MULTIPLE POSTTRANSCRIPTIONAL REGULATORY FEATURES CONTROL EXPRESSION OF ETHANOLAMINE UTILIZATION GENES IN ENTEROCOCCUS FAECALIS

Enterococcus faecalis is a Gram-positive bacterium that lives as a commensal organism in the mammalian gastrointestinal tract, but can behave as an opportunistic pathogen. Our lab discovered that mutation of the eutK gene attenuates virulence of E. faecalis in the C. elegans model host. eutK is part of the ethanolamine metabolic pathway which was previously unknown in E. faecalis. I discovered the presence of two unique posttranscriptional regulatory features that control expression of eut locus genes. The first feature I found is an AdoCBL riboswitch, a cis-acting RNA regulatory element that acts as a positive regulator of gene expression. The second feature I discovered is a unique two-component system, EutVW. The EutV response regulator contains an ANTAR family domain, which binds RNA to trigger transcriptional antitermination. I determined that induction of expression of several genes in the eut locus is dependent on ethanolamine, AdoCBL and the two-component system. AdoCBL and ethanolamine are both required for induction of eut locus gene expression. Additionally, I discovered eutG is regulated by a unique mechanism of antitermination. Both the AdoCBL riboswitch and EutV response regulator control the expression of the downstream gene eutG. EutV potentially acts through a novel antitermination mechanism in which a dimer of EutV binds to a pair of mRNA stem loops forming an antitermination complex. My data show a unique mechanism by which two environmental signals are integrated by two different posttranscriptional regulators to regulate a single locus.

Application of Cognitive Engineering Principles to the Redesign of a Dichotomous Identification Key for Parasitology

Dichotomous identification keys are used throughout biology for identification of plants, insects, and parasites. However, correct use of identification keys can be difficult as they are not usually intended for novice users who may not be familiar with the terminology used or with the morphology of the organism being identified. Therefore, we applied cognitive engineering principles to redesign a parasitology identification key for the Internet. We addressed issues of visual clutter and spatial distance by displaying a single question couplet at a time and by switching to the appropriate next couplet after the user made a choice. Our analysis of the original paper-based key versus the Web-based approach found that of 26 applicable cognitive engineering principles, the paper key did not meet 4 (15%) and partially met 11 (42%). In contrast, the redesigned key met 100% of 32 applicable cognitive engineering principles.

Application of Signal Advance Technology to Electrophysiology

Medical instrumentation used in diagnosis and treatment relies on the accurate detection and processing of various physiological events and signals. While signal detection technology has improved greatly in recent years, there remain inherent delays in signal detection/ processing. These delays may have significant negative clinical consequences during various pathophysiological events. Reducing or eliminating such delays would increase the ability to provide successful early intervention in certain disorders thereby increasing the efficacy of treatment. In recent years, a physical phenomenon referred to as Negative Group Delay (NGD), demonstrated in simple electronic circuits, has been shown to temporally advance the detection of analog waveforms. Specifically, the output is temporally advanced relative to the input, as the time delay through the circuit is negative. The circuit output precedes the complete detection of the input signal. This process is referred to as signal advance (SA) detection. An SA circuit model incorporating NGD was designed, developed and tested. It imparts a constant temporal signal advance over a pre-specified spectral range in which the output is almost identical to the input signal (i.e., it has minimal distortion). Certain human patho-electrophysiological events are good candidates for the application of temporally-advanced waveform detection. SA technology has potential in early arrhythmia and epileptic seizure detection and intervention. Demonstrating reliable and consistent temporally advanced detection of electrophysiological waveforms may enable intervention with a pathological event (much) earlier than previously possible. SA detection could also be used to improve the performance of neural computer interfaces, neurotherapy applications, radiation therapy and imaging. In this study, the performance of a single-stage SA circuit model on a variety of constructed input signals, and human ECGs is investigated. The data obtained is used to quantify and characterize the temporal advances and circuit gain, as well as distortions in the output waveforms relative to their inputs. This project combines elements of physics, engineering, signal processing, statistics and electrophysiology. Its success has important consequences for the development of novel interventional methodologies in cardiology and neurophysiology as well as significant potential in a broader range of both biomedical and non-biomedical areas of application.

