Theoretical and experimental studies of linkage disequilibrium in human populations

Linkage disequilibrium (LD) is defined as the nonrandom association of alleles at two or more loci in a population and may be a useful tool in a diverse array of applications including disease gene mapping, elucidating the demographic history of populations, and testing hypotheses of human evolution. However, the successful application of LD-based approaches to pertinent genetic questions is hampered by a lack of understanding about the forces that mediate the genome-wide distribution of LD within and between human populations. Delineating the genomic patterns of LD is a complex task that will require interdisciplinary research that transcends traditional scientific boundaries. The research presented in this dissertation is predicated upon the need for interdisciplinary studies and both theoretical and experimental projects were pursued. In the theoretical studies, I have investigated the effect of genotyping errors and SNP identification strategies on estimates of LD. The primary importance of these two chapters is that they provide important insights and guidance for the design of future empirical LD studies. Furthermore, I analyzed the allele frequency distribution of 26,530 single nucleotide polymorphisms (SNPs) in three populations and generated the first-generation natural selection map of the human genome, which will be an important resource for explaining and understanding genomic patterns of LD. Finally, in the experimental study, I describe a novel and simple, low-cost, and high-throughput SNP genotyping method. The theoretical analyses and experimental tools developed in this dissertation will facilitate a more complete understanding of patterns of LD in human populations. ^

Lobe specific Ca2+-calmodulin nano-domain in neuronal spines: a single molecule level analysis.

Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

An update on pathobiologic roles of anti-glycan antibodies in Guillain-Barré syndrome.

Anti-glycan antibodies directed against gangliosides are now considered the major immune effectors that induce damage to intact nerve fibers in some variants of the monophasic neuropathic disorders that comprise Guillain-Barré syndrome. Recent experimental studies elucidating the complexity of anti-glycan antibody-mediated pathobiologic effects on intact and injured nerves undergoing repair are discussed.

Identification of cadmium-induced mutations in a mammalian cell line

Epidemiological studies have shown cadmium to induce cancer in humans, while experimental studies have proven this metal to be a potent tumor inducer in animals. However, cadmium appears nonmutagenic in most prokaryotic and eukaryotic mutagenesis assays. In this study, we present the identification of mutations in normal rat kidney cells infected with the mutant MuSVts110 retrovirus (6m2 cells) as a result of treatment with cadmium chloride. The detection of these mutations was facilitated by the use of a novel mutagenesis assay established in this laboratory. The 6m2 reversion assay is a positive selection system based on the conditional expression of the MuSVts110 v-mos gene. In MuSVts110 the gag and mos genes are fused out of frame, thus the translation of the v-mos sequence requires a frameshift in the genomic RNA. In 6m2 cells this frameshift is accomplished by the temperature-dependent splicing of the primary MuSVts110 transcript. Splicing of MuSVts110, which is mediated by cis-acting sequences, occurs when 6m2 cells are grown at 33$\sp\circ$C and below, but not at 39$\sp\circ$C. Therefore, 6m2 cells appear transformed at low growth temperatures, but take on a morphologically normal appearance when grown at high temperatures. The treatment of 6m2 cells with cadmium chloride resulted in the outgrowth of a number of cells that reverted to the transformed state at high growth temperatures. Analysis of the viral proteins expressed in these cadmium-induced 6m2 revertants suggested that they contained mutations in their MuSVts110 DNA. Sequencing of the viral DNA from three revertants that constitutively expressed the P85$\sp{gag{-}mos}$ transforming protein revealed five different mutations. The Cd-B2 revertant contained three of those mutations: an A-to-G transition 48 bases downstream of the MuSVts110 3$\sp\prime$ splice site, plus a G-to-T and an A-to-T transversion 84 and 100 bases downstream of the 5$\sp\prime$ splice site, respectively. The Cd-15-5 revertant also contained a point mutation, a T-to-C transition 46 bases downstream of the 5$\sp\prime$ splice site, while Cd-10-5 contained a three base deletion of MuSVts110 11 bases upstream of the 3$\sp\prime$ splice site. A fourth revertant, Cd-10, expressed a P100$\sp{gag{-}mos}$ transforming protein, and was found to have a two base deletion. This deletion accomplished the frameshift necessary for v-mos expression, but did not alter MuSVts110 RNA splicing and the expression of p85$\sp{gag{-}mos}.$ Lastly, sequencing of the MuSVts110 DNA from three spontaneous revertants revealed the same G to T transversion in each one. This was the same mutation that was found in the Cd-B2 revertant. These findings provide the first example of mutations resulting from exposure to cadmium and suggest, by the difference in each mutation, the complexity of the mechanism utilized by cadmium to induce DNA damage. ^

Effects of butyrate on the colon cancer cell phenotype-chemosensitization and upregulation of proteins implicated in cancer progression

