No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 220-237).

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Jason B. Shear. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 177-188).

No description available.

The presence of harmful algal blooms (HAB) is a growing concern in aquatic environments. Among HAB organisms, cyanobacteria are of special concern because they have been reported worldwide to cause environmental and human health problem through contamination of drinking water. Although several analytical approaches have been applied to monitoring cyanobacteria toxins, conventional methods are costly and time-consuming so that analyses take weeks for field sampling and subsequent lab analysis. Capillary electrophoresis (CE) becomes a particularly suitable analytical separation method that can couple very small samples and rapid separations to a wide range of selective and sensitive detection techniques. This paper demonstrates a method for rapid separation and identification of four microcystin variants commonly found in aquatic environments. CE coupled to UV and electrospray ionization time-of-flight mass spectrometry (ESI-TOF) procedures were developed. All four analytes were separated within 6 minutes. The ESI-TOF experiment provides accurate molecular information, which further identifies analytes.

Bioanalysis is a subdivision of analytical chemistry and deals with biological analytes such as metabolites, proteins, nucleic acids, small molecules, virus particles and entire cells. The rationale of my thesis was to achieve two goals: (i) develop a set of ready to use methods (ii) which are capable providing exact concentrations of analytes as well as kinetic and thermodynamic parameters of their interactions. To investigate interactions between biomolecules special conditions are required which do not interfere with the course if biomolecule interactions. Establishing these conditions and optimization of separation and detection parameters can be tedious and can take longer than actual analysis of samples. I developed a variety of Capillary Electrophoresis – Mass Spectrometry (CE-MS) methods suitable for bionalalysis. CE-MS establishes a new paradigm that separation methods together with MS detection can be used as comprehensive kinetic tools. Most previous attempts to use chromatography and electrophoresis for studying nucleic acid interactions were restricted to assuming slow or no equilibrium between reactants. Kinetic CE (KCE) shows that non-zero kinetics and structural dynamics must be taken into account when separation happens. KCE-MS could be a valuable supplement to IM-MS due to the separation of ions in solution according to their size-to-charge ratio. These methods allowed to reveal new facts about biomolecules and added novel data to the bank of the mankind knowledge. For the best of my knowledge, kinetic parameters for TG2 and thrombin G-quadruplex folding were reported for the first time. I developed a homogeneous method to determine kon, koff and Kd of fast and weak noncovalent interactions between multiple unlabeled ligands (small molecule drugs) and an oligosaccharide (α- or β-cyclodextrin) simultaneously in one capillary microreactor. It has been shown for the first time that KCE can be used to separate and detect the slowly interconverting open and closed conformations of human TG2. It allowed the first direct measurement of the Kd value for calcium binding. Sixteen new substrates were discovered for three aminotransferases (AAT, BCAT, and DAAT). In addition, Viral qCE showed a feasibility to analyse both the count of intact viral particles and sample nucleic acid contamination.

This thesis examines the use of capillary electrophoresis for the study of several biomolecules and their interactions and viruses. The first two experimental chapters focus on its utility for thermodynamic and kinetic analysis of molecules. Chapter one focuses on the use of non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), to calculate the dissociation constant for the interaction between double stranded microRNA-122 and protein p19. NECEEM was used to calculate the rate constants (koff = 0.059 ± 0.013 s-1, kon = 0.0022 ± 0.0008 s-1M-1) and the dissociation constant between miR-122 and wild type p19 (Kd = 27 ± 9 nM). A new method was developed to calculate the rate constant koff, by using multiple electric fields; which resulted in a koff value of 0.072 ± 0.022 s-1. In chapter two, the dissociation constant, Kd, was determined between HIV trans-activation response element and nuclear protein TOE1. It was demonstrated that TOE1, more specifically peptides ER19 and ED35, were binding to TAR with Kd values of 4.08 ± 0.19 µM for ER19 and 7.43 ± 1.60 µM for ED35. The discovery of the peptides’ inhibitory action of viral replication at the transcription level is a significant step towards further elucidating mechanisms for host response to HIV-1 infection. The third chapter focuses on the use of capillary electrophoresis for studying vesicular stomatitis virus (VSV) and vaccinia virus (VV). A new method was developed for quantification of VSV, using dithiothreitol. Furthermore, CE was used to study the preservation of VSV by a previously selected aptamer construct (quadramer) during freeze-thaw cycles. It was found that the infectivity of quadramer and aptamer pool-protected virus was higher than pure virus after 60 freeze−thaw cycles. It was also found that adding quadramers to the virus without freezing (cycle 0) increased the virus infectivity by 30%. We also investigated the potency of a carbohydrate-based ice recrystallization inhibitor, N-octyl-D-gluconamide (NOGlc) for its ability to eliminate the cold chain and stabilize the potency of VV. Viral potency after storage at room temperature demonstrated that NOGlc conserved the infectivity of VV, during 40 days.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy