absence of E47. Not only were the in vivo long-term repopulating HSCs reduced by 3 fold in the bone marrow of E47 deficient mice, but also these HSCs displayed poor self-renewal efficiency by serial transplantation. The compromised self-renewal of E47 null HSCs appears to be associated with premature exhaustion due to over-proliferation under replication stress. The multipotent hematopoietic stem/progenitor cells from E47 deficient mice displayed a striking hyperproliferation following transplantation stress, and they exhibited increased susceptibility to in vivo challenge with a mitotoxic drug. Finally, loss of function and gain of function assays identified the cell cycle inhibitor p21 as a target gene of E47. Together, these observations suggested that E47 regulates the development and functional potential of multipotent hematopoietic subsets, probably through effects on p21-mediated cell cycle quiescence. These findings might provide novel mechanistic insights into hematopoietic damage repair and malignant transformation.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease with as yet unknown etiology and treatment. IPF lungs have a definitive gene expression signature, but the role of microRNAs in IPF has not been studied. MicroRNA genes are short, non-coding RNAs that function as post-transcriptional gene regulators. We hypothesized that microRNAs are differentially expressed in IPF and play a role in the pathogenesis of the disease. The microRNA expression profile of IPF lungs was characterized using microRNA microarrays. We identified 18 significantly decreased and 28 significantly increased microRNAs in samples obtained from patients with IPF compared to controls. Promoter analysis of the decreased microRNAs recognized a SMAD3 binding site in the promoter of let-7d. SMAD3 binding and responsiveness to TGF-β of the let-7d promoter were confirmed by SMAD3 ChIP, EMSA, luciferase assays and reduced expression of let-7d in response to TGF-β. In situ hybridization confirmed a significant reduction in let-7d expression in IPF lungs and localized it to alveolar epithelial cells in the control lungs. To study the role of let-7d, we determined the consequences of loss of function of let-7d by inhibiting its expression in lung epithelial cells. In-vitro inhibition of let-7d caused increases in epithelial mesenchymal transition (EMT) markers N-cadherin (CDH2), vimentin (VIM), alpha-smooth muscle actin (ACTA2) and high mobility group AT-hook 2 (HMGA2). HMGA2 is a proven let-7d target that was increased in IPF lungs and localized to alveolar epithelial cells. We reproduced our in vitro results in vivo. In mice, intratracheal administration of a let-7d inhibitor caused alveolar septal thickening, increases in collagen, ACTA2 and CDH2 and decreases in the epithelial markers, CDH1 and ZO1. We colocalized the mesenchymal markers FSP1 and ACTA2 with the epithelial marker SPC, indicative of EMT. Our results indicate a definitive role for microRNAs in IPF. The downregulation of let-7d in IPF and the pro-fibrotic effects of this downregulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Deciphering their mechanism of action of microRNAs has great public health significance. It would add new insight into the pathophysiology of the disease and aid in reducing the mortality by development of therapeutic interventions.

Prostate cancer (PC) remains a significant public health concern for men throughout the world. Identification of the environmental and genetic factors that predispose to PC for prevention or early intervention is a significant public health concern. PC is more prevalent in males of African descent living in the western hemisphere. Also, recent studies have shown high rates of PC in Afro-Caribbean populations. Men in the 40-79 year age range from the Caribbean island of Tobago have a 3 fold higher risk of developing prostate cancer than do Caucasians. We performed two candidate gene-based association studies to explore the genetic determinants for high risk of PC in Tobago population. In the first study, we performed an extensive study to identify sequence variation in the DC-SIGN gene, a candidate gene for PC, in the Tobago population, and carried out a case/control association study of DC-SIGN polymorphisms and PC susceptibility in the high-risk population of Tobago. We found a unique haplotype in the 5 proximal promoter of DC-SIGN that is associated with PC in Tobago. We further extended the association study to single gene polymorphisms likely to be in linkage disequilibrium with DC-SIGN. We also found that a single nucleotide polymorphism, rs4804806, which is located in the intragenic region between the DC-SIGN and DCSIGN-R genes (4228bp upstream of DC-SIGN), is significantly associated with elevated risk of PC in Tobago population. The function of rs4804806, and whether the risk associated with it is due to linkage disequilibrium between it and other causal variation remains to be explored. In the second study, we examined whether genetic variation in the androgen receptor (AR) and PSA genes was associated with risk of prostate cancer or with serum PSA levels in the Tobago population. Previous studies have proposed that the rs266882 G/A polymorphism, located in the androgen response element upstream of the PSA gene and the (CAG)n repeat polymorphism in DNA binding domain of the AR are associated with risk of PC and serum PSA levels, and those two genetic variants show evidence of genetic interaction. In order to examine whether those two genetic variants affect PC risk or PSA level in Tobago population, an association study was carried out in 167 PC cases and 320 controls. Association analysis with PC was conducted among all cases and controls for each locus individually, and also for both loci together, to test for interaction between them in determining risk of PC or PSA levels. Regression analysis was carried out for each locus and combined with serum PSA level in 320 controls only. Results from this study do not provide any association evidence with PC risk or PSA level for PSA rs266882 G/A genotypes or AR (CAG)n repeat length genotypes. The results from these two studies lend support to exploring genetic causes and prevention of prostate cancer.

