Public Health Relevance: The United States has 80 million employees with chronic pain resulting in annual losses of 61.2 billion dollars due to pain-related productive time lost. In addition, pain-related depression and inactivity reduce the quality of life. The development of effective analgesics is therefore important from a public health perspective. In this dissertation, the natural properties of herpes simplex virus (HSV-1) vectors are exploited to (i) develop an HSV-1 vector-based selection system that can potentially identify natural or chemical inhibitors of chronic pain and (ii) to test HSV-1 vector-expressed dominant negative PKCε (DNP) as a strategy to treat chronic pain. The vanilloid/capsaicin receptor (TRPV1) is a pro-nociceptive calcium ion channel that is upregulated in chronic pain. This occurs partly due to protein kinase C epsilon (PKCε)−mediated receptor phosphorylation. An HSV-1 vector expressing TRPV1 (vTT) was engineered and vTT-expressed TRPV1 functionality was confirmed.Treatment of vTT-infected cells with capsaicin or resiniferatoxin caused concentration-dependent Ca+2 influx, leading to cell-death and a dramatic reduction in infectious particle yield. TRPV1 antagonists, ruthenium red and SB-366791 reversed agonist-induced cell-death and rescued vTT growth, providing a basis for selection. Selection for antagonists was modeled using a mixed infection of vTT and vHG (capsaicin resistant control vector) and virus passage in the presence capsaicin. These experiments demonstrated that a single control vector particle was readily isolated from a population of 10^5 vTT particles. This approach can be used to identify antagonists from chemical or gene libraries and offers advantages of (i) a platform assay applicable to other ion channels and (ii) adaptability to high throughput formats. Dominant negative PKCε (DNP) was engineered into HSV-1 to create the vector, vHDNP. Following functional confirmation of vHDNP in U2OS, Vero cells and neurons, cobalt uptake showed a reduction of capsaicin sensitive vHDNP-transduced neurons. Electrophysiology confirmed this and also demonstrated a knockdown of TRPV1-PKCε coupling in nociceptive neurons. In-vivo studies of noxious heat-induced nocisponsive behavior in vHDNP-inoculated rats showed a subtle inhibition of withdrawal responses when compared with controls. In conclusion, HSV-1 expressed dominant negative PKCε is a viable strategy to specifically inhibit TRPV1 function in order to treat chronic pain.

The tumor suppressor IRF-1 has been gaining interest as a mediator of anticancer therapies and its role in mediating apoptosis and cell cycle arrest are currently being elucidated. Through the creation of recombinant adenoviral (Ad-) IRF-1 in our lab, we are in a unique position to study the underlying mechanisms of IRF-1 mediated tumor growth inhibition. First, we will further determine the role of IRF-1 in caspase-mediated apoptosis. Our work will examine the mechanism of IRF-1 activation of initiator caspase 8 and effector caspases 3 and 7 and the role of soluble factors. Our second course of study will delineate the role of IRF-1 mediated cell cycle effects and with a focus on G1 arrest and p21waf1cip1 upregulation. Our initial hypothesis that IRF-1 induces caspase 3/7 mediated apoptosis through a death receptor pathway in conjunction with the secretion of soluble factors in cancer was not supported by results obtained. We found that death ligands were not mediating IRF-1 growth inhibition; however we did find that the caspase cascade was clearly involved. Moreover, we have shown that caspase 8 activity is central in mediating IRF-1 apoptosis. While investigating the intrinsic pathway we made a novel discovery that IRF-1 localizes to the mitochondria. The significance of this finding is still under investigation. Studies of p21 knock down confirmed that IRF-1 utilizes p21 in p53 independent G1 cell cycle arrest. We hypothesized that cell cycle arrest would protect the cells from apoptosis but found that p21 up regulation by IRF-1 corresponded to caspase cleavage and that apoptosis was suppressed in our p21 knock down cell lines. We also found that the inhibitor of apoptosis, survivin may account for this effect. Finally, we show that IRF-1 growth inhibitory effects are directed to malignant and not normal breast cells. We show that this too may be linked to survivin which is commonly overexpressed in cancers and suppressed by IRF-1. Greater understanding of the mechanisms of IRF-1 cancer growth inhibition is significant to public health because it may allow better utilization and development of IRF-1 and agents that are mediated by IRF-1 in cancer treatment.

