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Propionic acidemia is an autosomal recessively inherited disorder of organic acid metabolism caused by deficient activity of propionyl CoA carboxylase. This enzyme is required for the catabolism of the odd-chain fatty acids, the side chain of cholesterol and the amino acids: isoleucine, methionine, and threonine. Although the clinical expression of this disorder is variable, affected children usually develop ketoacidosis and hyperammonemia which may progress to seizures, coma and possibly death. Some patients may exhibit vomiting, lethargy and hypotonia in the first few weeks of life whereas others may remain asymptomatic for months or even years.(1) The degree of residual enzyme activity in the tissues from these patients does not correlate to this apparent clinical heterogeneity. The relationship between the clinical variation observed among propionyl CoA carboxylase deficient patients and the corresponding genetic and biochemical make up, is not yet understood. The aim of this project is to study the biochemical and immunological properties of propionyl CoA carboxylase in liver homogenates and fibroblast samples from several patients with propionic acidemia, to gain a better understanding of the nature of the defect in this disorder. Previous comparisons of propionyl CoA carboxylase from fibroblasts of patients with this enzyme deficiency and unaffected individuals have suggested that propionyl CoA carboxylase is structurally altered in the patients with propionic acidemia. The fibroblast lines from these patients can be categorized into two major genetic complementation groups, pccA and pccBC based on the increase in activity observed in heterokaryons formed by pairwise cell fusions.(2) In addition, the defective propionyl CoA carboxylase can be differentiated biochemically from the normal enzyme and from each compIementation group.(3) These differences suggest that the aIterations in propionyl CoA carboxylase structure in each complementation group represent mutations in different subunits. In addition, mutations within a single gene resulting slightly different enzyme structures, would explain the clinical variation within a complementation group. Therefore, biochemical differences among mutant propionyl CoA carboxylases from the same c0mp1ementation group were investigated. To provide further evidence that propionic acidemia is the result of structural alterations in propionyl CoA carboxylase, immunologic techniques were used to determine if equal quantities of cross-reacting material (CRM) were present in liver and fibrobiast homogenates from propionyl CoA carboxyIase deficient patients from the various genetic complementation groups. Antiserum prepared against purified pig heart propionyl CoA carboxylase which cross-reacts with human propionyI CoA carboxyIase and another antiserum prepared against the human biotin-containing enzymes, were compared using immunotitration techniques. These tests demonstrated that there are equal quantities of cross-reacting material in the tissue homogenates of propionic acidemia patients.(4, 5) Investigations were undertaken to biochemically characterize and evaluate the heterogeneity within the pccBC genetic complementation group. This group was chosen because previous complementation and biochemical studies with fibroblasts from the pccBC subgroups have suggested the existence of interallelic complementation in this group. (6) Specific biochemical differences among propionyl CoA carboxylases from cells belonging to patients in this group could identify heterogeneity and characterize the complementation pattern. Normal and mutant propionyl CoA carboxylase from the pccBC complementation group were highly purified and their biochemical properties were compared using their isoelectric paint, thermostabiilty, and enzyme affinity for substrates. The properties of the purified enzymes were then compared with propionyl CoA carboxylase from other mutants in this group. These comparisons demonstrated biochemical heterogeneity within the pccBC complementation group.(7) The results provide compelling evidence that the defect in propionic acidemia represents a structural alteration of propionyl CoA carboxylase in the pccA and pccBC complementation groups. In addition, the biochemical heterogeneity demonstrated within the pccBC complementation group suggests that several different structural mutations, possibly of the same subunit, are involved that result in slightly different biochemical parameters for each mutant enzyme. These structural alterations may explain the complicated complementation map for the pccBC subgroups. Since interallelic complementation is based on the heteropolymer being slightly more active than the homopolymer, different structural alterations in the mutants may interfere with their ability to participate in interallelic complementation. Furthermore, biochemical differences within the group may be reflected in the clinical phenotype of the disease and may be an indicator of the clinical variation which has been observed in these patients.

Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Master of Sciences in the School of Informatics, Indiana University, August 2004 / Osteoporosis is the most common bone disease in United States and developed countries and a major public health threat for an estimated 44 million Americans. It is characterized by low bone mineral density and micro architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture, especially of hip, spine and wrist. Osteoporosis is multifactorial disease influenced by large number of environmental and genetic factors. Though a number of FDA approved drugs are available for treating this complex disease, a medication, which could specifically and effectively reverse symptoms of it is lackin. As the initial step for approaching disease treatment my current research focuses on locatin candidate genes on linkage regions for BMD on human chromosomes, which potentially can be used for developing novel targets and strategies for therapeutic interventions. We will also define the mouse homologs in the syntenic regions as basis for future studies involving animal models of disturbed BMD. An automated interface which will give information on human - mouse synteny between human marker intervals of interest was developed which will expedite future synteny studies.

