5 ABSTRACT The mitogen-activated protein kinase (MAPK) cascade is an essential member of the cell defense system against stressors. The capability and efficiency of the cell reactions to different stress signals depend on signal transduction pathway, where signals from MAPK kinase kinase (MAP3K) are transferred through phosphorylation to downstream MAPK kinase (MAP2K) and finally to MAPK. Apoptosis signal-regulating kinase 1 (ASK1) is a member of a MAP3K family and its activation and inhibition has a significant participation in a regulation of cell response to stress stimuli. The regulation of ASK1 has a strong influence in pathogenesis of several diseases, the excessive activation of human ASK1 or failure in the control of its function are associated with cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, infectious diseases, tumorigenesis, asthma, diabetes and ageing. The activity of ASK1 is regulated by its interaction with several proteins, the attention is focused on two physiological inhibitors, mammalian thioredoxin (TRX) and the 14-3-3 protein. ASK1 in its inactive form is inhibited by bonds formation with TRX and 14-3-3, however the explicit mechanism of this interaction is unclear due to the absence of structural data. This work is a part of an extensive research about...

Capillary ion analysis (CIA) is a form of capillary electrophoresis which uses the differential electrophoretic mobility of ions to perform a separation of an ionic mixture. Application of this technique for direct detection of potassium concentrations in human vitreous humor was the purpose of this investigation. CIA was performed using a Waters Quanta 4000 Capillary Electrophoresis System with a 745 Data Module using a 75 μm x 60 cm capillary and a run electrolyte of 67.7 mg hydroxyisobutyric acid (HIBA), 52.8 mg 18-crown-6-ether and 64 μL UV-CAT-1 reagent (4-ethylbenzylamine in a volume of 100 mL water (18 Mohm) with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: ammonium ion, 2.86 min; potassium, 3.24 min; calcium, 3.84 min; sodium, 3.98 min; barium (internal standard), 4.68 min; and lithium, 4.79 min. Correlation coefficients (r) between peak area ratios and concentration ranges of 2.5-144 mmole/L (100-1000 ppm) were from 0.9855 to 0.9999. Coefficients of variation (CV) ranged from 1.45 to 13.8% between days and from 1.38 to 9.43% within-day. Application of this methodology to twenty-five vitreous humor specimens from forensic cases was compared to analysis by ion-specific electrode for potassium concentration. Comparison of CIA to ion-specific electrode analysis of vitreous humor potassium concentrations revealed a correlation coefficient of 0.9642. CIA is applicable to forensic analysis of potassium concentration in forensic vitreous humor specimens. Quantitation of numerous cation concentrations is possible by direct CIA of vitreous humor.

Capillary ion analysis (CIA) is a form of capillary electrophoresis that uses the differential electrophoretic mobility of ions to perform a separation of an ionic mixture. Application of this technique for detection of lithium concentrations in plasma and tissues of Poecilia was the purpose of this investigation. CIA was performed using a 75 μm ID x 60 cm length fused-silica capillary and a run electrolyte of 67.7 mg hydroxyisobutyric acid (HIBA), 52.8 mg 18-crown-6-ether and 64 μL UV-CAT-1 reagent (4- methylbenzylamine) in a volume of 100 mL water (18 MΩ) with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: potassium, 2.98 min; calcium, 3.48 min; sodium, 3.60 min; barium (internal standard), 4.15 min and lithium, 4.26 min. Lithium and barium migration times were stable and reproducible. Correlation coefficients (r) between peak area ratios of lithium/barium for concentrations ranging from 0.1 to 2.0 mM were from 0.976 to 0.996. Coefficients of variation (CV) for lithium concentrations ranged from 4.07 to 15.71% between days and 4.38 to 7.76% within-day. Application of this methodology for determination of lithium concentrations in the plasma, brains and livers of fish dosed with lithium for 23 days are presented. CIA is applicable to analysis of lithium concentrations in biological fluids and tissues of fish.

