Obesity and type 2 diabetes are heterogeneous conditions caused by a combination of genetic and environmental factors. A number of candidate gene families have been identified that influence obesity- and diabetes-related traits, including the uncoupling proteins (UCPs) and fatty acids binding proteins (FABPs). The UCPs are mitochondrial transport proteins that promote proton leakage across the inner mitochondrial membrane, uncoupling oxidative phosphorylation from ATP production and releasing energy as heat. The FABPs are intracellular transporters of fatty acids that facilitate lipid metabolism and gene transcription regulation. The UCPs and FABPs influence energy metabolism, fuel substrate partitioning, glucose and lipid metabolism, and insulin action. In this study, we identified variation in UCP and FABP genes, explored the influence of that variation on phenotype through multi-locus analyses, and assayed the functional consequences of promoter variation on gene expression. Using the multi-locus analysis approach, we constructed regression models that explained a relatively large portion of the variation in phenotypes in comparison to the individual effects of single loci. Several of the models explained upwards of 10% of the variation in traits. This suggests that a multi-locus approach to studying complex disease is much more informative than considering single loci individually. In addition to the statistical analyses, functional studies were performed to assess the effects of promoter variation on gene expression. Variation in the FABP2 promoter region was associated with levels of promoter activity, suggesting a biological explanation for effects of this polymorphism on phenotype. This exploratory analysis identified a number of interesting multi-locus genetic effects on traits related to obesity and type 2 diabetes, suggesting that consideration of multiple gene effects is a more comprehensive approach to understanding complex disease.

Complex diseases such as type 2 diabetes, hypertension and psychiatric disorders have been major public health problems in US. In order to increase the power in the linkage analysis of complex traits, genetic heterogeneity has to be taken into account. During the past few years, several methods have been proposed for dealing with this issue by incorporating covariate information into the affected sib pair (ASP) analysis. However, it is still not clear how these approaches perform under different gene-environment (G x E) interactions. The covariate statistics evaluated in this study are: (1) mixture model; (2) general conditional-logistic model (LODPAL); (3) multinomial logistic regression models (MLRM under no dominance, no additive and min-max restriction); (4) extension of the maximum-likelihood-binomial approach (MLB); (5) ordered-subset analysis (OSA with three different rank orders: high-to-low, low-to-high and optimal-slice); (6) logistic regression modeling (COVLINK). Based on the chromosome-based approach, we have written simulation programs to generate data under various G x E models and disease models. We first define the empirical statistical significance thresholds using C2, the environmental risk factor, under the null hypothesis. We then evaluate the power of the covariate statistics when different covariates are used. We also compare the performance of the covariate statistics with the model-free methods (Sall and Spair). In all three G x E interaction models, most covariate methods perform better when using C1, the covariate with G x E interaction effect, than when using C2 or the random noise covariate C3, except for MLB and the low-to-high OSA method. Comparing with the model-free methods (using Sall as the baseline), mixture model and the high-to-low OSA method perform the best of the covariate statistics when using C1. However, when using C2 or C3, most covariate statistics provide less power than Sall. Only MLB has comparable power to Sall across all genetic models. According to our results, in different G x E interactions, one should apply the appropriate covariate statistic and include the suitable type of covariates carefully.

Inheritance of DNA methylation on imprinted genes depends on the Dnmt1 (cytosine-5-) methyltransferase protein. Methylation patterns on imprinted genes are maintained by oocytespecific Dnmt1o isoform at the 8-cell stage of preimplantation development. Methylation patterns in postimplantation embryos are maintained by the Dnmt1s isoform. To determine if Dnmt1s can functionally replace Dnmt1o, we expressed Dnmt1s in oocytes and discovered that Dnmt1s can maintain genomic imprints in the absence of Dnmt1o. However, the ability of Dnmt1s to maintain imprinting is dependant on the level of oocyte Dnmt1s. Though Dnmt1s and Dnmt1o have equivalent maintenance methyltransferase functions in oocytes, the unstable nature of oocyte Dnmt1s, in comparison to oocyte Dnmt1o, leads to levels lower than what are required to maintain methylation at the 8-cell stage. We also determined that in cloned embryos, Dnmt1o undergoes none of its expected trafficking to 8-cell stage nuclei. Instead, these embryos exhibit a mosaic pattern of Dnmt1s expression. Defects in intracellular trafficking of Dnmt1o and misexpression of Dnmt1s, along with the intrinsic instability of Dnmt1s, might contribute to iv aberrant DNA methylation in cloned embryos, thus raising concerns about the use of current cloning technologies for therapeutic cloning. Molecular mechanisms involved in the formation of ovarian teratomas were also analyzed. Unfertilized oocytes arrest at the MII stage of meiotic maturation. After fertilization, oocytes continue into cell division. Premature activation of MII oocytes without fertilization, can lead to ovarian teratoma formation. To better understand mechanisms governing the prevention of spontaneous oocyte activation, we investigated the molecular defects leading to formation of ovarian teratomas in the Tgkd mouse model. Tgkd is a transgene insertional mutation that leads to reduced levels of the Inpp4b protein in MII oocytes of hemizygous Tgkd females. Wildtype GV oocytes have less Inpp4b protein than MII oocytes, and a significant decrease in Inpp4b is also seen after fertilization. Also, the dependence of ovarian teratoma formation on the mouse strain, emphasizes the role of a strain-specific modifier on chromosome 6, possibly IP31. Thus, it is possible that oocyte Inpp4b normally suppresses spontaneous MII oocyte activation, possibly by reducing levels of IP3, an intermediate in the oocyte activation mechanism, that occurs following fertilization.

