Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. To investigate how mutant p53 impacts transcription, we studied how mutant p53 regulates vascular endothelial growth factor receptor 2 (VEGFR2), one of its strongest target genes that we identified through global gene expression profiling in mutant p53 expressing MDA 468 breast cancer cells. VEGFR2, the primary functional VEGF receptor and clinical target of bevacizumab, mediates endothelial cell neovascularization by promoting increased cellular proliferation, migration, and pro-survival signaling. In breast tumors, VEGFR2 is often aberrantly expressed on the breast tumor epithelia,which correlates with worse overall survival. We identify VEGFR2 as a mutant p53 transcriptional target in multiple breast cancer cell lines. Mutant p53 mediated upregulation of VEGFR2 mediates mutant 53 gain of function including increased cellular growth and migration. In humans, breast tumors with TP53 hotspot mutants have elevated VEGFR2 levels compared to tumors with loss of function mutations. The same class of tumors has significantly upregulated HIF1A and VEGFA compared to TP53 wild type tumors, indicating that mutant p53 containing breast tumors express a neoangiogenic gene signature that may intensify VEGFR2 autocrine signaling. A clinical trial suggests that TP53 mutated breast tumors may specifically respond to anti VEGF therapy, while TP53 wild type tumors may not respond. We suggest that mutant p53 containing breast tumors may be distinctively vulnerable to anti VEGF ntherapies. We investigated how mutant p53 impacts transcription of VEGFR2 using multiple techniques including scanning ChIP, micrococcal nuclease PCR, and in vivo DNase I footprinting by ligation mediated PCR. Mutant p53 was found to bind near the VEGFR2 transcriptional start site, causing the promoter to adopt a transcriptionally active conformation. Using SILAC mass spectrometry, we identified subunits of the SWI/SNF chromatin remodeling complex as mutant p53 interactors. Importantly, re ChIP and immunodepletion ChIP demonstrate that mutant p53 and SWI/SNF co-occupy the VEGFR2 promoter. Depletion of multiple SWI/SNF subunits reduced VEGFR2 RNA expression, and SWI/SNF is required for maximal mutant p53 promoter occupancy. Using RNA sequencing, we report that approximately half of all mutant p53 gene alteration impacts transcription of VEGFR2 as well as myriad other target genes by promoter remodeling through interaction with the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53 expressing tumors be uniquely susceptible to anti VEGF therapies, but restoration of SWI/SNF tumor suppressor function by targeting mutant p53 may have therapeutic potential. Mutant p53 interaction with the SWI/SNF complex may explain how mutant p53 modulates the expression of such a diverse set of genes.

HBV DNA replicates through a genomic RNA intermediate. The HBV reverse transcriptase lacks proof-reading activity, resulting in a much higher mutation rate for the HBV genome compared with other DNA viruses. HBV DNA thus is often present in quasi-species in an individual. One or more species may be favorably selected by factors like host immune clearance and use of antiviral drugs. / Hepatitis B virus (HBV) infected millions of people worldwide. Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). / In summary, this study developed and validated two platforms for (1) HBV mutation discovery; and (2) HBV mutation detection in viral quasi-species. These tools may be useful for research on HBV drug resistant mutations, clinical instructing and monitoring of antiviral treatment. / In this study, I have developed high-throughput methods for (1) discovery of novel HBV mutations; and (2) highly multiplexed detection of known HBV mutations, both in the background of HBV quasi-species. Patients undergoing long-term lamivudine treatment were used for mutation discovery. For mutation discovery in quasi-species, the MassCLEAVE(TM) technology, a method based on base-specific RNA cleavage and automated Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS), was used. I found that MassCLEAVE(TM) can be used to discover mutations present as minorities. Additionally, a synergistic effect was found between direct sequencing and MassCLEAVE(TM) in identifying minority mutations. Multi-PLEX, a method based on single nucleotide extension and automated MALDI-TOF MS, was used to develop a highly multiplexed assay for simultaneous detection of 60 HBV mutations including all functionally known HBV mutations and other frequently observed mutations during antiviral treatment with unknown functions. This multiplex assay was tested on a large cohort of single and multiple drug-resistant patients and was shown to be highly accurate in detecting HBV viral mutations in quasi-species. / Nucleotide and nucleoside analogues (NAs) are widely used for antiviral therapy by effectively suppressing viral DNA replication. However, long-term administration may select for drug-resistant mutant strains, leading to treatment failure and liver disease progression. A number of HBV mutations such as rtM204V/I, rtN236T and rtL180M within the HBV reverse transcriptase are known to confer drug resistance. Detection of these known mutations is useful genotypic markers for monitoring antiviral treatment. In addition, novel drug resistant mutations continue to be discovered. / by Luan, Ju. / Adviser: Chunming Ding. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

