published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

Clostridium thermocellum is a thermophilic bacterium that converts biomass to ethanol directly; however, high sensitivity of this bacterium toward ethanol limits its commercial utility. To elucidate the effect of ethanol on the growth of this bacterium a metabolite analysis of C. thermocellum was performed. The hypothesis of the project was that exogenous ethanol alters the metabolite profile of C. thermocellum. For metabolite analysis, capillary electrophoresis-electrospray ionization-mass spectrometry method (CE-ESI-MS) was developed due to highly polar and charged nature of metabolites. To increase the sensitivity of CE-ESI-MS, several parameters at the ESI interface were optimized. The application of 50% isopropanol as a sheath liquid increased sensitivity for metabolite analysis dramatically. Trimethylamine acetate (pH 10) was used as background electrolyte (BGE) due to its ability to separate the structural isomers of glucose phosphate. For metabolite sample preparation, novel methods for quenching and CE compatible metabolite extraction protocols were developed. Newly developed protocols were applied to metabolite analysis of wild type (WT) and ethanol adapted (EA) strains of C. thermocellum grown in batch cultures. Significant differences were found in key intracellular metabolites such as NAD+ and pyruvic acid. Intracellular concentrations of NAD+ were low in EA cells compared to WT cells and pyruvic acid was only detected in EA cells. To further understand the effect of ethanol on metabolite fluxes, WT and EA cells were grown in increasing concentrations of ethanol and the metabolite profile for each ethanol treatment was obtained. Significant changes were found in intracellular metabolite concentrations. Metabolic data showed that the glycolysis process in WT cells was obstructed due to exogenous ethanol which was evident from accumulation of G6P. On the other hand, no such accumulation of G6P was observed in the EA strain; however pyruvate began to accumulate in EA strain. These changes in intracellular metabolite concentrations due to perturbation of exogenous ethanol supported the hypothesis. Also, this investigation revealed a correlation between ethanol and metabolite profile changes and was able to explain a possible mechanism of growth inhibition of C. thermocellum which will certainly help genetic engineers to develop superior strains of C. thermocellum for commercial cellulosic ethanol production.

<p>Analysis of M-components is an important part of the diagnosis of monoclonal gammopathies and for the evaluation of disease response during treatment. In this project, two widely used electrophoresis methods and their corresponding immunotyping method were compared to evaluate the sensitivity of each method for the detection of small M-components. The project included 30 plasma samples from patients with identified M-components; 10 samples containing each IgG, IgA and IgM, respectively. All samples were diluted with normal EDTA plasma to achieve M-components of 5,00g/L. The samples were then serially diluted to achieve M-component concentrations of; 5,00, 2,50, 1,25, 0,63, 0,31 and 0,16g/L. All 180 samples were analysed with agarose gel electrophoresis and capillary electrophoresis. The dilutions above and below the detection level of each method were then analysed with immunofixation and immunosubtraction. The results showed good agreement between agarose gel electrophoresis and capillary electrophoresis in the highest concentrations of IgG and IgM. With agarose gel electrophoresis, IgA was detected in the same location as transferrin and the lowest concentration detected were therefore 1,25g/L. Besides the samples containing IgG, immunofixation was the most sensitive method.</p>

Development of chimeric, recombinant peptides, proteins and enzymes expands the availability of protein/enzyme–based tools for cellular analysis and new assay platforms. Ideal protein reagents for cellular analysis must translocate into a variety of cells with minimum cell damage, retain stability and biological activity within the cell during analysis, and provide a reliable, measurable signal. This work focused on development, characterization and utilization of chimeric recombinant peptide, protein and enzyme reagents for cellular analysis and immunoassays. A cell-penetrating, fluorescent protein substrate (PKAS) was developed to monitor intracellular protein kinase A activity in cells without the need for cellular transfection. PKAS translocated into HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity. Upon cellular loading, glucose dependent phosphorylation of PKAS was observed in both βTC-3 and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6 %) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4 %) in βTC-3 cells was with 3 mM glucose indicating a left-shifted glucose sensitivity. A cell-penetrating luciferase chimera (Luc-TAT) and a cell-penetrating phospholipid nanoshell entrapped luciferase (Luc-PPN) was constructed to monitor dynamic changes in intracellular ATP levels in mammalian cells. Upon cellular loading, the activity of Luc-TAT and Luc-PPN was monitored with time. Luc-TAT lost approximately 50% activity within one hour, and decreased rapidly over time. In contrast Luc-PPNs retain approximately 95% activity in 1 hour and 77% after 12 hours showing longer biological lifetime. Luc-PPNs were able to detect dynamic ATP changes in intact HeLa cells in the presence of KCN and NaN3. The bioluminescence returned to background levels within 8-10 minutes after treatment with KCN, whereas NaN₃ showed ~ 40% reduction. Two novel recombinant human parathyroid hormone (hPTH) analogs hPTHEGFP and hPTH-Cys were prepared to develop immunoassays for PTH detection in clinical samples. Initial experiments show promise for these analogs for use in CZELIF based immunoassays. The analogs present a number of distinctive advantages for clinical assays and can be used to develop several immunoassay platforms.

