published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

(Uncorrected OCR) Abstract Porphobilinogen (PBG) synthase condenses two molecules of aminolaevulinic acid (ALA) to form PBG in heme biosynthesis. The enzyme activity is sensitive to inhibition by heavy metals such as lead. It can act as a biological indicator of chronic lead POis~r\g to identify the risk group, especially in children, so that early treatment can be given to prevent possible permanent damages. A reversed-phase ion-pair HPLC analytical method for the assay of the PBG synthase activity based on detection of PBG production has been validated. A Hypersil CN column (150 x 4.6 mm; 5 urn) was employed together with a mixture of acetonitrile-40 mM phosphate buffer at pH 2.4 with 5 mM 1-heptanesulphonic acid (8:92, v/v). UV detection was performed at 240 nm. PBG was eluted as a spectrally pure peak resolved from its impurities in the methanol-inhibited enzyme reaction. The method was sensitive with a limit of quantitation of 2 ~M. The within-run and between-run precisions were 8.2% and 13.8% respectively. The recovery was 93.4 �7.1% (n=6). The preliminary reference range of the PBG synthase activities in the local pediatric population were from 21.5 to 26.3 ~mol/L RBC/min. Bland and Altman statistical analysis showed that the HPLC assay and the colorimetric assay could not be used interchangeably. The HPLC assay was an alternative way to assess the PBG synthase activities in the human erythrocyte samples. IV / abstract / toc / Medical Sciences / Master / Master of Medical Sciences

The development of analytical tools for investigating biological pathways on the molecular level has provided insight into diseases and disorders. However, many biological analytes such as glucose and inositol phosphate(s) lack the optical or electrochemical properties needed for detection, making molecular sensing challenging. Scintillation proximity assay (SPA) does not require analytes to possess such properties. SPA uses radioisotopes to monitor the binding of analytes to SPA beads. The beads contain scintillants that emit light when the radiolabeled analytes are in close proximity. This technique is rapid, sensitive and separation-free. Conventional SPA beads, however, are large relative to the cells and made of hydrophobic organic polymers that tend to aggregate or inorganic crystals that sediment rapidly in aqueous solution, thus limiting SPA applications. To overcome these problems, polystyrene core-silica shell nanoparticles (NPs) doped with pTP and dimethyl POPOP were fabricated to produce scintillation NPs that emit photons in the blue region of visible light. The developed scintillation particles are approximately 250 nm in diameter (i.e. 200 nm of core diameter and 10-30 nm of shell thickness), responsive to β-decay from tritium (³H) and have sufficient stability in the aqueous media. DNA hybridization-based SPA was performed to determine whether the scintillation NPs could be utilized for SPA applications. A 30-mer oligonucleotide was immobilized on the polystyrene core-silica shell NPs to give approximately 7.6 x 10³ oligonucleotide molecules per NP and ³H-labeled complementary strand was annealed to the immobilized strand. At the saturation point, increases in scintillation signal due to oligonucleotide binding to the NPs were about 9 fold compared to the control experiments in which no specific binding occurred, demonstrating that the scintillation NPs can be utilized for SPA. Along with the improved physical properties including smaller size and better stability in the aqueous system, the developed scintillation NPs could be potentially useful as biosensors in cellular studies.

Peptide receptor overexpression in diseased cells and tissues, including carcinomas provides an opportunity to develop therapeutics and imaging agents that selectively bind to such cells and tissues. This dissertation presents tools and processes that can be utilized to target melanocortin and cholecystokinin receptors through multivalent binding. In Chapter 2, improved synthesis and purification methods are described for the production of Eu-chelated probes that serve to evaluate the binding efficacy of multivalent molecules through competition binding assays. Specifically, a xylenol orange-based assay for quantification of unchelated metal ions was used to determine unbound metal ion contamination and the success of metal ion removal. The use of Empore™ chelating disks was determined to be the method of choice for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Applying new synthesis and purification strategies, the TRF probe Eu-DTPA-PEGO-CCK4 targeted to cholecystokinin receptors was synthesized (Chapter 2) and validated via saturation and competition binding assays (Chapter 4) using a HEK293 cell line overexpressing the human cholecystokinin 2 receptor. In Chapter 3, short and efficient syntheses of multivalent molecules targeted to melanocortin receptors based on three commercially available trigonal core scaffolds, phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane, are described. These constructs were designed to further test the 24 ±5 Å inter-ligand distance suggested in recent literature for multivalent binding to melanocortin receptors. The bioactivities of these compounds were evaluated using a competitive binding assay that employed HEK293 cells engineered to overexpress the human melanocortin 4 receptor. In the course of conducting these bioassays, novel in vitro binding assay protocols were established, which led to high repeatability and robustness of the bioassays compared to previous methods. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (~2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed in Chapters 3 and 6.