Objectives: Mammographic breast density (MBD) is a strong risk factor for breast cancer. It has been hypothesized that breast cancer susceptibility loci may also be associated with breast density. Recently, two genome-wide association studies identified a single-nucleotide polymorphism (SNP), rs2981582, in intron 2 of the FGFR2 gene to be associated with increased breast cancer risk. Further research revealed that intron 2 of FGFR2 contains estrogen receptor transcription factor binding sites. We examined associations of four FGFR2 SNPs (rs2981582, rs3750817, rs17542768 and rs1219643), hormone therapy (HT) use, and their interactions with MBD. Methods: We conducted a cross-sectional analysis using a subset of the Mammograms and Masses Study population. Subjects were 370 healthy postmenopausal Caucasian women. General linear models adjusted for covariates were used to evaluate the associations. Results: Overall, no statistically significant associations were observed between the four SNPs in FGFR2 and MBD. Duration of estrogen plus progestin use, but not duration of estrogen use, and HT status were statistically significantly associated with MBD in our study population. No statistically significant interactions between genotypes and HT use were observed. Conclusions: Our results suggest that the effects of the four evaluated FGFR2 polymorphisms on breast cancer risk are not mediated through MBD, and that the polymorphisms do not modify the effect of HT use on MBD. Implications for public health: Breast cancer is the most common cancer in women in the U.S. Identification of genes that influence MBD may provide insight into the biology of breast density and its effect on breast cancer, eventually leading to more effective prevention and treatment.

The incidence and mortality of brain tumors have not changed over the last 3 decades and pose a significant burden on the healthcare system of the United States. Temozolomide (TMZ) is the preferred chemotherapeutic agent for the treatment of brain tumors and base excision repair (BER) is a major mechanism for the repair of TMZ-induced DNA base lesions. BER inhibition, either by interrupting the delicate balance of the expression of key BER proteins or by chemical inhibitors, enhances cytotoxicity of chemotherapeutic DNA damaging agents such as TMZ. Understanding the mechanisms of enhanced cytotoxicity brought on by BER inhibition has great public health significance. By using DNA polymerase ß (Polß) deficiency as a model of BER inhibition, I report that DNA damage-induced cytotoxicity due to Polß deficiency triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR) or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. I offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. Resistance to TMZ is partially attributed to efficient repair of TMZ-induced DNA lesions. Using the human glioma cell lines LN428 and T98G, I report here that potentiation of TMZ via BER inhibition (methoxyamine (MX), the PARP inhibitors PJ34 and ABT-888 or depletion (knockdown) of PARG) is greatly enhanced by increasing BER initiation via over-expression of MPG. I also show that MX-induced potentiation of TMZ in MPG expressing glioma cells is abrogated by elevated-expression of the rate-limiting BER enzyme Polß, suggesting that cells proficient for BER readily repair AP sites even in the presence of MX. This study demonstrates that increased initiation of BER via MPG over-expression, together with inhibition of repair following initiation, further sensitizes glioma cells to alkylating agent TMZ, suggesting that the expression level of MPG might be used to predict the effectiveness of BER inhibition-induced potentiation of TMZ in glioma cells.

