Oligodeoxynucleotide synthesis using protecting groups and a linker cleavable under non-nucleophilic conditions

Oligodeoxynucleotides (ODNs) containing latent electrophilic groups can be highly useful in antisense drug development and many other applications such as chemical biology and medicine, where covalent cross-linking of ODNs with mRNA, protein and ODN is required. However, such ODN analogues cannot be synthesized using traditional technologies due to the strongly nucleophilic conditions used in traditional deprotection/cleavage process. To solve this long lasting and highly challenging problem in nucleic acid chemistry, I used the 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) function to protect the exo-amino groups on the nucleobases dA, dC and dG, and to design the linker between the nascent ODN and solid support. These protecting groups and linker are completely stable under all ODN synthesis conditions, but can be readily cleaved under non-nucleophilic and nearly neutral conditions. As a result, the new ODN synthesis technology is universally useful for the synthesis of electrophilic ODNs. The dissertation is mainly comprised of two portions. In the first portion, the development of the Dmoc-based linker for ODN synthesis will be described. The construction of the dT-Dmoc-linker required a total of seven steps to synthesize. The linker was then anchored to the solid support―controlled pore glass (CPG). In the second portion, the syntheses of Dmoc-protected phosphoramidites ODN synthesis monomers including Dmoc-dC-amidite, Dmoc-dA-amidite, Dmoc-dG-amidite are described. The protection of dC and dA with 1,3-dithian-2-yl-methyl 4-nitrophenyl carbonate proceeded smoothly giving Dmoc-dC and Dmoc-dA in good yields. However, when the same acylation procedure was applied for the synthesis of Dmoc-dG, very low yield was obtained. This problem was later solved using a highly innovative and environmentally benign procedure, which is expected to be widely useful for the acylation of the exo-amino groups on nucleoside bases. The reactions to convert the Dmoc-protected nucleosides to phosphoramidite monomers proceeded smoothly with high yields. Using the Dmoc phosphoramidite monomers dA, dC, dG and the commercially available dT, and the Dmoc linker, four ODN sequences were synthesized. In all cases, excellent coupling yields were obtained. ODN deprotection/cleavage was achieved by using non-nucleophilic oxidative conditions. The new technology is predicted to be universally useful for the synthesis of ODNs containing one or more electrophilic functionalities.

Genetic association studies under the population stratification, family pedigree and application to genome-wide association studies

This dissertation has three separate parts: the first part deals with the general pedigree association testing incorporating continuous covariates; the second part deals with the association tests under population stratification using the conditional likelihood tests; the third part deals with the genome-wide association studies based on the real rheumatoid arthritis (RA) disease data sets from Genetic Analysis Workshop 16 (GAW16) problem 1. Many statistical tests are developed to test the linkage and association using either case-control status or phenotype covariates for family data structure, separately. Those univariate analyses might not use all the information coming from the family members in practical studies. On the other hand, the human complex disease do not have a clear inheritance pattern, there might exist the gene interactions or act independently. In part I, the new proposed approach MPDT is focused on how to use both the case control information as well as the phenotype covariates. This approach can be applied to detect multiple marker effects. Based on the two existing popular statistics in family studies for case-control and quantitative traits respectively, the new approach could be used in the simple family structure data set as well as general pedigree structure. The combined statistics are calculated using the two statistics; A permutation procedure is applied for assessing the p-value with adjustment from the Bonferroni for the multiple markers. We use simulation studies to evaluate the type I error rates and the powers of the proposed approach. Our results show that the combined test using both case-control information and phenotype covariates not only has the correct type I error rates but also is more powerful than the other existing methods. For multiple marker interactions, our proposed method is also very powerful. Selective genotyping is an economical strategy in detecting and mapping quantitative trait loci in the genetic dissection of complex disease. When the samples arise from different ethnic groups or an admixture population, all the existing selective genotyping methods may result in spurious association due to different ancestry distributions. The problem can be more serious when the sample size is large, a general requirement to obtain sufficient power to detect modest genetic effects for most complex traits. In part II, I describe a useful strategy in selective genotyping while population stratification is present. Our procedure used a principal component based approach to eliminate any effect of population stratification. The paper evaluates the performance of our procedure using both simulated data from an early study data sets and also the HapMap data sets in a variety of population admixture models generated from empirical data. There are one binary trait and two continuous traits in the rheumatoid arthritis dataset of Problem 1 in the Genetic Analysis Workshop 16 (GAW16): RA status, AntiCCP and IgM. To allow multiple traits, we suggest a set of SNP-level F statistics by the concept of multiple-correlation to measure the genetic association between multiple trait values and SNP-specific genotypic scores and obtain their null distributions. Hereby, we perform 6 genome-wide association analyses using the novel one- and two-stage approaches which are based on single, double and triple traits. Incorporating all these 6 analyses, we successfully validate the SNPs which have been identified to be responsible for rheumatoid arthritis in the literature and detect more disease susceptibility SNPs for follow-up studies in the future. Except for chromosome 13 and 18, each of the others is found to harbour susceptible genetic regions for rheumatoid arthritis or related diseases, i.e., lupus erythematosus. This topic is discussed in part III.

WATER USE AND SYSTEM RELIABILITY UNDER DIESELGENERATOR AND SOLAR PHOTOVOLTAIC POWERED PUMPING SYSTEMS: A CASE STUDY OF SOLLA TOGO

Access to improved potable water sources is recognized as one of the key factors in improving health and alleviating global poverty. In recently years, substantial investments have been made internationally in potable water infrastructure projects, allowing 2.3 billion people to gain access to potable water from 1990-2012. One such project was planned and installed in Solla, Togo, a rural village in the northern part of the country, from 2010-2012. Ethnographic studies revealed that, while the community has access to potable water, an estimated 45% of the village’s 1500 residents still rely on unprotected sources for drinking and cooking. Additionally, inequality in system use based on income level was revealed, with the higher income groups accessing the system more regularly than lower income groups. Cost, as well as the availability of cheaper sources, was identified as the main deterrent from using the new water distribution system. A new water-pricing scheme is investigated here with the intention of making the system accessible to a greater percentage of the population. Since 2012, a village-level water committee has been responsible for operations and maintenance (O&M), fulfilling the community management model that is recommended by many development theorists in order to create sustainable projects. The water committee received post-construction support, mostly in the form of technical support during system breakdowns, from the Togolese Ministry of Water and Sanitation (MWSVH). While this support has been valuable in maintaining a functional water supply system in Solla, the water committee still has managerial challenges, particularly with billing and fee collection. As a result, the water committee has only received 2% - 25% of the fees owed at each private connection and public tap stand, making their finances vulnerable when future repairs and capital replacements are necessary. A new management structure is proposed by the MWSVH that will pay utilities workers a wage and will hire an accountant in order to improve the local management and increase revenue. This proposal is analyzed under the new water pricing schemes that are presented. Initially, the rural water supply system was powered by a diesel-generator, but in 2013, a solar photo-voltaic power supply was installed. The new system proved a fiscal improvement for the village water committee, since it drastically reduced their annual O&M costs. However, the new system pumps a smaller volume of water on a daily basis and did not meet the community’s water needs during the dry season of 2014. A hydraulic network model was developed to investigate the system’s reliability under diesel-generator (DGPS) and solar photovoltaic (PVPS) power supplies. Additionally, a new system layout is proposed for the PVPS that allows pumping directly into the distribution line, circumventing the high head associated with pumping solely to the storage tank. It was determined that this new layout would allow for a greater volume of water to be provided to the demand points over the course of a day, meeting a greater fraction of the demand than with the current layout.