Semiconductor nanocrystals, otherwise known as Quantum dots (Q dots), are a new type of fluorophore that demonstrates many advantages over conventional organic fluorophores. These advantages offer the opportunity to improve known immunofluorescent methods and immunofluorescent biosensors for rapid and portable bacterial detection in water. The detection of the micro organism Escherichia coli O157:H7 by attenuation of a fluorophore’s signal in water was evaluated alone and in the presence of another bacterial species. A sandwich immunoassay with antibodies adhered to SU-8 as a conventional comparison to our novel attenuation detection was also conducted. The assays were tested for concentration determination as well as investigation for cross reactivity and interference from other bacteria and from partial target cells. In order to immobilize the capture antibodies on SU-8, an existing immobilization self-assembly monolayer (SAM) for glass was modified. The SAM was composed of a silane ((3-Mercaptopropyl) trimethoxysilane (MTS)) and hetero-bifunctional cross linker (N-γ-maleimidobutyryloxy succinimide ester (GMBS)) was utilized in this procedure. The SU-8 surface was activated using various acids baths and oxygenated plasma, and it was determined that the oxygenated plasma yielded the best surface attachment of antibodies. The use of direct fluorophore signal attenuation for detection of the target E. coli resulted in the lowest detectable population of 1x10¹ cfu/mL. It was not specific enough for quantitative assessment of target concentration, but could accurately differentiate between targeted and non-targeted species. The sandwich immunofluorescent detection on SU-8 attained the lowest detectable population of 1x10⁴ cfu/ml. The presence of Klebsiella pneumoniae in solution caused some interference with detection either from cross reactivity or binding site blocking. Partial target cells also caused interference with the detection of the target species, mainly by blocking binding sites so that differences in concentration were not discernable. The signal attenuation not only had a better lowest detectable population but also had less measurement error than the sandwich immunoassay on SU-8 which suffered from non-uniformed surface coverage by the antibodies.

Epidemiological studies have linked exposure to elevated levels of airborne particulate matter with increased incidences of several types of respiratory disease, hospital admissions and morbidity. Millions of tons of airborne particulate matter are generated and released into the atmosphere each year. However, particulate matter resulting from the combustion of fuel oil and coal are of particular concern, because they are generally composed of small particles that can easily penetrate deep into the lungs, and can contain significant concentrations of toxic transition metals, such as zinc, iron and vanadium. Pulmonary toxicity (i.e. damage caused to lung tissues) of particulate matter is currently evaluated via time-consuming in-vivo testing, or via in-vitro testing. Compared to in-vivo testing, in-vitro testing offers significant advantages in terms of time savings and sample throughput. Unfortunately, the number of in-vitro testing methods are currently very limited, and do not allow a thorough investigation of the mechanisms of particulate matter toxicity. In light of these issues, the goals of the study described here were three-fold: *To adapt several in-vitro toxicity assays currently used in other applications to use in measuring particulate matter toxicity on lung cell layers; *To use these adapted assays to quantify the toxicity of numerous types of oil and coal ashes with varying particle sizes and transition metal concentrations, and; *To use the same assays to quantify the toxicities of several transition metals found in coal and oil ashes to better understand their relative contributions to overall particulate matter toxicity. Three colorimetric in-vitro assays were chosen for adaptation, and proved effective in measuring adverse cellular response to particulate matter exposure. Particle size was shown to have a large effect on the overall cytotoxicity of particulate matter; fine (less than 2.5 μm aerodynamic diameter) particles proved substantially more toxic than coarse (larger than 2.5 μm aerodynamic diameter) particles. Dose-response experiments measuring the toxic effects of the transition metals zinc, vanadium and iron revealed that zinc was the most toxic; a concentration of 0.6 mM caused a 50% drop in cellular metabolism, compared to 3 mM and 4 mM for vanadium and iron respectively.