Peptide nucleic acids: Mechanism of tight binding and application in artificial nuclease

Peptide nucleic acids (PNA) are mimics of nucleic acids with a peptidic backbone. Duplexes and triplexes formed between PNA and DNA or RNA possess remarkable thermal stability, they are resistant to nuclease cleavage and can better discriminate mismatches. Understanding the mechanism for the tight binding between PNA and oligonucleotides is important for the design and development of better PNA-based drugs.^ We have performed molecular dynamics (MD) simulations of 8-mer PNA/DNA duplex and two analogous duplexes with chiral modification of PNA strand (D- or L-Alanine modification). MD simulations were performed with explicit water and Na$\sp{+}$ counter ions. The 1.5-ns simulations were carried out with AMBER using periodic boundary and particle mesh Ewald summation. The point charges for PNA monomers were derived from fitting electrostatic potentials, obtained from ab initio calculation, to atomic centers using RESP. Derived charges reveal significantly altered charge distribution on the PNA bases and predict the Watson-Crick H-bonds involving PNA to be stronger. Results from NMR studies investigating H-bond interactions between DNA-DNA and DNA-PNA base pairs in non-polar environment are consistent with this prediction. MD simulations demonstrated that the PNA strand is more flexible than the DNA strand in the same duplex. That this flexibility might be important for the duplex stability is tested by introducing modification into the PNA backbones. Results from MD simulation revealed dramatically altered structures for the modified PNA-DNA duplexes. Consistent with previous NMR results, we also found no intrachain hydrogen bonds between O7$\sp\prime$ and N1$\sp\prime$ of the neighboring residues in our MD study. Our study reveals that in addition to the lack of charge repulsion, stronger Watson-Crick hydrogen bonds together with flexible backbone are important factors for the enhanced stability of the PNA-DNA duplex.^ In a related study, we have developed an application of Gly-Gly-His-(Gly)$\sb3$-PNA conjugate as an artificial nuclease. We were able to demonstrate cleavage of single stranded DNA at a single site upon Ni(II) binding to Gly-Gly-His tripeptide and activation of nuclease with monoperoxyphthalic acid. ^

Single nucleotide polymorphism (SNP) selection for genotype-phenotype association studies

With hundreds of single nucleotide polymorphisms (SNPs) in a candidate gene and millions of SNPs across the genome, selecting an informative subset of SNPs to maximize the ability to detect genotype-phenotype association is of great interest and importance. In addition, with a large number of SNPs, analytic methods are needed that allow investigators to control the false positive rate resulting from large numbers of SNP genotype-phenotype analyses. This dissertation uses simulated data to explore methods for selecting SNPs for genotype-phenotype association studies. I examined the pattern of linkage disequilibrium (LD) across a candidate gene region and used this pattern to aid in localizing a disease-influencing mutation. The results indicate that the r2 measure of linkage disequilibrium is preferred over the common D′ measure for use in genotype-phenotype association studies. Using step-wise linear regression, the best predictor of the quantitative trait was not usually the single functional mutation. Rather it was a SNP that was in high linkage disequilibrium with the functional mutation. Next, I compared three strategies for selecting SNPs for application to phenotype association studies: based on measures of linkage disequilibrium, based on a measure of haplotype diversity, and random selection. The results demonstrate that SNPs selected based on maximum haplotype diversity are more informative and yield higher power than randomly selected SNPs or SNPs selected based on low pair-wise LD. The data also indicate that for genes with small contribution to the phenotype, it is more prudent for investigators to increase their sample size than to continuously increase the number of SNPs in order to improve statistical power. When typing large numbers of SNPs, researchers are faced with the challenge of utilizing an appropriate statistical method that controls the type I error rate while maintaining adequate power. We show that an empirical genotype based multi-locus global test that uses permutation testing to investigate the null distribution of the maximum test statistic maintains a desired overall type I error rate while not overly sacrificing statistical power. The results also show that when the penetrance model is simple the multi-locus global test does as well or better than the haplotype analysis. However, for more complex models, haplotype analyses offer advantages. The results of this dissertation will be of utility to human geneticists designing large-scale multi-locus genotype-phenotype association studies. ^

The application of principal component analysis on human development indicators: Temporal approach from 1999 to 2010

