This dissertation develops technology for microfluidic point-of-care immunoassay devices. This research (2004–2007) improved microfluidic immunoassay performance by reducing reagent consumption, decreasing analysis time, increasing sensitivity, and integrating processes using a lab-on-a-chip. Estimates show that typical hospital laboratories can save $1.0 million per year by using microfluidic chips. Our first objective was to enhance mixing in a microfluidic channel, which had been one of the main barriers to using these devices. Another goal of our studies was to simplify immunoassays by eliminating surfactants. Manufacturers of latex immunoassays add surfactants to prevent non-specific aggregation of microspheres. However, these same surfactants can cause false positives (and negatives) during diagnostic testing. This work, published in Appendix A (© 2006 Elsevier) shows that highly carboxylated polystyrene (HCPS) microspheres can replace surfactants and induce rapid mixing via diffusion in microfluidic devices. Our second objective was to develop a microfluidic device using fiber optics to detect static light scattering (SLS) of microspheres in Appendix B (© 2007 Elsevier). Fiber optics were used to deliver light emitting diode (LED) or laser light. A miniature spectrometer was used to measure 45° forward light scattering collected by optical fiber. Latex microspheres coated with PR3 proteins were used to test for the vasculitis marker, anti-PR3. No false negatives or positives were observed. A limit of detection (LOD) of 50 ng mL⁻¹ was demonstrated. This optical detection system works without fluorescence or chemiluminescence markers. It is cost effective, small, and re-usable with simple rinsing. The final objective in this dissertation, published in Appendix C (© 2007 Elsevier), developed a multiplex immunoassay. A lab-on-a-chip was used to detect multiple antibodies using microsphere light scattering and quantum dot (QD) emission. We conjugated QDs onto microspheres and named this configuration “nano-on-micro” or “NOM”. Upon radiation with UV light, strong light scattering is observed. Since QDs also provide fluorescent emission, we are able to use increased light scattering for detecting antigen-antibody reactions, and decreased QD emission to identify which antibody is present.

The problem of finding targeted medicine is a central problem in chemotherapy. From this point of view the ubiquitin-proteasome system is a highly promising object in the pharmaceutical approach. Proteasome plays a critical role in cellular protein degradation, cell cycle and apoptosis regulation. Proteasome inhibitors are substances blocking the actions of proteasome. Cancer cells are more sensitive to inhibition of the ubiquitin-proteasome system than normal cells. Therefore proteasome inhibitors have the potential to be successfully used in the cancer treatment. The study aimed to test various substances to identify possible proteasome inhibitors with the IncuCyteTM FLR image system and fluorometric microculture cytotoxicity assay. Using the IncuCyte FLR method allows for detecting changes in the molecular processes of living cells. To make proteasome inhibition visible the model cell line MelJuSoUbG76V-YFP is used which helps to detect alterations in proteasome activity by means of the yellow fluorescent protein enrichment in cells as a response to proteasome inhibition. Fluorometric microculture cytotoxicity assay is a method for the determination of cytotoxicity in human tumor cells. The study showed that substance #25 possessed a proteasome inhibitory capacity in a dose-dependent manner as demonstrated with the IncuCyte FLR image system. According to the fluorometric microculture cytotoxicity assay, substance #1 was the most stable and toxic. Substances #2 and #185 had selective toxicity against cancer cells and lower effects against normal cells. Combining IncuCyte FLR and fluorometric microculture cytotoxicity assay allows finding substances which act as proteasome inhibitors with high toxic effect.