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules. Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).

Fumonisins, mycotoxins produced by the fungus, Fusarium moniliforme, which grows on maize, are a major worldwide agricultural problem. Consumption of contaminated maize feeds causes a wide variety of toxic effects in animals depending on the species of animal. In humans, high concentrations of fumonisins have been shown to correlate with increased incidence of oesophageal cancer (OC). Most analyses for fumonisins are done using high performance liquid chromatography (HPLC) which requires time-consuming extraction and clean-up prior to preparation of a fluorescent derivative. Enzyme-linked immunosorbent assays (ELISA), which are sensitive and specific, are a viable alternative but commercially available antibodies and kits are extremely expensive. Polyclonal antibodies against fumonisin B, (FB,) were raised in chickens and rabbits; all animals produced antibodies from week 2 onwards, the highest titre was at week 8 from one of the chickens. Cross-reactivities with FB, analogues were checked. A sensitive, quantitative competitive indirect ELISA (CI-ELISA) was developed and optimised; range 0.2 to 20 ng/ml (in buffer), detection limit 0.2 nglml (in buffer), intra-assay coefficient of variation (CV) was 5.33 % and inter-assay 7.04%. This method was adapted to analyse human plasma and urine samples. After removal of proteins by boiling, the range of recoveries of FBI were 94.7% toI12.4% at 4 ng/ml; and 94.6% to 108.7% at 8 ng/ml. Blood and urine samples from patients with OC (40 plasma, 17 urine), controls (21 plasma, 12 urine) and patients with other forms of cancer (20 plasma, 10 urine) were collected from hospitals in the Durban Metropolitan area and analysed for fumonisins. Detectable levels (>0.4 nglml) were found in 86.9% of plasma samples and 94.9% of urine samples. Statistical evaluation showed a highly significant difference between plasma results for OC and controls (p<0.000 1) but no significant difference between the urine results. Comparison of other forms of cancer and controls showed no significant differences for either the plasma or the urine samples. However, there was a highly significant difference between the OC and other forms of cancer results for both plasma (p<0.005) and urine (p<0.05) samples. Some samples (9 plasma, 8 urine) were checked by HPLC. For plasma samples there was correlation between the ELISA and HPLC methods (r = 0.656, p<0.005) but not for urine samples.

Septins are an evolutionarily conserved family of proteins that have been implicated in a multitude of cellular processes. Septin 5 is mainly expressed in the nervous system and it has been linked to regulated secretion through its binding to the SNARE protein syntaxin 1. However, the exact mechanism of septin 5 function in localized exocytosis remains unknown. Over-expression of septin 5 is known to lead to lower levels of secretion in HIT-T15 cells. Interestingly, in the current study, the knock-down of septin 5 also results in reduced levels of regulated secretion in PC12 cells, suggesting a more complex role of septin 5 that includes both negative and positive effects on exocytosis. Septin 5 knock-down data point to a possibility of septin 5 facilitating formation of a tether between the vesicles and their site of secretion.

Septins are an evolutionarily conserved family of proteins that have been implicated in a multitude of cellular processes. Septin 5 is mainly expressed in the nervous system and it has been linked to regulated secretion through its binding to the SNARE protein syntaxin 1. However, the exact mechanism of septin 5 function in localized exocytosis remains unknown. Over-expression of septin 5 is known to lead to lower levels of secretion in HIT-T15 cells. Interestingly, in the current study, the knock-down of septin 5 also results in reduced levels of regulated secretion in PC12 cells, suggesting a more complex role of septin 5 that includes both negative and positive effects on exocytosis. Septin 5 knock-down data point to a possibility of septin 5 facilitating formation of a tether between the vesicles and their site of secretion.