by Kwok Fung, Lo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 89-97). / Acknowledgement --- p.i / Table of Content --- p.ii / Abstract --- p.vi / List of Abbreviation --- p.viii / List of Tables and figures --- p.ix / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The immunoglobulin --- p.1 / Chapter 1.1.1 --- Immunoglobulin structure --- p.1 / Chapter 1.1.2 --- Immunogloblulin genes --- p.1 / Chapter 1.1.3 --- Immunogloblulin gene recombination --- p.3 / Chapter 1.1.4 --- Antibody diversity --- p.4 / Chapter 1.1.4.1 --- Imprecise joining --- p.5 / Chapter 1.1.4.2 --- N region addition --- p.5 / Chapter 1.1.4.3 --- Somatic mutation --- p.6 / Chapter 1.2 --- Hypermutation --- p.6 / Chapter 1.2.1 --- Features of hypermutation --- p.6 / Chapter 1.2.2 --- Germinal centre & Affinity maturation --- p.8 / Chapter 1.2.3 --- Mutational Hotspots --- p.9 / Chapter 1.2.4 --- Intrinsic characteristics of hypermutation --- p.10 / Chapter 1.2.5 --- Models for the mechanism of hypermutation --- p.11 / Chapter 1.2.5.1 --- DNA replication --- p.11 / Chapter 1.2.5.2 --- DNA repair --- p.12 / Chapter 1.2.5.3 --- Gene conversion --- p.13 / Chapter 1.2.5.4 --- Transcription --- p.15 / Chapter 1.2.5.5 --- Homologous recombination of reverse transcribed mRNA --- p.16 / Chapter 1.2.5.6 --- Transcription-coupled repair --- p.17 / Chapter 1.3 --- Scope of investigation --- p.17 / Chapter Chapter 2 --- Material and Method --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.2 --- Methods (first generation of LMPCR) --- p.21 / Chapter 2.2.1 --- Animal and cell lines --- p.21 / Chapter 2.2.2 --- Oxazolone antigen immunization --- p.21 / Chapter 2.2.2.1 --- Preparation of Bordetella pertussis --- p.21 / Chapter 2.2.2.2 --- Coupling of phenyloxazolone with CSA or BSA --- p.22 / Chapter 2.2.2.3 --- Preparation of aluminium hydroxide adjuvant --- p.23 / Chapter 2.2.2.4 --- Mice immunization --- p.23 / Chapter 2.2.3 --- Detection of anti-phOx antibody by enzyme-linked immunosorbent assay (ELISA) --- p.24 / Chapter 2.2.3.1 --- Reagents --- p.24 / Chapter 2.2.3.2 --- Assay procedure --- p.24 / Chapter 2.2.4 --- Extraction of genomic DNA (mice/cell line) --- p.25 / Chapter 2.2.4.1 --- Reagents --- p.25 / Chapter 2.2.4.2 --- Isolation of DNA from cell line (NQ2.12.4 & NQ5.4.3) --- p.25 / Chapter 2.2.4.3. --- DNA extraction from mice --- p.26 / Chapter 2.2.5 --- Ligation-mediated polymerase chain reaction (LMPCR) --- p.26 / Chapter 2.2.5.1 --- Procedure --- p.26 / Chapter 2.2.5.1.1 --- First primer extension --- p.29 / Chapter 2.2.5.1.2 --- Ligation --- p.29 / Chapter 2.2.5.1.3 --- PCR amplification --- p.30 / Chapter 2.2.5.1.4 --- Labelling of LMPCR product --- p.30 / Chapter 2.2.6 --- Marker preparation --- p.31 / Chapter 2.2.7 --- Polyacrylamide gel electrophoresis --- p.32 / Chapter 2.2.7.1 --- Reagents --- p.32 / Chapter 2.2.7.2 --- Procedure --- p.32 / Chapter 2.2.8 --- Southern blot hybridization --- p.33 / Chapter 2.2.8.1 --- Reagents --- p.33 / Chapter 2.2.8.2 --- DNA blotting --- p.34 / Chapter 2.2.8.3 --- Preparation of 32P labelling DNA probe --- p.34 / Chapter 2.2.8.4 --- Prehybridization and Hybridization --- p.35 / Chapter 2.2.9 --- Simplified protocol for the first generation of LMPCR --- p.36 / Chapter 2.3 --- Method (second generation of LMPCR) --- p.37 / Chapter 2.3.1 --- Excess linker removal --- p.37 / Chapter 2.3.1.1 --- Exonuclease III Treatment --- p.37 / Chapter 2.3.1.2 --- Mung bean nuclease Treatment --- p.37 / Chapter 2.3.1.3 --- Chroma Spin Treatment --- p.37 / Chapter 2.3.2 --- HindIII digestion after LMPCR --- p.38 / Chapter 2.3.3 --- DNA sequencing --- p.38 / Chapter 2.3.3.1 --- Cloning of amplified sequences to M13mpl9 plasmid --- p.38 / Chapter 2.3.3.2 --- Plaque hybridization --- p.39 / Chapter 2.3.3.3 --- Preparation of single-stranded templates --- p.39 / Chapter 2.3.3.4 --- Sanger dideoxy sequencing of single-stranded DNA --- p.40 / Chapter 2.3.4 --- Simplified protocol for second generation of LMPCR --- p.41 / Chapter Chapter 3 --- First generation of LMPCR --- p.42 / Chapter 3.1 --- General design --- p.42 / Chapter 3.1.1 --- LMPCR protocol and its modification --- p.42 / Chapter 3.1.2 --- Oligonucleotide design --- p.44 / Chapter 3.1.3 --- Experimental design --- p.48 / Chapter 3.2 --- Result --- p.50 / Chapter 3.2.1 --- Anti-phOx Ig level in normal and immunized mice --- p.50 / Chapter 3.2.2 --- LMPCR analysis of the sense strand of VkOxl --- p.50 / Chapter 3.2.2.1 --- Overall patterns of the LMPCR signals --- p.50 / Chapter 3.2.2.2 --- Southern hybridization --- p.50 / Chapter 3.2.2.3 --- Distribution of signals --- p.57 / Chapter 3.2.2.4 --- LMPCR analysis of the VkOxl-Jk5 anti-phOx transgene --- p.61 / Chapter 3.2.2.5 --- Effect of the number of cells carrying the VkOxl-Jk5 gene on LMPCR --- p.61 / Chapter 3.2.3 --- LMPCR analysis of the antisense strand of VkOxl --- p.64 / Chapter 3.3 --- Discussion --- p.64 / Chapter Chapter 4 --- Second generation of LMPCR --- p.72 / Chapter 4.1 --- Introduction(experi mental modification) --- p.72 / Chapter 4.1.1 --- Tagging the specific LMPCR products by addition of a Hin dIII site in the linker --- p.72 / Chapter 4.1.2 --- "Removal of excess linker, OXUH" --- p.72 / Chapter 4.1.2.1 --- Exonuclease III treatment --- p.73 / Chapter 4.1.2.2 --- Chroma spin treatment --- p.73 / Chapter 4.1.2.3 --- Mung Bean Nuclease treatment --- p.75 / Chapter 4.1.3 --- Other modifications in LMPCR --- p.75 / Chapter 4.2 --- Results --- p.75 / Chapter 4.2.1 --- Effect of including Exonuclease III treatment --- p.75 / Chapter 4.2.2 --- Effect of including Mung Bean Nuclease treatment --- p.76 / Chapter 4.2.3 --- Effect of including Chroma spin treatment --- p.76 / Chapter 4.2.4 --- Strand break positions detected at the sense strand --- p.76 / Chapter 4.2.5 --- DNA sequence analysis of the antisense strand LMPCR products --- p.82 / Chapter 4.3 --- Discussion --- p.84 / References --- p.89 / Appendix I --- p.98