Breast cancer is the most common noncutaneous form of cancer among women in the US. In recent years, the overall mortality rate has declined, yet there still exists a significant racial disparity in the incidence and mortality between African American and Caucasian women. While numerous hypotheses have been proposed to explain this difference, few offer a biological explanation. There is a well established association between estrogens and breast cancer risk, and the ratio of two estrogen metabolites, 2-hydroxyestrone (2OHE1) and 16á-hydroxyestrone (16OHE1), has been implicated as a marker of breast cancer risk. Many studies have also assessed the relationship between endogenous estrogens and mammographic density. Mammographic density is one of the strongest predictors of breast cancer risk, but the mechanism by which it influences this risk remains unknown. Nonetheless, few have examined mammographic density in relation to the 2OHE1:16OHE1 estrogen metabolite ratio (EMR). Research suggests that the Cytochrome P450 1B1 (CYP1B1) gene may also mediate breast cancer risk, as this gene is very active in estrogen metabolism. In fact, the Leu432Val polymorphism has reportedly been associated with urinary levels of the 2OHE1:16OHE1 EMR. The objective of this study was to investigate some of the relationships found among the 2OHE1: 16OHE1 EMR, CYP1B1 Leu432Val polymorphism, mammographic density, race, and breast cancer risk. The 2OHE1:16OHE1 EMR was associated with both breast cancer risk and the CYP1B1 Leu432Val polymorphism, yet, no association with breast cancer risk and this polymorphism was observed. This suggests that if the CYP1B1 Leu432Val polymorphism alters breast cancer risk, it does so through variations in the 2OHE1:16OHE1 EMR. When taking race into account, no association between mammographic density and the 2OHE1:16OHE1 EMR was observed. In culture, evidence was found to suggest that the 2OHE1:16OHE1 EMR is influenced by subcellular effects or other intrinsic factors (i.e. genetic variation), as passage number was the only significant contributor to the 2OHE1:16OHE1 EMR. The results of this study have great public health significance, as it provides a better understanding of the risk factors, including racial differences, and etiology of breast cancer, which will ultimately lead to better prevention and treatment for all women.

Cancers and infectious diseases are becoming a growing public health problem in the world today. The importance of my work for public health is in detecting these diseases earlier and more accurately, potentially leading to better therapies and higher survival rates for patients. Current diagnostic techniques focus on detecting antibodies from serum, gene expression and miRNA profiles of tumor tissues and, more recently, in the bodily fluids of patients. This dissertation shows a novel technique that makes use of small microvesicles called exosomes and the microRNAs (miRNAs) they carry for the potential diagnosis of cancer. Exosomes are small (40-100 nm) membrane-bound vesicles that are created from the inverse budding of the multivesicular endosome and originate from a variety of tumor types. Exosomes can be easily purified from cell cultures and serum of patients and have recently been shown to carry small non-coding RNAs called miRNAs. In the first part of this study, I developed techniques that enabled us to increase the amount of our exosome and total RNA starting material before proceeding to use these as potential diagnostics for head and neck cancers. Although the use of exosomes to diagnose diseases is not novel, the use of miRNAs present in tumor-derived exosomes is a new approach. In the final two chapters, I discuss the use of exosomes to diagnose KSHV viral infections as well as head and neck cancer. Increasing the accuracy and reducing the amount of starting material needed for these studies would provide a non-invasive technique to detect viral infections and cancers. This would help in providing earlier therapeutic treatments and help to increase the longevity and quality of life of patients.

Pluripotent stem cells have the capability to undergo unlimited self-renewal and differentiation into all somatic cell types. They have acquired specific adjustments in the cell cycle structure that allow them to rapidly proliferate, including cell cycle independent expression of cell cycle regulators and lax G1 to S phase transition. However, due to the developmental role of embryonic stem cells (ES) it is essential to maintain genomic integrity and prevent acquisition of mutations that would be transmitted to multiple cell lineages. Here we show that several modifications in DNA damage response of ES cells accommodate dynamic cycling and preservation of genetic information. ATM-dependent checkpoint signaling cascade is activated after irradiation of ES cells, and induces G2/M, but not G1/S cell cycle arrest. The absence of a G1/S cell cycle arrest promotes apoptotic response of damaged cells before DNA changes can be fixed in the form of mutation during the S phase, while G2/M cell cycle arrest allows repair of damaged DNA following replication. Human ES cells express higher level of DNA repair proteins, and rely on homologous recombination to repair double strand breaks. Radiation does not lead to long-term loss of pluripotency, since irradiated ES cells show transient decrease in the level of pluripotency factor transcripts, while protein levels remains stable. One week after irradiation, ES cells retain capacity to differentiate into three germ layers and form teratomas in immunocompromised mice. Similarly to ES cells, induced pluripotent stem (iPS) cells are poised to proliferate and exhibit extreme sensitivity to DNA damage, lack of G1/S cell cycle arrest, and express high level of DNA repair genes, suggesting that DNA damage responses are controlled by developmental state of the cell. Public health significance of this study originates in great promise that human ES and iPS cells hold in cell replacement therapies. Since human ES, and particularly iPS, cells represent potential source of cells for clinical and pharmaceutical applications, the DNA damage response pathways that maintain genomic integrity need to be studied in greater detail.