Phosphorylcholine (PC) based phospholipid bilayers have been explored as coating materials for various substrates due to their inherent resistance to non-specific protein adsorption. Phospholipids have been used for coatings in capillary electrophoresis (CE) to suppress electroosmotic flow (EOF) and to obtain better separation of proteins. Here, a series of investigations geared towards developing highly stable phospholipid based biomimetic stationary phases for chromatographic separations was performed.Fluid phospholipid bilayers lack the desired chemical and physical stability to serve as long-term coatings. In this work, highly stable phospholipid coatings generated via crosslinking polymerization of bis-SorbPC monomers were investigated. Reproducible EOF and migration times for model proteins were obtained for coated capillaries that were kept at room temperature for up to two months. Furthermore, the effects of surfactants, pH and capillary inner diameter (i.d.) on the stability of the lipid coating were investigated.In an alternate approach, stabilized phospholipid coatings for capillary electrophoresis were investigated via formation of hybrid monolayers. The capillary surface was chemically modified with a cyano group followed by deposition of phospholipid monomers. In this approach, marked enhancements in coating stability were attained with commercially available reagents. The hybrid coating was utilized for protein separations and gave efficiencies comparable to non-stabilized lipid coated capillaries.Fused silica capillaries were modified with phospholipid bilayers that were chemically tuned to introduce specific affinity binding agents, while minimizing nonspecific protein adsorption to the capillary wall. The wall of fused silica was functionalized with DOGS-NTA-Ni2+ lipid to present binding sites inside the capillary for 6xHis-tagged proteins. Fluorescence microscopy and changes in electrophoretic mobility were used to follow the interaction of the model proteins with the functionalized silica surface.The structural similarity of lipid vesicles to cell membranes made them attractive in developing stationary phases for both liquid chromatography and capillary electrophoresis to study interactions between analytes and phospholipid membranes. Stabilized PLB coated silica microspheres were prepared via polymerization of lipid monomers and displayed enhanced stability to extended storage and organic solvent. These highly stable microspheres, while minimizing nonspecific protein adsorption, were also functionalized with DOGS-NTA-Ni2+ and effectively bind 6xHis-EGFP proteins.

5-aminolevulino rūgštis (ALA) – provaistas naudojamas fotodinaminėje (FDT) terapijoje, protoporfirino IX prekursorius hemo biosintezės kelyje. Manoma, kad FDT vienas iš selektyviausių odos vėžio gydymo metodų. Nors ALA naudojimas FDT yra daug žadantis tiek terapiniu tiek komerciniais požiūriais, gydytojai vis dar sunkiai priimą šį gydymo metodą. Perkutaninės vaistinės formos dažnai kritikuojamos dėl nepakankamos skvarbos per raginį sluoksnį į odą ir dėl nepakankamo stabilumo. Skvarbai pagerinti yra kelios priemonės: skvarbos stiprikliai (dimetilsulfoksidas (DMSO), oleino rūgštis (OR) ir kt.), ALA derivatizacija į esterius, raginio sluoksnio pašalinimas ir kt. Dideli kiekiai DMSO, ar OR žymiai pagerina skvarbą, tačiau oda gali būti negrįžtamai pažeista. ALA esterių naudojimas vienas iš sėkmingesnių būdų įvesti ALA į odą, tačiau irgi nevisada sėkmingas. Kai kurios ligos pasireiškia jau su pažeistu raginiu sluoksniu (pvz. aktininė keratozė), šiuo atvejų gerai skvarbai užtikrinti nebūtina atlikti jokių papildomų farmacinės formos ruošimo, ar terapinių procedūrų. Viena iš buvusių opiausių ALA farmacinių formų problemų – stabilumas tiek farmacinių formų saugojimo metu, tiek atliekant ekstrakciją biocheminių tyrimų metų. Daugumos autorių nuomone ALA stabilumas užtikrinamas sumažinant pH bent iki 5. Tačiau per daug sumažinus galima sukelti odos paraudimus, ar net pažeidimus, be to mažas pH slopina PpIX biosintezę. Pastaruoju metu yra nemažai atliktų tyrimų prasiskverbusiam ALA... [toliau žr. visą tekstą] / 5-amineluvulinic acid (ALA) is a prodrug that is used in pfotodynamic therapy (PDT). It is a precursor of protoporphyrin IX (PpIX) in heme biosynthesis pathway. It is known, that FDT is promising treatment method according to both commercial and therapeutical aspects. Topical pharmaceutical forms are being criticised due to insufficient penetration to skin and ALA instability. Penetration enhancers such as dimethylsulfoxide (DMSO) or oleic acid (OA) could be used in order to improve penetration to skin. ALA derivatization to obtain more lipopfilic esters and stratum corneum disruption procedures could also be used for penetration improvement. Some skin lessions provide disrupted SC barrier (actinic keratosis). In this case, ALA penetration to skin is often sufficient to obtain therapeutical effect. Various authors state that ALA to be stable at pH 5 or lower, but too low pH can result in skin erythema, or more seriuos damage, moreover low pH inhibits PpIX biosynthesis. Nowadays there are numerous articles about ALA in skin penetration studies. Mostly, the HPLC method method might be epmloyed with a requirement of common derivatization reactions for amino acid determination. Liquid scintillation may also be employed in penetration studies, but it is quite expensive and require radioactive substances for quantitative determination. There are only few methods available for direct analysis. Ion pairing, ion exchange chromathography and capillary electrophoresis (CE) may be used... [to full text]