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disease in which motor neurons selectively degenerate, leading to paralysis and death. Rare causal mutations in FUS and TARDBP implicated RNA binding proteins and RNA metabolism in ALS disease mechanisms. The absence of faithful animal models has impeded precise understanding of the impact of ALS mutations on all functions of ALS-associated proteins. In my graduate studies, I used a novel, animal model of FUS-ALS to explore gain of function disease mechanisms and observed specific, aberrant interactions between mutant FUS and other RNA binding proteins including hnRNP U. Genetic experiments indicate loss of hnRNP U is toxic to motor neurons, suggesting mutant FUS toxicity may result from hnRNP U sequestration and loss of function. In a parallel series of experiments, I also generated novel knock-in mouse models of ALS expressing pathogenic TARDBP mutations to address the flaws of existing model systems and to study the functional consequences of disease-related mutations. We demonstrate that the ALS mutant alleles TDP-43M337V and TDP-43G298S are fully functional and are insufficient to cause age-dependent motor neuron pathology, indicating that physiological levels of mutant TDP-43 are alone insufficient to initiate disease. This model enables future exploration of the interaction between genetic and environmental factors that lead to TDP-43 toxicity in ALS and related disorders. Collectively, our findings suggest a gain of function mechanism of toxicity in which mutations and aging, with other factors, alter the local concentration of RNA binding proteins, leading motor neurons to degenerate.