Background and Objective. Ever since the human development index was published in 1990 by the United Nations Development Programme (UNDP), many researchers started searching and corporative studying for more effective methods to measure the human development. Published in 1999, Lai’s “Temporal analysis of human development indicators: principal component approach” provided a valuable statistical way on human developmental analysis. This study presented in the thesis is the extension of Lai’s 1999 research. ^ Methods. I used the weighted principal component method on the human development indicators to measure and analyze the progress of human development in about 180 countries around the world from the year 1999 to 2010. The association of the main principal component obtained from the study and the human development index reported by the UNDP was estimated by the Spearman’s rank correlation coefficient. The main principal component was then further applied to quantify the temporal changes of the human development of selected countries by the proposed Z-test. ^ Results. The weighted means of all three human development indicators, health, knowledge, and standard of living, were increased from 1999 to 2010. The weighted standard deviation for GDP per capita was also increased across years indicated the rising inequality of standard of living among countries. The ranking of low development countries by the main principal component (MPC) is very similar to that by the human development index (HDI). Considerable discrepancy between MPC and HDI ranking was found among high development countries with high GDP per capita shifted to higher ranks. The Spearman’s rank correlation coefficient between the main principal component and the human development index were all around 0.99. All the above results were very close to outcomes in Lai’s 1999 report. The Z test result on temporal analysis of main principal components from 1999 to 2010 on Qatar was statistically significant, but not on other selected countries, such as Brazil, Russia, India, China, and U.S.A.^ Conclusion. To synthesize the multi-dimensional measurement of human development into a single index, the weighted principal component method provides a good model by using the statistical tool on a comprehensive ranking and measurement. Since the weighted main principle component index is more objective because of using population of nations as weight, more effective when the analysis is across time and space, and more flexible when the countries reported to the system has been changed year after year. Thus, in conclusion, the index generated by using weighted main principle component has some advantage over the human development index created in UNDP reports.^

PIM KINASE INHIBITION: SINGLE AGENT AND COMBINATION APPROACHES IN B-CELL LYMPHOMA

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are Ser/Thr/Tyr kinases. They modulate B-cell development but become oncoproteins and promote cancer development once overexpressed. Containing three isoforms, Pim-1, -2 and -3 are known to phosphorylate various substrates that regulate transcription, translation, cell cycle, and survival pathways in both hematological and solid tumors. Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma. Elevated Pim kinase levels are common in MCL, and it negatively correlates with patient outcome. SGI-1776 is a small molecule inhibitor selective for Pim-1/-3. We hypothesize that SGI-1776 treatment in MCL will inhibit Pim kinase function, and inhibition of downstream substrates phosphorylation will disrupt transcriptional, translational, and cell cycle processes while promoting apoptosis. SGI-1776 treatment induced moderate to high levels of apoptosis in four MCL cell lines (JeKo-1, Mino, SP-53 and Granta-519) and peripheral blood mononuclear cells (PBMCs) from MCL patients. Phosphorylation of transcription and translation regulators, c-Myc and 4E-BP1 declined in both model systems. Additionally, levels of short-lived Mcl-1 mRNA and protein also decreased and correlated with decline of global RNA synthesis. Collectively, our investigations highlight Pim kinases as viable drug targets in MCL and emphasize their roles in transcriptional and translational regulation. We further investigated a combination strategy using SGI-1776 with bendamustine, an FDA-approved DNA-damaging alkylating agent for treating non-Hodgkin’s lymphoma. We hypothesized this combination will enhance SGI-1776-induced transcription and translation inhibition, while promoting bendamustine-triggered DNA damage and inducing additive to synergistic cytotoxicity in B-cell lymphoma. Bendamustine alone resulted in moderate levels of apoptosis induction in MCL cell lines (JeKo-1 and Mino), and in MCL and splenic marginal zone lymphoma (a type of B-cell lymphoma) primary cells. An additive effect in cell killing was observed when combined with SGI-1776. Expectedly, SGI-1776 effectively decreased global RNA and protein synthesis levels, while bendamustine significantly inhibited DNA synthesis and generated DNA damage response. In combination, intensified inhibitory effects in DNA, RNA and protein syntheses were observed. Together, these data suggested feasibility of using Pim kinase inhibitor in combination with chemotherapeutic agents such as bendamustine in B-cell lymphoma, and provided foundation of their mechanism of actions in lymphoma cells.

Cellular Trafficking of Single and Multistage Vectors

Nanomedicine is an innovative field of science which has recently generated many drug delivery platforms with exciting results. The great potential of these strategies rely on the unique characteristics of the devices at the nano-scale in terms of long time circulation in the blood stream, selective accumulation at the lesions sites, increased solubility in aqueous solutions, etc. Herein we report on a new drug delivery system known as a multistage system which is comprised of non-spherical, mesoporous silicon particles loaded with second stage nanoparticles. The rationally designed particle shape, the possibility to modulate the surface properties and the degree of porosity allow these carriers to be optimized for vascular targeting and to overcome the numerous biological barriers found in drug delivery. In this study we investigated the intra and inter cellular trafficking of the multistage system in endothelial cells bringing evidence of its bio-compatibility as well as its ability to perform multiple intra and inter cellular tasks. Once internalized in cells, the multi-particle construct is able to dissociate, localizing in different subcellular compartments which can be targeted for exocytosis. In particular the second stage nanoparticles were found to be secreted in microvesicles which can act as mediators of transfer of particles across the endothelium and between different endothelial and cancer cells.