Soil pollution in recent years has emerged as an issue of great environmental concern. Contamination of soil by improper disposal or spillage of petrochemicals and products containing petroleum hydrocarbons is one of such pollution cases highly reported. To remediate petroleum contaminated soil, A DC powered electrokinetic reactor was used with biosurfactants as an enhancement for the remediation process. To begin with, studies were made under voltage variations of 10 V and 30 V with an electrode spacing of 185 mm. Biosurfactant with its producing microbes and biosurfactant free cells were introduced in the soil chamber after which the reactor was left to run for 10 days under the electric field. The technology was able to achieve the highest oil recovery of 75.15 % from the soil in 96 hours at 30 V. With other factors remaining constant, the reactor was also operated under a constant voltage of 30 V with configurations of fixed electrodes spacings of 335 mm, 260 mm,185 mm and continuous approaching electrodes at 335 mm, 260 mm and 185 mm. The current in the electrolyte was highest with the least electrode distance of 185 mm. The increase in current led to a direct proportional increase in the electroosmotic flow towards the cathode leading to increased coalescence of the oil from the soil as compared to the other electrode distances. The analysis of the results showed reduction in the total carbon content in the soil with viable oil recovery rates for all the electrode distances with 185 mm being the most effective in both oil recovery and degradation. The reactor was further operated with amended biosurfactant concentrations of 28 g/L, 56 g/L and 84 g/L to enhance the recovery of oil from the soil and aid in biodegradation of the remaining oil by hydrocarbon degrading microbes. The highest oil recovery of 83.15 % was obtained with the biosurfactant concentration of 56 g/L showing that the hyper increase in concentration of the biosurfactants is not necessary to have an efficient process. In all experiments the microorganisms were able to survive under the electro-halo-thermal environment in the reactor and degraded the remaining hydrocarbons to acceptable amounts in the environment. The bacteria were however affected by the constantly changing pH in all experiments. The presence of biosurfactants was so significant in aiding oil recovery and increasing bioavailability of hydrocarbons to the microbes. Production of biosurfactants in the reactor followed up by kinetic suggestions of the processes in the bioelectrokinetic reactor should be studied in future. / Dissertation (MEng (Environmental Engineering))--University of Pretoria, 2019. / Chemical Engineering / MEng (Environmental Engineering) / Unrestricted

In this study, electrophoretic mobilities of native and two types of trypsin digested sarcoplasmic reticulum vesicles have been determined by microelectrophoresis using a Doppler Electrophoretic Light Scattering Analyzer to investigate the influence of hydrodynamic drag, caused by the Ca2+, Mg2+ -ATPase protruding from the surface of native sarcoplasmic reticulum vesicles. After the prolonged digestion (protein:trypsin ratio of 20 for 3 hours at 25°C), the ATPase was cleaved and removed from the sarcoplasmic reticulum membrane as shown with SDS gel electrophoresis and an ATPase activity assay. Ionic strength and pH dependence of mobility showed a nearly pH independent increase in initial surface charge density after prolonged digestion. Adsorption isotherms for native, short (protein:trypsin ratio of 200 for 2 minutes at 25°C), and prolonged digested sarcoplasmic reticulum vesicles were recorded for TPhP+ (tetraphenylphosphonium), PCP- (pentachlorophenol), and Ca2+, and fitted to the Langmuir adsorption model. The most important result from the adsorption isotherms is that adsorption of the three ions did not increase significantly after prolonged digestion. From this it can be concluded that hydrodynamic drag does not have a measurable influence on electrophoretic mobility of sarcoplasmic reticulum vesicles and therefore cannot account for the big differences in mobility between sarcoplasmic reticulum vesicles and a comparable artificial membrane system (phosphatidylcholine/phosphatidylserine liposomes), which were observed in this lab earlier. A thermodynamic analysis of adsorption was done for PCP- adsorption to phosphatidylcholine liposomes, TPhP+ adsorption to phosphatidylcholine liposomes, and TPhP+ adsorption to sarcoplasmic reticulum vesicles, by recording adsorption isotherms at 10°C, 25°C, 40°C, and 55°C. The adsorption of PCP- to phosphatidylcholine liposomes was clearly driven by enthalpy. In contrast, the adsorption of TPhP+ to phosphatidylcholine liposomes and sarcoplasmic reticulum vesicles was characterized by a positive enthalpy and a still larger negative entropy term. The thermodynamic analysis of ion adsorption shows that the driving forces of adsorption are very similar for sarcoplasmic reticulum vesicles and the chosen artificial membrane system (phosphatidylcholine liposomes) in spite of the significant lower adsorption of biological membranes compared to artificial membrane systems.

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Forensic toxicology is a critical field in which scientific techniques are employed in order to establish the presence or absence of pharmacological substances and/or their metabolites within an individual. The results of such analyses can have legal implications, and toxicology has a number of important applications, including post-mortem investigations, workplace drug testing, therapeutic drug monitoring, and impaired driving studies. The focus of this specific body of work is on the use of toxicology in the detection and quantification of drugs of abuse –specifically opioids - in biological samples. In recent years, there has been a surge in opioid abuse and the need for forensic toxicology labs to process samples from such cases quickly and accurately continues to increase. As a result, it is imperative to research different techniques and technologies that can be applied in toxicology to improve efficiency of sample processing while still remaining sensitive and specific. Many toxicology laboratories today use immunoassay techniques for screening, and utilize a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification. While these methods are established and reliable, the need to analyze an increasing number of samples in a more efficient time frame is essential, and with that, the need to develop and validate new analytical methods. This study sought to validate the use of Microchip Capillary Electrophoresis-Tandem Mass Spectrometry (CE-MS/MS) as a method for detecting and quantifying a panel of fourteen opioids. The experiments were run using a ZipChip (908 Devices, Boston, MA) as the separation scheme, which contains a small capillary where analytes are separated out by electrophoretic mobility - dictated largely by size and charge. These analytes were then ionized by electron spray ionization (ESI) at the end of the chip, and then detected, fragmented, and analyzed in a SCIEX 4500 Triple Quadrupole Mass Spectrometer (Framingham, MA). The analytical run time of the method evaluated was two and half minutes per sample. Calibration curves were run and the method was assessed for a number of validation parameters, including bias, precision, limit of detection, common analyte interferences, matrix interferences, and carryover, as recommended by the American Academy of Forensic Sciences Standards Board. The fourteen drugs and metabolites looked at in this study were 6-monoacetylmorphine, buprenorphine, codeine, dihydrocodeine, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidine (EDDP), fentanyl, heroin, methadone, morphine, naloxone, norfentanyl, oxycodone, oxymorphone, and tramadol. All standards were ordered from Cerilliant (Round Rock, TX), as well as deuterated internal standards used for quantification purposes. This study showed that as the method currently stands, it can reliably detect this panel of opioids at limits of detection between 1 and 15 ng/mL, with the exception of buprenorphine and morphine, for which the method appeared less sensitive. While some applications desire higher sensitivity than this, this level of detection could be very useful as a screening technique that is quick and also far more specific than current immunoassay screening techniques, and provide the additional advantage of quantification for samples at slightly higher concentrations. Quality control samples at 100 ng/mL and 150 ng/mL generally showed consistent results and acceptable levels of bias and precision, indicating that the method can be used to reliably quantify this panel of opioids at those concentrations. In addition, interference signals detected during analysis of other common analytes often encountered with opioids were negligible, with the exception of heroin and norfentanyl. Analysis of ten lots of urine for blank matrix interferences also demonstrated low potential for interference, with the exception of heroin. Finally, there was no evidence of significant carryover between samples, or interference from the deuterated internal standards. While some potential instrumentation issues such as mass spectrometer calibration prompt further study, the method shows promise for future use as a high throughput analysis tool in forensic toxicology labs. CE-MS/MS has the added benefit of not only faster run times, but significantly less sample consumption per run, and additionally, less sample preparation. CE is a viable separation scheme for metabolites and forensic applications, and could make large impacts as an effective way to analyze toxicological samples.