Protein biomarkers circulating in blood hold the promise of improved diagnosis, prognosis and follow-up of treatment of disease via minimally invasive procedures. For the discovery and validation of such biomarkers, methods are needed that can facilitate parallel, highly specific and in-depth analysis of the blood proteome. The work presented in this thesis intends to develop and apply such assays, building on the concept of the proximity ligation assay (PLA). In paper I, I present an easy and non-expensive alternative for the conjugation of oligonucleotides to antibodies via biotin-streptavidin-biotin interaction. This approach can be used when large sets of antibodies and/or oligos need to be validated for their performance as probes in PLA reactions. In paper II, a solid-phase variant of PLA (SP-PLA) for the detection and quantification of proteins in blood is presented. SP-PLA exhibited an improved limit of detection compared to commercial ELISA assays by two orders of magnitude. In addition SP-PLA exhibited a broader dynamic range by at least one order of magnitude and required only 5 μl of sample, rendering the method very well suited for analyses of precious bio-banked material. Last but not least, SP-PLA was used to validate the diagnostic potential of GDF-15 as a biomarker for cardiovascular disease in a set of cardiovascular disease patients and healthy controls. Paper III discusses the development of a multiplex SP-PLA (MultiPLAy) for the simultaneous detection of 36 proteins in just 5 μl of sample. MultiPLAy exhibited an improved LOD when compared to state-of-the-art bead-based sandwich assays. Most importantly, we observed only a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that much higher levels of multiplexing will be possible. The assay was used to identify putative biomarkers in sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD). Subsequent multivariate analysis revealed previously known diagnostic biomarkers. Furthermore, we successfully applied next-generation sequencing as a readout for the protein assays, allowing for the first time digital recording of protein profiles in blood. In paper IV, we investigated the suitability of prostasomes as blood biomarkers in patients with prostate cancer using a newly developed PLA assay (4PLA) that utilizes five binders for the detection of complex target molecules. The assay successfully detected significantly elevated levels of prostasomes in blood samples from prostate cancer patients prior to radical prostatectomy, compared to controls and men with benign biopsy results.

Increased reactive oxygen species (ROS) have been reported to be at the centre of various diseases. Although several reports have implicated elevated levels of ROS in the pathogenesis of diabetes mellitus, the early detection of ROS is still not attainable. This limitation causes difficulty in the early diagnosis of ROS related disorders. The presence of high levels of ROS was reported to result in differential expression of antioxidant genes involved in protecting cells from their deleterious effects. Among the antioxidant genes that are expressed, it was postulated that expression of metallothioneins (MTs) are also induced. MTs are low molecular weight, cysteine-rich proteins involved in metal homeostasis and reported to harbour antioxidant function. The aim of this investigation was to explore MTs as biomarkers for elevated levels of ROS in whole blood of type 2 diabetic (T2D) individuals. The level of ROS in diabetic, non-diabetic as well as individuals at risk of developing T2D was determined via the use of biochemical assays. Real-Time PCR was utilised to analyse the expression of MTs and the presence of MT proteins was analysed via the ELISA. In this study it was observed that diabetic individuals had elevated levels of ROS. However, no significant difference in the expression of MTs and the presence of MT proteins between the diabetic and non-diabetic individuals was observed. In vitro experimental conditions indicated that MT expression is induced by elevated levels of ROS. In pathological conditions the ROS-dependent induction of MT expression needs to be elucidated further. It therefore can be suggested that MTs can not yet be utilised as biomarkers for the detection of elevated levels of ROS in pathological conditions with ROS aetiology. This investigation also highlights the fact that blood is not an optimal medium in which this objective can be attained. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2005.

POPs are a group of chemicals that have been extensively studied over the last few years. The main reason that these chemicals have received so much scientific attention is the myriad of negative effects they have on the environment and human health. The properties that cause the deleterious effects include a high molecular stability, rendering them highly persistent. Added to this is the lipophilic and hydrophobic nature of the compounds. POPs will thus tend to bio-accumulate and bio-magnify in the environment, causing a direct threat to humans and wildlife. To address this threat, the Stockholm Convention on Persistent Organic Pollutants, under the supervision of United Nations Environment programme (UNEP), was initiated and became legally binding on 17 May 2004. All countries, including South Africa, which ratified this agreement, will be expected to monitor and regulate the formation of POPs. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) are all members of the dioxin-like family of POPs. This family of chemicals pose serious health threats such as carcinogenic effects and negative effects on reproduction. These substances, with the exception of PCBs, are formed unintentionally as by-products of industrial and thermal processes. One of the main sources of dioxin-like chemicals is medical waste incinerators. In this project the area surrounding a medical waste incinerator was monitored using a bio-assay technique. The determination of dioxin concentrations is usually preformed by chemical analysis, however, bio-assays have proven themselves to be a cheaper and time-saving screening method. The Toxic Equivalency Quotient (TEQs) determined through bio-assays can support chemical analysis in determining biologically-relevant risk assessments since bio-assay data has ecotoxicological relevance. These assays represent an integrated biological response to chemical pollutants, where biological effects are accounted for which is not possible in chemical analyses. One of the bio-assays used in the determination of the dioxin-like chemical TEQ is the H411 E reporter gene bio-assay. This assay is based on the Ah-receptor mediated toxicity of dioxin-like chemicals. Using this technique the TEQs for areas surrounding an active incinerator were determined, to indicate the distribution of these substances. The TEQs for the soil samples collected ranged between nondetectable and 154 ngTEQ/kg. There was no clear distributional pattern and the total organic carbon content in the soil did not seem to play a crucial role in the distribution of dioxin-like chemicals. Although a decrease in soil tillage showed a corresponding increase in TEQ. The predominant wind direction was taken into account but no correlation could be seen. However, meteorological parameters such as the ambient temperature and low precipitation in the area may have contributed to lower TEQ values. Cytotoxicity excluded data points and the phenomenon has to be addressed. High TEQ values in a residential area where free-range chickens are raised pose a serious concern to the level of dietary dioxin-like chemical intake. Eggs in the area could theoretically contain between 2.75 and 28.75 pgTEQ/g egg fat. Further studies are needed to determine how much dioxin-like chemicals are being transferred to humans through the consumption of free-range eggs / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006.