Mutation of repetitive DNA by repeat-induced point mutation (RIP) is a process that occurs in many filamentous fungi of the Ascomycota during the sexual cycle. Concurrently, direct DNA repeats are often deleted by homologous recombination at high frequency during the sexual cycle. Thus, the processes of RIP and deletion compete to either mutate or remove repetitive DNA from the genome of filamentous fungi during sexual cycles. Both processes contribute to genome streamlining by controlling proliferation of transposable elements and by limiting expansion of gene families. While the genetic requirements for deletion by homologous recombination are well known, the mechanism behind the specific detection and mutation of repetitive DNA by RIP has yet to be elucidated as only a single gene essential for RIP, rid, has been identified. We have developed Fusarium graminearum as a model organism for the study of RIP by showing that it mutates repetitive DNA frequently during the sexual cycle and that the mutations due to RIP are dependent on rid. Further, we have sequenced a genetic mapping strain of F. graminearum (00-676-2) and identified 62,310 single nucleotide polymorphisms (SNPs) compared to the reference strain (PH-1). The SNP map will be useful for quickly mapping new mutants by bulk segregant analysis and high-throughput sequencing for which bioinformatic tools were specifically developed. The groundwork has thus been laid for identification of novel RIP mutants in F. graminearum, which being homothallic has a major advantage for identification of recessive mutations. We used a forward genetics approach to shed light on the mechanism of RIP in Neurospora crassa. Two rrr mutants that dominantly r��educe R��IP and r��ecombination were characterized and identified as different mutated alleles of the same gene, rrr-1[superscript L496P] and rrr-1[superscript G325N] by bulk segregant analysis and high-throughput sequencing. Bioinformatic characterization suggests RRR-1 belongs to a previously uncharacterized group of dynamin-like proteins, which are generally involved in membrane fission and fusion. RRR-1-GFP localizes to the nuclear membrane, but not DNA, suggesting it affects RIP and recombination frequency indirectly by altering nuclear membrane dynamics during sexual development and thereby altering temporal aspects of RIP and recombination. We used a reverse genetics approach to determine whether high frequency RIP and homologous recombination of repetitive DNA during the sexual cycle are linked mechanistically or spatio-temporally. We tested strains where genes important for deletion by homologous recombination were knocked out and found all to be completely RIP competent except mre11, which, while sterile in homozygous deletion crosses, displayed lower RIP frequency in heterozygous crosses. This suggests that mre11 has roles in homologous recombination as well as non-homologous end joining may be important for RIP. Collectively, this work developed methods for efficiently mapping mutations and identified a novel protein that reduces RIP and recombination frequency but did not identify any mechanistic link between the two processes. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Dec. 10, 2012 - Dec. 10, 2014