Inflammatory autoimmune diseases like Crohn's Disease (CD) and Rheumatoid Arthritis (RA) share some of the same single nucleotide polymorphisms (SNPs) in protein tyrosine phosphatase non-receptor types 2 and 22 (PTPN2/22), which contribute to their pathogenesis. In clinical CD and RA samples, PTPN2:rs478582 and PTPN22:rs2476601 were found to exacerbate a number of inflammatory processes associated with CD and RA. To confirm the role of these SNPs in CD and RA pathogenesis, CRISPR-Cas9 was used to induce the SNPs in T-cells. Cells were also treated with the naturally occurring polyamine, spermidine, to restore PTPN2/22 function and reverse the inflammatory effects of the SNPs. The PTPN2 SNP decreased PTPN2 expression by 3.2-fold and the PTPN22 SNP decreased PTPN22 expression by 2.4-fold compared to WT T-cells. Proliferation increased by 10.2-fold in PTPN2 SNP cells and 8.4-fold in the PTPN22 SNP cells compared to WT cells. Both SNPs increased secretion levels IFN-γ and TNF-α. Additionally, 80.32% of PTPN2 SNP cells and 85.82% of PTPN22 SNP cells were activated compared to 70.48% of WT T-cells. Spermidine treatment increased PTPN2/22 expression in all cell types and was dose-dependent in cells with either SNP. Proliferation levels decreased by approximately 5-fold in spermidine treated PTPN2 SNP cells and 3.1-fold in PTPN22 SNP cells treated with 10 µM of spermidine and 12.0-fold when treated with 20 uM. IFN-γ and TNF-α secretion levels decreased with spermidine treatment in all cell groups. Lastly, T-cell activation decreased to 51.39% of PTPN2 SNP cells and 46.36% of PTPN22 SNP cells when treated with 10 µM of spermidine. These findings may explain the therapeutic response seen in CD and RA patients prescribed polyamines. This study confirms the inflammatory role of the PTPN2/22 SNPs and displays the anti-inflammatory and restorative property of spermidine for its potential therapeutic use in CD and RA patients with genetic polymorphisms.

16S ribosomal RNA (rRNA) gene sequences are commonly analyzed for taxonomic and phylogenetic purposes because they contain variable regions that help distinguish genera. However, intra-genus classification is difficult due to high sequence similarity among closely related species. The biological impact of nucleotide variants in 16S variable regions are often unknown and hence their sequence differences are weighted evenly during classification, which provides poor species identity confidence. In this dissertation, I determined that analysis of intra-genus 16S allelic variants can provide species information and that nucleotide changes in 16S rRNA variable regions can impact ribosome quality. In one study, I analyzed ribosomal gene sequences, including 16S variable regions, to identify microbes that can spoil different retail draft beers. Based on relative sequence abundance changes of variable region sequences, I determined that certain bacteria preferred growth on draft lines rather than beers. Sequences of certain species were consistently detected at ratios indicative of their 16S gene copies, suggesting they came from specific strains. In a second study, I computationally interrogated 16S variable sequences in closely related genera Escherichia and Shigella and discovered that certain species could be differentiated. I demonstrated that Escherichia coli ribosomes were compromised when they carried 16S rRNA with these species-informative nucleotides, suggesting that variable region nucleotides may be constrained to respective species. In a third study, metagenomic sequencing was used to identify organisms that resided on cables submerged off the coast of Florida. Relative abundances of DNA for putative polymer-degrading organisms reduced over time and DNA for putative polymer-degrading enzymes were present at low relative abundance. Altogether, this dissertation shows the capabilities of DNA-based microbial identification and suggests that acknowledgment 16S alleles can improve intra-genus bacterial classification.

Glioblastoma Multiforme (GBM) is the most lethal subtype of glioma (brain tumor), with a 5-year survival rate of merely 5.6% post diagnosis1. The traditional study of glioblastoma has investigated the role of multiple genes in advancing its progression, including the upregulation of Epidermal Growth Factor Receptor (EGFR). However, there is at yet little research into the epigenetic factors that control EGFR, both in the precursor astrocytes and in glioblastomas themselves2. EGFR and its regulation may play a significant role in the progression and development of GBM from astrocytes. Through modification of genomic pathways as observed in GBM, our lab generated an in vitro glioblastoma model that is representative of the pathways modulated in glioblastoma. The purpose of this study was to investigate the modulation of epigenetic factors that occurs upon conversion from astrocyte to glioblastoma-like cells using epigenetic and expression analysis methods, in hopes of revealing potential therapeutic venues for future study. The study revealed that activating marks present in astrocytes were in fact downregulated in the glioblastoma cells leading to decreased expression of wild-type EGFR mRNA. Further exploration may provide more clues as to why this modulation occurs.