Colon cancer is the second leading cause of cancer mortality in the U.S. Surgery is the only truly effective human colon cancer (HCC) therapy due to marked intrinsic drug resistance. The inefficacy of therapies developed for metastatic HCC suggests that advances in colon cancer chemoprevention and chemotherapy will be needed to reduce HCC mortality. The dietary fiber metabolite butyrate (NaB) is a candidate cancer chemopreventive agent that inhibits growth, promotes differentiation and stimulates apoptosis of HCC cells. Epidemiological and experimental studies suggest that dietary fiber protects against the development of HCC, however, recent large prospective trials have not found significant protection. ^ The first central hypothesis of this dissertation project is that the diversity of phenotypic changes induced by NaB in HCC cells includes molecular alterations that oppose its chemopreventive action and thereby limit its efficacy. We investigated the effect of NaB on the expression/activity of epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) in HCC HT29 cells. NaB treatment induced a 13-fold increase in EGFR expression in concert with its chemopreventive action in vitro, i.e., induction of growth suppression and G1 arrest, apoptosis and a differentiated phenotype. NaB-induced EGFR was active based on multiple lines of evidence. The EGFR was: (1) heavily phosphorylated at Tyrosine (P-Tyr); (2) associated with the cytoskeleton; (3) localized at the cell surface, and activated in response to EGF; and (4) NaB treatment of the cells induced activation of the EGFR effector Erk1/2. NaB treatment also induced a 7-fold increase in COX-2 expression. The NaB-induced COX-2 was active based on significantly increased PGE2 production. ^ The second central hypothesis is that NaB treatment would render HCC cells more chemosensitive to chemotherapy agents based on the increased apoptotic index induced by NaB. NaB treatment chemosensitized HT29 cells to 5-FU and doxorubicin, despite increases in the expression of P-glycoprotein and a related drug resistance protein (MRP). ^ These results raise the intriguing possibility that the chemopreventive effects of fiber may require concomitant treatment with EGFR and/or COX-2 inhibitors. Similarly, NaB may be a rational drug to combine with existing chemotherapeutic agents for the management of advanced HCC patients. ^

Malaria prevention in Kenyan women of childbearing age

The objective of this program is to reduce malaria incidence in Kenya. Malaria poses a large public health challenge in Kenya, and although public health efforts have traditionally been focused on treatment of infected patients, due to increased drug resistance and lack of drug-adherence, prevention strategies are needed. This program targets Kenyan women, the likely caretakers in the home, and promotes malaria prevention behaviors through health education. ^ A planning group will be assembled and a needs assessment will be performed, verifying risk factors and conditions associated with malaria, as well as personal and external determinants. Behavioral and environmental outcomes will be determined, and performance objectives for each outcome will be established. Matrices of change objectives will be created, and detailed methods and strategies will be linked to each change objective. Program elements include media, education, and incentives. All materials used in this program will be subjected to pre-test to ensure cultural relevance and fidelity. Matrices of change objectives will be created for program adopters and implementers, as well as correlating methods and strategies associated with each change objective. Performance objectives will also be compiled for program maintainers. A program evaluation plan will follow "Pre-Post Comparison Group" design. Outcome evaluation and process evaluation will be conducted. The sample population will be screened based on age and gender so as to maintain comparability to the target population. Measurements will be taken before the program to establish baseline, directly following the program to determine short-term effects, and three months after the program is completed to determine long-term effects. ^ One limitation of this program is selection bias, due to the nature of quasi-experimental studies. Thorough screening prior to sample selection will minimize selection bias and ensure group homogeneity. Another limitation is attrition, and this will be minimized where possible through the use of incentives. In cases where loss to follow-up is not avoidable, such as death or natural disasters, the attrition effect will be estimated using structural equation modeling after reviewing the sample size, differential attrition and total attrition. ^ This intervention is based heavily on health promotion theories, but it is important to remember that in the field, the program plan will likely include only the necessary practical strategies. The target population, Kenyan women of childbearing age, will be significant in decreasing the malaria disease burden in Kenya.^

Vitamin A supplementation in very low birth weight infants

PURPOSE: In United States, the percentage of Extremely Low Birth Weight (ELBW) born for year 2006 was 0.8% (approximately 32,000 babies) & Very Low Birth Weight (VLBW) 1.48% (1). ELBW babies account for nearly half (49%) of the infant mortality for United States. Very Low birth weight infants are at a significant risk for high mortality and morbidity due to their multi system involvement and predisposition to lung prematurity and impaired immune function. One of the common causes cited is Vitamin A deficiency (2, 3).The purpose of this study is to look at published literature on Vitamin A supplementation in very low birth weight (VLBW) infants. ^ RESEARCH DESIGN: Systematic review of literature of published articles meeting the pre-defined criteria. ^ PROCEDURE: Studies included in this review were those which looked at very low birth weight infants defined as birth weight<1500gms. All experimental studies were reviewed. Studies looking at the effect of Vitamin A supplementation in comparison with a placebo or by itself in varying dosing regimens as an intervention were reviewed. Vitamin A deficiency and its manifestations were of interest. We used key words such as "very low birth weight", "mortality", "Vitamin A", "retinol" and "supplementation" in our search. ^ RISKS & POTENTIAL BENEFITS: We do not see any potential risks associated with this study. The potential benefit is recommendation for future studies based on the review of literature available currently. ^ IMPORTANCE OF KNOWLEDGE THAT MAY REASONABLY BE EXPECTED TO RESULT: The systematic review of literature of all experimental studies in VLBW infants showed uniform correlation of parenteral Vitamin A dosing and high plasma concentrations achieved. The recommended dosage for use is 5000 IU 3 times/week given intramuscularly for 4 weeks to prevent CLD. Higher doses have not shown benefit, with a potential for toxicity, while lower doses are inadequate. There is no role of use of Vitamin A in closure of patent ductus arteriosus & reducing mortality. However, it is important to state that the number of studies done so far is limited with small sample sizes. There is a need in the future for experimental studies to ascertain the role of Vitamin A to improve outcomes in VLBW. Atleast, one more RCT should be conducted using the dosage recommended above to make this a standard practice.^