Nearly 45% of oral squamous cell carcinomas (OSCC) are characterized by amplification of chromosomal band 11q13, which occurs by breakage-fusion-bridge (BFB) cycle mechanism. The first step in this cycle involves loss of distal 11q. Consequently, critical genes involved in the DNA damage response pathway, MRE11A, ATM, H2AFX and CHEK1 are lost in the step preceding 11q13 amplification. We hypothesized that this loss on distal 11q may lead to a diminished DNA damage response in OSCC. Characterization of OSCC using fluorescence in situ hybridization revealed partial loss of MRE11A, ATM, H2AFX and CHEK1 in all cell lines with 11q13 amplification and in additional cell lines without 11q13 amplification. Quantitative microsatellite analysis and loss of heterozygosity studies confirmed this loss. Quantitative PCR and immunoblotting revealed reductions in RNA and protein expression of MRE11A, ATM and H2AX. All cell lines with distal 11q loss exhibited aberrant gamma-H2AX foci and increased chromosomal instability to ionizing radiation. Surprisingly, distal 11q loss also correlated with reduced sensitivity to ionizing radiation. Although the literature attributes poor prognosis in OSCC to 11q13 gene amplification, our results suggest that distal 11q deletions may be equally if not more significant. We observed an upregulation of the ATRCHEK1 pathway in a subset of OSCC with loss of the G1 cell cycle checkpoint. We hypothesized that this upregulation protects OSCC from premature chromatin condensation or mitotic catastrophe (cell death) by enhancing the S phase and G2 phase checkpoints. In OSCC, we observed a gain in ATR gene copy number; whereas CHEK1 is partially lost at the gene level. However, we observed that both ATR and CHEK1 are overexpressed in a subset of OSCC with loss of the G1 cell cycle checkpoint. Inhibition of ATR or CHEK1 with caffeine or with the respective siRNAs results in an increased susceptibility of OSCC to DNA damaging agents. Thus, inhibition of the ATRCHEK1 pathway in OSCC may aid the current therapeutic modalities used in the treatment of OSCC. The public health significance of our studies involves the development and use of distal 11q loss and ATRCHEK1 upregulation as biomarkers for OSCC.

Stem cells offer the hope of curing a variety of ailments such as diabetes, Parkinsons disease, myocardial infarct, muscular dystrophy and spinal cord injuries. In this regard, a detailed understanding of the origin and behavior of stem cells is invaluable to the advancement of public health. The adult human adipose tissue (hWAT) is an attractive and convenient source of therapeutic cells for use in the clinical setting. Previous studies have demonstrated that the stromal vascular compartment within hWAT contains multipotent cells, called adipose stem cells (ASC). However, the identity and anatomic distribution of ASC or progenitors within hWAT remain unclear. We addressed this issue through an a priori identification of different cell subsets within the hWAT stroma, by visualization of cells in their native state within the resident tissue, and analysis of their immunohistochemical profile. Endothelial cells, pericytes, as well as non-vascular cells from adult subcutaneous abdominal fat were separated and sorted to homogeneity based on CD34, CD146 and CD45 antigen expression. We first tested the adipogenic potential of the different purified stromal cell populations. A higher level of leptin mRNA, as much as a 20-fold difference, was observed in pericytes and the non-vascular cell fractions when compared to endothelial cells. High levels of leptin expression were maintained even after extensive expansion of the cells in culture. Additionally, we found a reserve of brown adipocyte progenitors within the adult fat tissue vasculature, among pericytes, which challenges the notion that uncoupling protein-1 (UCP-1) expressing cells are confined to fetal and early human life. We also herein describe a precedently unsuspected role of adipose-derived pericytes as human muscle progenitors. When transplanted into cardiotoxin-injured NOD-SCID mouse muscles, pericytes generated a significantly higher number of myofibers than the other hWAT stromal cell populations. Quantitatively, the myogenic potential of adipose-derived pericytes was similar to that of a population of robustly myogenic cells within the skeletal muscle of adult humans, the myogenic-endothelial cells. The long-term culture of hWAT pericytes did not diminish their capacity for myogenic differentiation. These results suggest that human adipose tissue is a viable alternative tissue source to skeletal muscle for muscle cell-mediated therapy.