Natural products have been a source of many pharmaceutical drugs and a number of drugs that are currently used in the treatment of cancer are derivatives of compounds originally isolated from natural products. There is evidence that extracts of Tulbaghia violacea can be used to treat cancer. The activation of apoptosis in cancer cells is a target for the development of novel anti-cancer drugs since one of the characteristics of cancer cells is resistance to apoptosis due to the deregulation of biochemical pathways leading to apoptosis. In fact, many current anti-cancer drugs exert their effects through the activation of apoptosis. Previous studies showed that extracts of T.violacea induce apoptosis in cancer cells and one study reported on the isolation of a compound (methyl-ԃ-D-glucopyranoside), which is responsible for the pro-apoptotic activity of the T.violacea extract. Therefore the aim of this study was to investigate the anti-cancer activity of methyl-ԃ-Dglucopyranoside and extracts prepared from T.violacea. In this study the pro-apoptotic activity of methyl-ԃ-D-glucopyranoside and extracts prepared from T.violacea were investigated on a panel of human cancer cell lines, which included HepG2, MCF7, H157, HT29 and the non-cancerous cell line, KMST6. The induction of apoptosis was evaluated by flow cytometry using several bioassays which measures biochemical events (caspase activation, phosphatidylserine externalisation and reactive oxygen species (ROS) production that is associated with the induction of apoptosis. The results demonstrated that the effects of methyl--D-glucopyranoside on cultured cells are transient and that the cells recover from the effects of methyl--D-glucopyranoside. This suggested thatmethyl-ԃ-D-glucopyranoside is not the compound responsible for the pro-apoptotic bioactivity in the T.violacea extract. This study also showed that cytotoxic and pro-apoptotic bioactivity of the leaf-extract was significantly higher in comparison to the tuber-extract. The bioactivity of the organic solvent extracts (dichloromethane, hexane, methanol and 50% methanol/water) of T.violacea leaves was also significantly higher than water extracts of T.violacea leaves. A comparison of the different organic extracts prepared from the T.violacea leaves showed that the highest activity was observed for the dichloromethane and hexane extracts. In an effort to identify the bioactive compound(s) the dichloromethane extract was subjected to Versaflash® column chromatography. However, due to problems experienced with the solubility of the dichloromethane sub-fractions, these compounds could not be tested for their bioactivity. Palmitone (16-hentriacontanone) was identified as one of the major compounds present in the dichloromethane sub-fractions. This compound was previously shown to have anticonvulsant bioactivity but there is no evidence in the literature that it has anti-cancer or pro-apoptotic activities. Fingerprinting of the methanol extract showed the presence of long chain fatty acid derivatives, flavonoids and allicin derivatives in the methanol extract. Although, this study failed to isolate the pro-apoptotic bioactive compound(s) present in the extracts of T.violacea, it confirmed that extracts of this plant induce apoptosis in cultured human cancer cell lines.