Learning What Works: Demonstrating Practice Effectiveness with Children and Families Through Retrospective Investigation

Intensive family preservation services (IFPS), designed to stabilize at-risk families and avert out-of-home care, have been the focus of many randomized, experimental studies. Employing a retrospective “clinical data-mining” (CDM) methodology (Epstein, 2001), this study makes use of available information extracted from client records in one IFPS agency over the course of two years. The primary goal of this descriptive and associational study was to gain a clearer understanding of IFPS service delivery and effectiveness. Interventions provided to families are delineated and assessed for their impact on improved family functioning, their impact on the reduction of family violence, as well as placement prevention. Findings confirm the use of a wide range of services consistent with IFPS program theory. Because the study employs a quasi-experimental, retrospective use of available information, clinical outcomes described cannot be causally attributed to interventions employed as with randomized controlled trials. With regard to service outcomes, findings suggest that family education, empowerment services and advocacy are most influential in placement prevention and in ameliorating unmanageable behaviors in children as well as the incidence of family violence.

Looking Inside The Black Box

Intensive family preservation services (IFPS), designed to stabilize at-risk families and avert out-of-home care, have been the focus of many randomized, experimental studies. The emphasis on "gold-standard" evaluation of IFPS has resulted in fewer "black box" studies that describe actual IFPS service patterns and the fidelity with which they adhere to IFPS program theory. Intervention research is important to the advancement of programs designed to protect the safety of children, improve family functioning, as well as prevent out-of-home placement. Employing a retrospective “clinical data-mining” (CDM) methodology, this exploratory study of Families First, an IFPS program, makes use of available information extracted from client records to describe interventions and service patterns provided over a two year period. This study uncovers actual IFPS service patterns, demonstrates IFPS program fidelity, as well as reveals the usefulness of CDM as a social work research methodology. These findings are particularly valuable for program planning and treatment, policy development and evidence-based practice research.

Evaluation of PRESAGE® dosimeters for brachytherapy sources and the 3D dosimetry and characterization of the new AgX100 125I seed model

With continuous new improvements in brachytherapy source designs and techniques, method of 3D dosimetry for treatment dose verifications would better ensure accurate patient radiotherapy treatment. This study was aimed to first evaluate the 3D dose distributions of the low-dose rate (LDR) Amersham 6711 OncoseedTM using PRESAGE® dosimeters to establish PRESAGE® as a suitable brachytherapy dosimeter. The new AgX100 125I seed model (Theragenics Corporation) was then characterized using PRESAGE® following the TG-43 protocol. PRESAGE® dosimeters are solid, polyurethane-based, 3D dosimeters doped with radiochromic leuco dyes that produce a linear optical density response to radiation dose. For this project, the radiochromic response in PRESAGE® was captured using optical-CT scanning (632 nm) and the final 3D dose matrix was reconstructed using the MATLAB software. An Amersham 6711 seed with an air-kerma strength of approximately 9 U was used to irradiate two dosimeters to 2 Gy and 11 Gy at 1 cm to evaluate dose rates in the r=1 cm to r=5 cm region. The dosimetry parameters were compared to the values published in the updated AAPM Report No. 51 (TG-43U1). An AgX100 seed with an air-kerma strength of about 6 U was used to irradiate two dosimeters to 3.6 Gy and 12.5 Gy at 1 cm. The dosimetry parameters for the AgX100 were compared to the values measured from previous Monte-Carlo and experimental studies. In general, the measured dose rate constant, anisotropy function, and radial dose function for the Amersham 6711 showed agreements better than 5% compared to consensus values in the r=1 to r=3 cm region. The dose rates and radial dose functions measured for the AgX100 agreed with the MCNPX and TLD-measured values within 3% in the r=1 to r=3 cm region. The measured anisotropy function in PRESAGE® showed relative differences of up to 9% with the MCNPX calculated values. It was determined that post-irradiation optical density change over several days was non-linear in different dose regions, and therefore the dose values in the r=4 to r=5 cm regions had higher uncertainty due to this effect. This study demonstrated that within the radial distance of 3 cm, brachytherapy dosimetry in PRESAGE® can be accurate within 5% as long as irradiation times are within 48 hours.