As cyanobacteria (also known as blue green algae) produce a range of cyclic peptides which are highly toxic, capillary electrophoresis and associated techniques have been investigated to assess their applicability for toxin monitoring in the water bodies of kwaZulu Natal, South Africa. Capillary electrophoresis (CE) is a technique in which charged molecules can be efficiently separated in a buffer solution within a capillary tube under the influence of a strong electric field. Two CE modes, namely capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were initially evaluated using a laboratory-built CE instrument. The former mode lacked selectivity due to the similar charge to size ratio of the algal toxins. However, with the latter mode, incorporation of a surfactant (sodium dodecyl sulphate) into the buffer, produced sufficient resolution between components. Parameters including surfactant concentration, buffer ionic strength, buffer pH and operating voltage were systematically optimized to separate the four algal toxins under investigation (microcystin YR, microcystin LR, microcystin RR and nodularin). The optimum separation conditions were: 30 mM borax, 9 mM sodium dodecyl sulphate, pH 9.18, 30 kV applied voltage, 10 s hydrodynamic injection, 70 cm x 50 Ilm Ld. bare fused silica capillary (LEFF 40 cm) and UV detection at 238 nm. Under these conditions, typical detection limits were in the low ng/IlL range (14.13 ng/IlL for microcystin LR to 29.85 ng/ILL for nodularin). The MECC method was evaluated in terms of migration time precision, efficiency and resolution, peak area and normalised peak area precision. Standard deviation values for retention times acquired using replicate electrokinetic injections ranged from 0.018 to 0.054 and 0.069 to 0.148 for hydrodynamic injections. Normalised peak area precision for replicate hydrodynamic injections were in the range 84 to 97 % RSD, while improved % RSD values of 11.5 to 18.7 were achieved for electrokinetic injections. Due to poor precision resulting from the lack of automation on the laboratory built CE system, poor correlation between increasing concentration and a corresponding change in normalised peak areas were achieved. The MECC method developed was applied to the analysis of an algal scum extract to illustrate the technique. A general problem with CE is that it suffers from poor detection sensitivity. Hence in this study, alternative injection modes, sample concentration strategies and alternative detection techniques were investigated in an attempt to improve detection limits for algal toxins. Using optimized electrokinetic injection conditions, detection limits were five to ten times better than those obtained with hydrodynamic injections. On-line sample concentration methods were partially successful. Field amplified back and forth MECC in which analyte injected in the entire column volume and subsequently focused in a narrow band by manipulating the electric field, resulted in an enormous sensitivity enhancement that ranged from 197 times for microcystin RR to 777 times for microcystin YR when compared to hydrodynamic injections. Field amplified sample stacking (FASI) was ineffective for toxin preconcentration, while electro-extraction produced detection limits ranging from 0.27 ng/J.tL for microcystin YR to 1.08 ng/J.tL for microcystin RR. Solid phase extraction, in which analytes are first trapped and concentrated on HPLC material in a cartridge and then eluted in a more concentrated form for injection, was found to be practical only in the offline mode. A concentration detection limit of less than 0.002 ng/J.tL was obtained. Attempts with on-line solid phase extraction failed due to problems associated with coupling the cartridge with the separation capillary. Finally, laser induced fluorescence (LIF) detection was investigated as an alternative to UV detection. Unfortunately, the algal toxins were not amenable to LIF detection because tagging with the fluorescent moiety, fluorescein isothiocyanate (FITC), was prevented by the stereochemistry of these cyclic peptides. A comparative study between HPLC and MECC revealed that the former displayed poor efficiency peaks and long analysis times for toxin analysis. However HPLC was superior in terms of retention time precision (0.12 to 0.64 % RSD) and area precision (1.78 to 2.86 % RSD). Mass detection limits for MECC (0.0142 to 0.0603 ng) were far superior to those achieved by HPLC (0.55 to 1.025 ng). In addition to HPLC and MECC, a preliminary investigation of micro-high performance liquid chromatography (J.tHPLC) and capillary electrochromatography (CEC) for the analysis of algal toxins was made using 50 J.tm Ld. capillary columns packed in-house, with reverse phase HPLC packing material. / Thesis (M.Sc.)-University of Natal, 1997.