Interactions and heterogeneity play a vital role in the miscommunication between genotype and phenotype in complex diseases. Detection of genes that influence the risk of common, complex disorders involves many statistical and computational challenges. This led us to investigate and compare the common methods of linkage analysis in complex diseases. We applied various methods of linkage analysis on the simulated dataset from the Genetic Analysis Workshop (GAW) 14. As the disease modeled in this dataset resembled a qualitative disorder, we employed methods such as nonparametric linkage scans, association studies of susceptibility regions, and conditional studies (conditioning on a previously identified susceptibility locus). The goal of this project was to study the efficiencies and inadequacies of various methods in detecting interactions and heterogeneity in the simulated dataset. The methods used on this dataset showed very low percentage in the detection of interactions. We attribute this unsatisfactory performance of these methods mostly to the low prevalence of interactions in the imaginary populations studied. We also propose various ways of improving the power in these analyses like considering haplotype studies instead of targeting single markers and increasing the range of the flanking markers around regions of high LOD scores. Public Health Importance: Understanding the complexities involved in the genetics of diseases will provide new insight for disease prevention and health promotion. For over twenty years, public health agencies have focused more and more on newborn screening programs to detect and prevent rare genetic disorders. But common complex disorders pose a bigger problem because of their unique characteristics like heterogeneity, gene-gene interactions, multiple susceptible loci, incomplete penetrance, phenocopy and presence of environmental risk factors. By comparing common methods of linkage analysis in complex disorders in the simulated dataset of Genetic Analysis Workshop (GAW) 14, our study aims to come up with a better understanding of how heterogeneity and interaction work in the context of a whole genome genetic analysis. It is also expected to lay a foundation on which future public health researchers will be able to expand on our work.

Most of the complex diseases have major public health concern in United States. Linkage analysis helps to map disease genes, and we have proposed linkage statistics that give higher power in real data scenario where the true family structure might not be known. In linkage analysis with affected related pairs (ARP), stated familial relationships are usually assumed to be correct, thus misspecified relationships can lead to either reduced power or false-positive evidence for linkage. In practice, studies either discard individuals with erroneous relationships or use the best possible alternative pedigree structure. We have developed several linkage statistics that model the relationship uncertainty by properly weighting over possible true relationships. We consider ARP data for a genome-wide linkage scan. Simulation study is performed to assess the proposed statistics, and compare them to maximum likelihood statistic (MLS) and Sall LOD score using true and discarded structures. We have simulated small and large pedigree datasets with different underlying true and apparent relationships, and typed for 367 microsatellite markers. The results show that two of our relationship uncertainty linkage statistics (RULS) have power as high as MLS and Sall using the true structure. Also, these two RULS have greater power to detect linkage than MLS and Sall using the discarded structure. Thus, our RULS provide a statistically sound and powerful approach for dealing with the commonly encountered problem of relationship errors. To apply RULS on a real data, we have used Otitis Media with effusion (OME) data from Caucasian families. OME is an infection causing fluid in the middle ear, and is the most common cause of hearing loss among young children. We have recruited subjects (with history of tympanostomy tube insertion) and their families (parents and affected/unaffected siblings). Genotyping was done using Affymetrix 10K SNP chip technology, and out of 1584 enrolled individuals (322 families), 1191 (305 families) are genotyped at this date. We performed nonparametric multipoint linkage analysis using conservative dataset. The preliminary results show suggestive linkage peaks on chromosomes 2, 7 and 10, the highest being on chromosome 7 (rs2014450, 153cM) with Sall LOD score of 2.08 (p-value 0.001).

