Interpreting DNA profiles manually can potentially call into question subjectivity between analysts who may interpret specific results differently. There are multiple features of a DNA profile that can complicate interpretation, which include allelic dropout and drop-in, allele sharing, and polymerase chain reaction (PCR) artifacts, which can all confound manual interpretation of DNA profiles. Difficulties in interpretation of DNA profile evidence can also be caused by degradation of the DNA itself, which can be caused by various environmental factors. Over the last 15 years, developments in DNA profile interpretation using probabilistic genotyping software (PGS) have been made in order to assist in the complicated task of interpreting and deconvoluting a challenging mixture. Among these PGSs is DNA-View® Mixture Solution™, a continuous-model program that is based on stochastic modeling. In this research, Mixture Solution was used to provide statistical analyses on DNA mixtures that were subject to various levels of degradation, through the assignment of a likelihood ratio (LR) to the mixture profile. The LR would either support the hypothesis that the person of interest (POI) contributed to the mixture, or support the contrary hypothesis, that the POI was not one of the contributors. Mixtures were prepared at four different contributor ratios with varying combinations of three levels of degradation: no degradation, partial degradation, and full degradation, using controlled heating to systematically degrade the DNA template prior to amplification. Using two hypothesis tests, Mixture Solution was used to compute LRs for each of the mixtures with a variety of defined POIs. Results showed that Mixture Solution successfully generated appropriate LRs for all 20 mixtures in this research, with no Type I errors that falsely excluded a known contributor from a mixture via an LR less than one. Even with the greatest level of degradation and at the most disproportionate ratio of contributors, Mixture Solution was able to assign an LR to each contributor that confirmed their presence in the mixture. When the DNA of a POI was subjected to degradation, decreases in the LR values were observed when compared to the values computed for undegraded DNA from the same POI. However, in all mixtures, Mixture Solution was able to assign an LR with “moderate support” or higher to each of the POIs in both tests, regardless of the level of degradation.

Ribozymes are small RNA molecules that cleave specific RNA molecules enzymatically. Ribozymes can be designed to target specific RNA molecules, thereby modulating gene expression. A hammerhead ribozyme was designed to cleave inhibin $\alpha$ subunit mRNA. This ribozyme was synthesized as double-stranded oligodeoxynucleotides and cloned into plasmid pGEM-4Z. In vitro transcripts of this cloned ribozyme and a cloned rat inhibin $\alpha$ subunit cDNA were incubated together both at 50$\sp\circ$C and at 37$\sp\circ$C. Northern blot analysis revealed two extra bands of the expected size representing the cleavage products. The cleavage was specific. No cleavage was observed when in vitro antisense transcripts of rat inhibin $\alpha$ subunit cDNA were incubated with the ribozyme. Since our goal is to down-regulate inhibin production using ribozyme, a DNA construct carrying the ribozyme was constructed to generate transgenic mice. The ribozyme was connected to the 3$\sp\prime$ end of the E. coli lacZ gene, and was placed under the control of the H-2K$\sp{\rm b}$ gene promoter. Two transgenic mouse lines were established from one transgenic founder, because the injected DNA integrated at two sites, which segregated independently from each other. In one line lacZ expression was detected in tissues including the cornea, the snout, the tongue, the palm of paws, the ventral midline, the pituitary, the dorsum near the spinal cord, on day 13 embryos. Northern blot analysis indicated that ribozyme expression was detected in adult spleen. These results demonstrated that (1) this engineered ribozyme cleaved inhibin $\alpha$ subunit transcripts in vitro, suggesting that there were no secondary structures which interfere with the cleavage activity of the ribozyme; (2) two transgenic mouse lines carrying the ribozyme were established, and expression of the ribozyme was determined.

A protein’s intracellular location is an integral part of its function. Mislocalization caused by mutations within a protein can lead to protein dysfunction and leave an individual susceptible to diseases such as type II diabetes, certain cancers, and Resistance to Thyroid Hormone Syndrome (RTH). Such is the case for the thyroid hormone receptor α1 (TRα1), as certain mutations in TRα1’s ligand binding domain can cause RTH. TRα1 binds to thyroid hormone (T3) and acts as a transcription factor in the nucleus. As such, maintenance of TRα1’s nucleocytoplasmic shuttling and intranuclear dynamics is essential to ensure proper protein function. Prior studies have demonstrated that certain RTH TRα1 mutants have an increased affinity to one of TRα1’s coregulators, nuclear corepressor 1 (NCoR1), and form a more stable complex compared to wild type TRα1. This finding suggests that the intranuclear mobility of RTH TRα1 mutants would decrease relative to their wild-type counterpart, and that nucleocytoplasmic shuttling would be impacted due to having one of several nuclear export motifs (NES-H12) compromised. The major aim of this thesis was to evaluate the intranuclear dynamics of RTH TRα1 mutants E403X, Ala382ProfsX7, and F397fs406X in response to increased levels of NCoR1. The intranuclear dynamics of each mutant RTH TRα1 was evaluated in human cells using fluorescence recovery after photobleaching (FRAP), and statistically analyzed for a significant difference compared to wild type TRα1. A mutant NCoR1 that lacks TR interaction domains, NCoRΔID, was used to control for wild type NCoR1’s increased binding affinity to RTH TRα1 mutants. While overexpressing NCoR1 did not significantly impact RTH TRα1 mutant intranuclear mobility, NCoRΔID overexpression significantly decreased RTH-F397fs406X intranuclear mobility, with RTH-E403X showing a similar trend. Interestingly, RTH-A383PfsX7 did not appear to be affected by NCoRΔID. As a second measure of intracellular dynamics, the nucleocytoplasmic shuttling ability of RTH-A383PfsX7 and wild type TRα1 was evaluated using heterokaryon assays. Despite having a compromised or missing NES-H12, RTH-A383PfsX7 maintained shuttling capabilities qualitatively similar to those of wild type TRα1. These findings offer new insight into RTH TRα1’s molecular phenotype and warrant further investigation into the interaction between NCoR1/ NCoRΔID with TRα1 and RTH-E403X, Ala382ProfsX7, and F397fs406X, as well as evaluation of RTH-E403Xand F397fs406X’s nucleocytoplasmic shuttling.

Note: faint and missing pages

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.