The influence of microparticles on electroosmotic flow was investigated experimentally and numerically. Experiments were conducted using four different particle types of varying chemical composition, surface charge and polarity. Each particle type was tested at five different volume fractions ranging from 0.001 – 0.025. With a constant applied electric field, positively charged particles enhanced the electroosmotic flow by as much as 850%. The enhancement depended on particle composition, size and concentration. For negatively charged particles, the bulk electroosmotic flow was retarded with the largest reductions being 35%. This occurred for the greatest negative paricle concentration studied. A final experimental study utilizing a single volume fraction and particle type was conducted using microtube inner diameters of 100 – 300 micrometers. It was found that the effective electroosmotic mobility decreases with increasing microtube diameter. A numerical study of microparticle influenced electroosmotic flow was also conducted for positively and negatively charged particles. A Galilean transformation was employed in which the particles were held stationary. A moving wall model was utilized to account for the particle velocity and the wall-induced electroosmotic flow. The particle-induced electroosmotic flow was also accounted for. A range of particle velocities were imposed in order to study the flow physics for a range of potential flows. Scenarios were run for a single tube diameter of 100 micrometers and a single particle diameter of 1.7 micrometers. Volume fractions of 0.001, 0.0075 and 0.025 were tested for both positively and negatively charged particles. At least two particle charges were studied for each volume fraction and polarity. Comparisons of the trends in the numerical model are qualitatively compared with the trends in the experimental data. The numerical and experimental data demonstrated similar trends. For positively charged particles, an increase in volume fraction showed a nonlinear increase in the average bulk flow velocity. For negatively charged particles an increase in volume fraction showed a nonlinear decrease in the average bulk flow velocity.

<p>Capillary electrophoresis is a high resolution microseparation technique that is increasingly being recognized as a physical tool to characterize biomolecular interactions, where dynamic complexation of analytes with discrete additives is used to resolve complex mixtures of solutes, including enantiomers. Despite the wide interest in developing high-throughput screening platforms for drug discovery or disease prognosis, little emphasis has been placed on enhancing "pre-analysis steps" that are often the most crucial component determining the overall performance of a method. Off-line sample pretreatment protocols for complex biological samples are often time-consuming and not amenable for automation. The major goal of this thesis is the development of a single-step analytical platform by CE for targeted metabolites that integrate several different sample pretreatment processes during separation, which can also be used to characterize the thermodynamic parameters associated with covalent and non-covalent interactions. Two distinct projects in this thesis have been examined involving boronic acid-polyol and protein-cyclic nucleotide interactions that illustrate the concept of integrating sample pretreatment with chemical analysis based on dynamic complexation-capillary electrophoresis.</p> <p>The first project consists of a new strategy for enhancing target selectivity when using 3-nitrophenylboronic acid as an electrokinetic probe in dynamic complexation-capillary electrophoresis. The differential migration of ternary boronate ester complexes permits the selective analysis of micromolar levels of UV-transparent polyol stereoisomers in urine samples that is applicable to single-step screening of in-born errors of sugar metabolism, such as galactosemia. In the second project, the impact of ligand binding on protein stability is assessed by dynamic ligand exchangeaffinity capillary electrophoresis with laser-induced native fluorescence detection. This is a convenient yet rapid format for comparative thermodynamic studies of a regulatory subunit of protein kinase involving different cyclic nucleotide analogues without off-line sample pretreatment, since ligand exchange and protein unfolding processes are integrated incapillary during electromigration.</p> / Thesis / Master of Science (MSc)