Approximately one in four pregnant women experience one or more miscarriages, making spontaneous abortion the most common pregnancy complication, and of public health importance. Recurrent spontaneous abortion (RSA) can be defined as the loss of two or more pregnancies and affects 1% of couples. This prevalence is higher than expected by chance, suggesting some couples have an underlying systemic cause for RSA. We have chosen to study two immunological aspects of pregnancy loss. The first involves maternal defense against infection in terms of predicted mannose binding lectin (MBL) plasma levels. The second approach is to analyze the human leukocyte antigen-G (HLA-G) gene, which is believed to play a role in maternal recognition of paternal antigens at the maternal-fetal interface. The case population included women having two or more clinically recognized spontaneous abortions as well as having unknown etiology for RSA. Controls subjects were selected from healthy primiparous women with no history of miscarriage. Cases and controls were genotyped for five MBL single nucleotide polymorphisms (SNPs). Both populations genotyped were in Hardy-Weinberg equilibrium, at all five sites. Fishers exact test of cases and controls was not significant at each of the five sites, p-values > 0.05. No association was observed between MBL genotypes or predicted MBL plasma levels and risk of RSA, or presence of live birth and recurrent pregnancy loss, among women with unexplained RSA. Using the same population, the HLA-G promoter region and 3 untranslated region (UTR) was sequenced in cases and controls. Twenty-three SNPs were observed with a minor allele frequency >0.02 in the promoter region. Linkage disequilibrium was detected throughout 1400 base pairs of the promoter region that were sequenced. While SNP data revealed allele frequency differences between cases and controls, haplotype data proved even more beneficial; one haplotype potentially predicting increased risk of RSA, while the other potentially protecting against risk of RSA. Finally, cases had a higher frequency of individuals homozygous for the 14 base pair insertion in the 3 UTR.

Cardiovascular disease (CVD) is a major public health concern. It is the Nations leading killer for both men and women of all racial and ethnic groups. CVD is responsible for about 1 million deaths each year in the United States. Health-related behaviors such as smoking, lack of physical activity and poor nutritional habits, as well as, many genetic factors contribute to its high incidence. Many of the genetic factors have been linked to high cholesterol, high blood pressure, obesity, diabetes and cardiac arrhythmias leading to stroke or sudden cardiac death. CVDs associated with ventricular arrhythmias are most severe. Among these is the Brugada syndrome also known as Sudden Unexpected Death Syndrome or SUDS. In 1992, the Brugada syndrome was classified as a distinct clinical heart condition characterized by an apparent right bundle branch block and ST segment elevation in the right precordial electrocardiogram (ECG) leads V1-V3. It is the most common cause of sudden cardiac death in South Asian men who are less than 50 years of age and have no underlying cardiac disease. Currently the only effective treatment for the disease is the Implantable Cardioverter Defibrillator (ICD) surgically placed in the patients chest. The genetic basis for the Brugada syndrome has been linked to mutations in the SCN5A gene that codes for the alpha-subunit of the cardiac sodium channel. Recently, a missense mutation has been discovered in a novel gene that causes the Brugada syndrome. The novel gene is named the Glycerol-3-phosphate Dehydrogenase 1-Like (GPD1L) gene. Preliminary studies suggest a direct relationship between the GPD1L mutation and a decrease in cellular sodium current. Transgenic murine models are useful tools for understanding the molecular function of novel genes. Transgenic constructs of the wild type and mutant GPD1L gene were generated and used for the production of transgenic mice. The mice were produced by pronuclear injection at the University of Pittsburgh Transgenic facility. These mice will provide an in vivo approach to study the GPD1L gene and create the first Brugada syndrome mouse model for cardiovascular disease studies.