Background: As the prevalence of obesity has steadily increased, it has rapidly emerged as a major public health concern due to a high risk of morbidity and mortality. Rare missense and nonsense mutations in the melanocortin-4 receptor (MC4R) gene are a cause of genetic forms of severe obesity, and targeted disruption of the mouse MC4R leads to obesity. The role of variation at the MC4R locus in influencing interindividual variation in body size and composition in the general population is controversial. Objective: To test the hypothesis that polymorphic variation at the MC4R locus is significantly associated with measures of adiposity in the general population. Methods: Two single nucleotide polymorphisms, -4599 T>G and -4850 T>C, in the 5¡¯-flanking region of MC4R were verified by resequencing in 16 individuals and genotyped in the larger sample by fluorescence polarization. 1,099 healthy, non-Hispanic white volunteers, age 30-54 years, were recruited from the Pittsburgh community. A medical and demographic history was collected and anthropomorphic measures were determined. ANOVA was used to assess the relationship between genotype and metabolic parameters. Results: BMI was greater in participants having a -4599 G allele (GG + TG genotypes) than among TT homozygotes (p < 0.03), and this association was of similar magnitude in both men (BMI 28.2 vs. 27.4) and women (BMI 26.5 vs. 25.9). Nominally defined overweight (BMI ¡Ý 27) also varied significantly (Chi-square = 6.874, p < 0.04) across -4599 genotypes (GG: 52.4%; TG: 46.8%, TT: 40.6%). A similar relationship was seen for the -4850 T>C SNP (CC: 53.2%; TC: 48.0%; TT: 41.4%; Chi-square = 6.256, p < 0.05). Finally, subjects with any -4599 G allele had significantly higher weight (177.0 vs. 172.8; p < 0.05) and percent of body fat (29.8% vs. 28.8%; p < 0.04). Greater waist circumference was also significantly associated with both the -4599 G allele (36.3 vs. 35.6; p < 0.04) and the -4850 C allele (36.4 vs. 35.6; p < 0.05). Conclusion: Common variation in the 5¡¯-flanking region of the MC4R gene is significantly associated with measures of adiposity in men and women in the general population.

Among the public health relevant disorders, Type 1 Diabetes (T1D) is a degenerative disease affecting almost 2 million Americans. It is characterized by the loss of insulin-producing b-cells due to a T cell-mediated autoimmune response. The risk to develop T1D is HLA associated. HLA-DQ8-DR4 has been identified as the most prevalent HLA haplotype in the Caucasian T1D population. Although DQ8 has been demonstrated to be the primary genetic determinant of disease susceptibility, its predisposing effect is likely modulated by the expression of closely linked DR4 alleles. As one of hypotheses to explain the role of DR4 molecules in T1D etiology, the peptide competition model holds that DR4 competes to bind diabetogenic peptides with DQ8 and thus affects DQ8-restricted autoreactive CD4 T cell responses. However, the evidence of the competition is insufficient due to the lack of detection reagents and the difficulty of segregating the expression of DR4 from DQ8. In this study, we investigated the competition of peptides derived from Glutamic Acid Decarboxylase 65 (GAD65) a putative b-cell autoantigen. A panel of DQ8-restricted T cell lines was generated to serve as detection reagents to evaluate the peptide occupancy of DQ8. After demonstrating that a single peptide derived from GAD65 could bind both HLA-DQ8 and HLA-DR4, we compared CD4 T cell responses elicited by antigen presenting cells expressing DQ8 alone with those expressing DQ8 and DR4 simultaneously. Results indicated that the co-expression of HLA-DR4 diminished DQ8-restricted T cell responses. In addition, distinct DR4 subtypes were demonstrated to affect DQ8-restricted T cell responses differently, suggesting the variable degrees of peptide competition potentials. Taken together, this study provides the evidence that DR4 is able to compete for peptides with DQ8. The outcome of this competition decreases DQ8-restricted CD4 T cell responses, which may hence contribute to a peripheral tolerance mechanism and explain the modulating role of DR4 to the DQ8-conferred T1D susceptibility.