The goal of this project was to reduce processing time for forensic DNA testing without incurring significant added costs and/or the need for new instrumentation, while still generating high quality profiles. This was accomplished by: 1) extraction normalization using the ChargeSwitch® Forensic DNA Purification Kit such that a small range of DNA concentrations was consistently obtained, eliminating the need for sample quantification and dilution; 2) developing fast PCR protocols for STR primer sets using shorter amplification methods, low volume reactions and non-fast thermal cyclers; and 3) developing a quicker 3130xl Genetic Analyzer detection method using an alternative polymer/array length combination. Extraction normalization was achieved through a reduction in bead quantity, thereby forcing an increase in bead binding efficiency. Four products (AmpliTaq Gold® Fast PCR Master Mix, KAPA2G™ Fast Multiplex PCR Kit, SpeedSTAR™ HS DNA Polymerase and Type-it Microsatellite PCR Kit) were evaluated for low volume (3μl) fast PCR on a 384-well Veriti® thermal cycler with the Identifiler primer set. KAPA2G™ was selected for 3μl fast PCR protocols using PowerPlex 16 HS and Identifiler Plus primer sets (42-51min), as well as 5μl and 6μl Identifiler fast reactions on a 9700 thermal cycler (51-60min). Alternative detection (POP-6™/22cm) achieved 24-28min run times, but with decreased resolution as compared to traditional POP-4®/36cm detection for alleles >200bp; however, 1bp resolution was still obtainable for alleles <300bp. These modifications resulted in robust databasing processes with up to a 37% reduction in processing time for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated (3μl fast PCR reactions) and generated high quality STR profiles with ≥90% pass rates.

Heparin is a complex mixture of sulfated polysaccharides derived from animals and one of the oldest drugs in use. While an efficacious anticoagulant, heparin is beset by side effects and pharmacokinetic difficulties. Low molecular weight heparins (LMWH) are made by depolymerizing unfractionated heparin (UFH) and present improvements in these areas. However, they still retain a phenomenally high level of complexity due to their polydispersity and the introduction of non-native structural features. This makes the structural characterization LMWHs a daunting task. This work details the development of a novel capillary electrophoretic (CE) method for fingerprinting LMWHs. Since their complexity normally results in a nearly featureless electropherogram, polyalkylamines were used as a resolving agents to yield highly resolved and reproducible fingerprints characteristic of the LMWH being investigated. Linear polyamines of resolved LMWH in a manner dependent on chain length and charge density, while cyclic polyamines were incapable of resolution. Longer length glycosaminoglycans such as UFH and chondroitin sulfate were not successfully fingerprinted as they lacked run to run consistency. Further investigation into the mode of polyamine binding showed that they bound to LMWH via a two site binding model, indicating the presence of specific sites on LMWH that tightly bind polyamines. Upon the saturation of these sites, the polyamines continue to interact via general electrostatic binding. Pentaethylenehexamine was also able to separate the known contaminant oversulfated chondroitin sulfate from UFH. In July of 2010, the US food and drug administration approved a generic for the widely used LMWH enoxaparin, a questionable move due to the difficulties of proving the equivalence of such a complex mixture. A comparison of the brand and generic batches of enoxaparin using the fingerprinting method revealed striking similarities, bolstering the generic’s claim of equivalency and providing a protocol for the evaluation of other biosimilar LMWHs. This is the first work utilizing CE in developing high resolution fingerprints of LMWH. It presents a noteworthy method for quality assessment of LMWH and provides the basis for designing other small molecule probes for the analysis of complex glycosaminoglycans.