Essential hypertension (EH) is a major risk factor for cardiovascular disorders, the leading cause of death in the United States. Given its great public health impact, it is crucial to understand the genetic basis of EH. EH is highly heterogeneous and to use an intermediate phenotype of EH, sodium lithium countertransport (SLC), will provide substantial advantage for disease genes discovery. We proposed two approaches to explore the genes for SLC. The first study examined the relationship between SLC and a positional candidate gene, SLC34A2, which is linked to SLC in baboon. We sequenced gene SLC34A2 in baboon and human. Strong homology was established in exonic organization and sequence between human and baboon SLC34A2 genes and extensive variation in both species was identified. Association studies between SLC and SLC34A2 were carried out in 1856 RFHS phase II individuals and 634 baboons. Significant association of SLC with human SNP rs3775909 (p=0.03) in SLC34A2 and haplotype block 2 (p<0.005) were observed. Strong evidence for association of SLC with SLC34A2 was found for baboon SNP Asn136Asn (p=0.0001). Consistent findings in two different species implied that SLC34A2 may be one of the genes involved in SLC. However, linkage analyses conditional on genotypes of baboon Asn136Asn suggest that Asn136Asn is not the primarily functional site for SLC. We conclude that SLC34A2 is associated with SLC, though it may not be the major effect gene. In second study, we integrated gene expression micrarray with linkage analysis to search for genes for SLC. Two independent microarrays (U133A and U133_plus_2.0) were used to identify the differentially expressed genes in high verse low SLC groups. Five genes, IER3, ARHGAP15, CD47, CDKAL1 and PRKRA, were among top 1% of differentially expressed genes in both arrays and also mapped to linkage region for SLC in RFHS Phase II population. A follow-up association study for IER3 shows that SNP rs8512 is significantly associated with SBP (p=0.002) and DBP (p=0.0008), and SNP rs2284174 has marginal association with SLC (p=0.055) and SBP (p=0.085). In conclusion, we identified some interesting susceptible genes for SLC by combining gene expression profiling and linkage study.

Background: Populations of African ancestry have greater bone strength and lower osteoporotic fracture risk than other ethnic groups but there is little information about skeletal health among individuals of African heritage. Methods: Univariate, bivariate and multivariate analytical methods under the variance components framework were employed to dissect the genetic and environment determinants for DXA and pQCT measured bone strength related phenotypes. Our analyses were performed on phenotypic and genotypic data on 471 individuals aged 18+ from 8 large, multigenerational Afro-Caribbean families. Results: The major conclusions of this study are that (1) compared to Caucasians and African Americans, Afro-Caribbeans have the highest peak areal BMD and slowest bone loss rate, but heritabilities of many bone strength related traits are similar among different populations, and (2) genes and environmental factors differentially affect trabecular versus cortical traits, and also BMD versus bone size. These conclusions are supported by differences in heritability and genetic correlation estimates among these bone categories, differential effects of environmental risk factors, as well as associations with different candidate genes. We also evaluated the capability of two multivariate analysis methods for uncovering underlying genetic factors using both simulated and real family data. We concluded Factor Analysis behaves better for both simulated and real data compare to Principal Component Analysis. The residual strategy increases the probability that composite phenotypes detect underlying genetic components if no gene-environment interaction is involved. And most importantly, composite phenotypes from multivariate analysis demonstrated their capabilities to capture more and stronger association signals in real data analysis. Public health significance: Our work has identified the facts that environmental risk factors and genetic determinants may differentially affect various bone compartments and types of bone phenotypes. This information will contribute to the understanding of the underlying genetic architecture of osteoporosis and hence lead to better methods of treatment and prevention of the disease.