Virulent Trypanosoma cruzi, and the non-pathogenic Trypanosoma conorhini and Trypanosoma rangeli are protozoan parasites with divergent lifestyles. T. cruzi and T. rangeli are endemic to Latin America, whereas T. conorhini is tropicopolitan. Reduviid bug vectors spread these parasites to mammalian hosts, within which T. rangeli and T. conorhini replicate extracellularly, while T. cruzi has intracellular stages. Firstly, this work compares the genomes of these parasites to understand their differing phenotypes. Secondly, genome architecture of T. cruzi is examined to address the effect of a complex hybridization history, polycistronic transcription, and genome plasticity on this organism, and study its highly repetitive nature and cryptic genome organization. Whole genome sequencing, assembly and comparison, as well as chromosome-scale genome mapping were employed. This study presents the first comprehensive whole-genome maps of Trypanosoma, and the first T. conorhini strain ever sequenced. Original contributions vii to knowledge include the ~21-25 Mbp assembled genomes of the less virulent T. cruzi G, T. rangeli AM80, and T. conorhini 025E, containing ~10,000 to 13,000 genes, and the ~36 Mbp genome assembly of highly virulent T. cruzi CL with ~24,000 genes. The T. cruzi strains exhibited ~74% identity to proteins of T. rangeli or T. conorhini. T. rangeli and T. conorhini displayed greater complex carbohydrate metabolic capabilities, and contained fewer retrotransposons and multigene family copies, e.g. mucins, DGF-1, and MASP, compared to T. cruzi. Although all four genomes appear highly syntenic, T. rangeli and T. conorhini exhibited greater karyotype conservation. T. cruzi genome architecture studies revealed 66 maps varying from 0.13 to 2.4 Mbp. At least 2.6% of the genome comprises highly repetitive repeat regions, and 7.4% exhibits repetitive regions barren of labels. The 66 putative chromosomes identified are likely diploid. However, 20 of these maps contained regions of up to 1.25 Mbp of homology to at least one other map, suggestive of widespread segmental duplication or an ancient hybridization event that resulted in a genome with significant redundancy. Assembled genomes of these parasites closely reflect their phylogenetic relationships and give a greater context for understanding their divergent lifestyles. Genome mapping provides insight on the genomic evolution of these parasites.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / We have constructed a database of hydroxyl radical (•OH) cleavage patterns of DNA in order to investigate the relationship between the sequence of a DNA molecule and its three-dimensional structure. The hydroxyl radical cuts DNA at every nucleotide, with the amount of cutting proportional to the solvent accessible surface area (SASA) of the deoxyribose hydrogen atoms. Cleavage fragments are quantified by a fluorescence sequencer, followed by normalization and deposition into the database. Our database currently contains 151 DNA sequences with lengths ranging from 35 to 41 nucleotides. These data have enabled us to develop some general rules regarding the sequence-dependence of DNA structure as well as to predict the cleavage pattern of any given DNA sequence with remarkable precision. Using this prediction algorithm, it is possible to construct structural maps of entire genomes. As there are many examples of DNA binding proteins with highly degenerate binding sites, the use of structural information to locate these sites may be helpful. There also exists other signals, including the signal for nucleosome positioning, which have no apparent consensus, making it likely that the structure of DNA is of critical importance. We have developed algorithms to identify regions of conserved structure using •OH cleavage intensity as a proxy. Within a set of DNase I hypersensitive sites (DHS) obtained from the ENCODE Consortium, we were able to identify a stretch of 12 nucleotides for which the structural conservation is much greater than the sequence conservation. These sites have been dubbed Conserved •OH Radical Cleavage Signatures, or CORCS. Upon further analysis, these CORCS were found to be 17-fold enriched for DHS as compared to shuffled elements. Through the continued analysis of hydroxyl radical cleavage data and development of algorithms to employ the data in biologically meaningful ways, we hope to further our understanding of the relationship between DNA sequence and structure, and how the local structural heterogeneity of genomic DNA contributes to biological function. / 2031-01-02

<p>Maintaining high genetic diversity within and among wildlife populations is an important component to the management of threatened species. Population genomics utilizes recent advancements in high-throughput next-generation sequencing to obtain genome-wide data that can yield deeper perspectives on intraspecific genetic variation and elucidate evolutionary significant units that may require conservation management or augmentation. The semi-aquatic Blanding’s Turtle (<i>Emydoidea blandingii</i>) has experienced drastic population declines in North America due in large part to anthropogenic activities. This species is listed as threatened or endangered across most of its range. A population genomic study can help to understand the status of this species and guide future management practices. Hence, a population genomic analysis was conducted using 3RAD to discover and analyze SNPs across the range using samples from Nebraska, Indiana, Michigan, Ohio, and Nova Scotia,. Range-wide analysis used 8,602 SNPs while analysis within the Great Lakes region used 7,893 SNPs. High amounts of missing data were found across all individuals and loci. Low levels of genetic variation relative to other turtle species were detected both across the range and within the Great Lakes region. Minimal population structure was detected range-wide via clustering and admixture analyses; however, a signal of population differentiation was detected among Nebraska, Nova Scotia, and the Great Lakes. Clustering and differentiation analyses focused on the Great Lakes region found a signal of population structure and differences between the Lake Michigan and Lake Erie watershed. These results may prove useful for conservation management of Blanding’s Turtle populations, particularly related to efforts using translocation or head-starting practices.</p>

Thesis (Ph. D.)--Pennsylvania State University, 1994. / Includes bibliographical references.