The primary goals of our research are to understand the virology and enzymology of human T-cell leukemia virus type I (HTLV-I) that will lead to the development of treatments for patients infected with HTLV-I. HTLV-I is an oncogenic virus of the Retroviridae family and is the causative agent of adult T-cell leukemia/lymphoma (ATL), tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). HTLV-I has been classified as a dangerous emerging pathogen by the Centers for Disease Control and Prevention with at least 20 million people infected with the virus. This is a significant problem because there are currently no effective treatments to control HTLV-I infection and prevent or treat HTLV-I induced ATL and TSP/HAM. The protease is necessary for retroviral maturation and replication and is, therefore, an attractive target for inhibitor design. Investigation of peptide mimetic compounds incorporating the tetrahedral intermediate of aspartyl protease catalyzed cleavage are crucial for the development of lead inhibitors. Compounds containing statine, 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), or hydroxyethylamine (HEA) are presented in this work. The best compound was a statine-based inhibitor, which had a Ki = 29 +/- 4 nM and 88% inhibition against an HTLV-I protease native substrate in a FRET assay.

Thesis (M.S.)--University of North Carolina Wilmington, 2009. / Title from PDF title page (February 17, 2010) Includes bibliographical references (50-54)

published_or_final_version / Pathology / Master / Master of Philosophy

Apolipoprotein E (ApoE) is a plasma protein that plays a prominent role in lipid metabolism and cholesterol transport. The gene is polymorphic: three alleles, ε2, ε3, and ε4 code for three different protein isoforms, E2, E3, and E4 respectively, each of which has different genetic implications. Carriers of ε2 and ε4 alleles have shown greater susceptibility to diseases such as lipid metabolism problems, cardiovascular disease, and Alzheimer’s disease. Although ε4 is the ancestral form of the gene, the most common allele in the human population currently is the ε3 allele. The three isoforms of ApoE generally occur in all populations at different frequencies. However, the frequencies of the various alleles have not been examined in the Midwest. The purpose of this project was to determine if a correlation existed between frequencies of ApoE mutation and heritage in Midwest individuals by using real-time PCR. It was hypothesized that this method could be an alternative and cost effective method to sequence an individual’s genome for personalized healthcare purposes. In 2000, Dr. Vann and her research assistants collected saliva samples from approximately 300 anonymous volunteers participating in the UniverCity fair hosted by Ball State University. The samples were catalogued in an Excel database with their corresponding information (gender, self- reported lineage). From this database, 50 samples were randomly selected for this study and the DNA was screened by real-time PCR for isoforms of ApoE. A handful of individuals with altered alleles were identified. The specific number of each isoform and the genotype of each individual were determined. Only one of the 50 individuals resulted in a non-wild type haplotype. A confirmatory melt curve analysis of this major heritage group A individual resulted in a homozygous E4 genotype. Unfortunately, a correlation between frequencies of ApoE mutation and heritage in Midwest individuals could not be inferred based on one differing individual. Thus, we did not have sufficient non-wild type haplotypes to permit us to amplify the variable regions of maternally inherited mitochondrial DNA for sequencing. Those sequences could have been aligned with the Cambridge Reference Sequence (Mitomap 2006) to determine maternal lineage or haplotype, which could then be correlated with self-reported lineage, and the presence of specific isoforms of ApoE. / Department of Biology

Chromosomal rearrangements play a major role in the evolution of eukaryotic genomes. Genomic aberrations are also a hallmark of many tumors and are associated with a number of hereditary diseases in humans. The presence of repetitive sequences that can adopt non-canonical DNA structures is one of the factors which can predispose chromosomal regions where they reside to instability. Palindromic sequences (inverted repeats with or without a unique sequence between them) that can adopt hairpin or cruciform structures are frequently found in regions that are prone for gross chromosomal rearrangements (GCRs) in somatic and germ cells in different organisms. Direct physical evidence was obtained that double-strand breaks (DSBs) occur at the location of long inverted repeats, a triggering event for the genomic instability. However, the mechanisms by which palindromic sequences lead to chromosomal fragility are largely unknown. The overall goal of this research is to elucidate the mechanisms of DSB and GCR generation by palindromic sequences in yeast, Saccharomyces cerevisiae.