Prostate cancer is a public health concern, particularly to the African-American community. African-American men are disproportionately affected and experience prostate cancer rates about 60% more often than white Americans. It is the second leading cancer in men and it is second only to lung cancer in cancer related deaths. This is a significant public health problem. This paper will focus on the gene Interleukin 6 signal transducer isoform 1 (IL6ST), on chromosome 5q11.2, with polymorphisms relating to gp130 [rs 3730293 and rs 3729960]; both single nucleotide polymorphisms result in an amino acid change. The change is Glycine to Arginine for rs 3729960 which is located in exon 14 and Isoleucine to Threonine for rs 3730293 which is located in exon 10. Human IL-6 signaling requires the 80kDa IL-6R receptor, which is responsible for IL-6 specificity and the 130kDa glycoprotein, gp130, the signal transduction subunit for signal transduction to occur. IL-6 acts through its receptors which are polymorphic, and subsequently may cause it to have different functionality. A population based case/control study was conducted to evaluate the influence of gp130 snps on prostate cancer risk in an Afro-Caribbean male population of Tobago. This population was chosen because of the high prevalence of prostate cancer. A subset of 1000 samples taken from a study done by Bunker et al, was genotyped by PCR and fluorescence polarization methods. Statistical analysis yielded all estimated allele frequencies to be in Hardy Weinberg Equilibrium. Chi square analysis of the cases and controls yielded no significant association of the gp130 snps and prostate cancer risk.

Schizophrenia is a debilitating disorder characterized by disturbances in thought with lifetime prevalence of one percent. The public health burden of schizophrenia due to medical care, social disability, and co-morbid conditions is substantial. Genetic variation, viral infection, or interaction of the two could influence schizophrenia risk. An understanding of these disease pathways could lead to strategies for prevention and treatment of schizophrenia. We used a positional approach to identify schizophrenia candidate genes that could interact with cytomegalovirus and herpes simplex viruses (HSV). We focused on three groups of genes: TNF and MICB near D6S2672, which was associated with schizophrenia and CMV in our previous studies; IL1â, IL1RN, and IL10, immune related genes associated with schizophrenia in published articles; and IL-18, IL18BP, IL18RAP, IL12A, and IL12B, positional candidate genes in the IL-18 pathway. We used multiple case-control and family-based samples to test these hypotheses. We comprehensively sequenced TNF, and genotyped eight SNPs in a case-control sample. We detected no significant associations. We used a dual-luciferase expression assay to quantify TNF expression driven by common promoter haplotypes. Differences in TNF expression did not correlate with schizophrenia. To localize the D6S2672 association, we genotyped 26 SNPs spanning 100kb in a case-control sample. Based on suggestive associations, we selected five SNPs to assay among additional samples. A SNP in MICB was associated with schizophrenia in these samples. The opposite allele was associated with HSV1 in two non-schizophrenia groups. We used comprehensive sequencing data to select tag SNPs at IL1â, IL1RN, IL10, and IL-18 pathway genes. Tag SNPs were evaluated in a case-control sample. In IL1â, IL1RN, and IL10 significant associations were not detected. However, meta-analysis of rs16944 (IL1â 511) studies suggests modest, but significant, risk for schizophrenia in Caucasian samples. In IL-18 pathway genes, a IL18RAP SNP was associated with schizophrenia, the opposite allele was associated with HSV1. Identified associations with schizophrenia may be due to host gene-virus interaction. These genetic variants could clarify which patients are vulnerable to viral infection. Treatment or prevention may be feasible, if our results are confirmed. Further replicate studies are warranted, as are functional studies of associated polymorphisms.

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.