Natural products have been a source of many pharmaceutical drugs and a number of drugs that are currently used in the treatment of cancer are derivatives of compounds originally isolated from natural products. There is evidence that extracts of Tulbaghia violacea can be used to treat cancer. The activation of apoptosis in cancer cells is a target for the development of novel anti-cancer drugs since one of the characteristics of cancer cells is resistance to apoptosis due to the deregulation of biochemical pathways leading to apoptosis. In fact, many current anti-cancer drugs exert their effects through the activation of apoptosis. Previous studies showed that extracts of T.violacea induce apoptosis in cancer cells and one study reported on the isolation of a compound (methyl-ԃ-D-glucopyranoside), which is responsible for the pro-apoptotic activity of the T.violacea extract. Therefore the aim of this study was to investigate the anti-cancer activity of methyl-ԃ-Dglucopyranoside and extracts prepared from T.violacea. In this study the pro-apoptotic activity of methyl-ԃ-D-glucopyranoside and extracts prepared from T.violacea were investigated on a panel of human cancer cell lines, which included HepG2, MCF7, H157, HT29 and the non-cancerous cell line, KMST6. The induction of apoptosis was evaluated by flow cytometry using several bioassays which measures biochemical events (caspase activation, phosphatidylserine externalisation and reactive oxygen species (ROS) production that is associated with the induction of apoptosis. The results demonstrated that the effects of methyl--D-glucopyranoside on cultured cells are transient and that the cells recover from the effects of methyl--D-glucopyranoside. This suggested thatmethyl-ԃ-D-glucopyranoside is not the compound responsible for the pro-apoptotic bioactivity in the T.violacea extract. This study also showed that cytotoxic and pro-apoptotic bioactivity of the leaf-extract was significantly higher in comparison to the tuber-extract. The bioactivity of the organic solvent extracts (dichloromethane, hexane, methanol and 50% methanol/water) of T.violacea leaves was also significantly higher than water extracts of T.violacea leaves. A comparison of the different organic extracts prepared from the T.violacea leaves showed that the highest activity was observed for the dichloromethane and hexane extracts. In an effort to identify the bioactive compound(s) the dichloromethane extract was subjected to Versaflash® column chromatography. However, due to problems experienced with the solubility of the dichloromethane sub-fractions, these compounds could not be tested for their bioactivity. Palmitone (16-hentriacontanone) was identified as one of the major compounds present in the dichloromethane sub-fractions. This compound was previously shown to have anticonvulsant bioactivity but there is no evidence in the literature that it has anti-cancer or pro-apoptotic activities. Fingerprinting of the methanol extract showed the presence of long chain fatty acid derivatives, flavonoids and allicin derivatives in the methanol extract. Although, this study failed to isolate the pro-apoptotic bioactive compound(s) present in the extracts of T.violacea, it confirmed that extracts of this plant induce apoptosis in cultured human cancer cell lines.

A modified yeast one-hybrid (MY1H) system has been developed for in vivo investigation of simultaneous protein-protein and protein:DNA interactions. The traditional yeast one-hybrid assay (Y1H) permits examination of one expressed protein targeting one DNA site, whereas our MY1H allows coexpression of two different proteins and examination of their activity at the DNA target. This single-plasmid based MY1H was validated by use of the DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. The MY1H system could be used to examine proteins that contribute inhibitory, repressive, coactivational or bridging functions to the protein under investigation, as well as potential extension toward library screening for identification of novel accessory proteins. After development and validation of the MY1H with the p53/LTAg/53BP2 system, we applied the MY1H system to investigate the DNA binding activities of heterodimeric proteins, the bHLH/PAS domains of AhR and Arnt that target the xenobiotic response element (XRE). The AhR/Arnt:XRE interaction, which served as our positive control for heterodimeric protein binding of the XRE DNA site, showed negative signals in initial MY1H experiments. These false negative observations were turned into true positives by increasing the number of DNA target sites upstream of the reporter genes and increasing the number of activator domains fused to the two monomers. This methodology may help trouble-shooting false negatives stemming from unproductive transcription in yeast genetic assays, which can be a common problem. In the study of XRE-binding proteins, two bHLHZ-like hybrid proteins, AhRJunD and ArntFos were designed and coexpressed in the MY1H and yeast two-hybrid (Y2H) systems; these proteins comprise the bHLH domains of AhR and Arnt fused to the leucine zipper (LZ) elements from bZIP proteins JunD and Fos, respectively. The in vivo assays revealed that in the absence of the XRE DNA site, heterodimers and homodimers formed, but in the presence of the nonpalindromic XRE, only heterodimers bound to the XRE and activated reporter transcription. The present results provide valuable information on DNA-mediated protein heterodimerization and specific DNA binding, as well as the relationship between protein structure and DNA-binding function.

A modified yeast one-hybrid (MY1H) system has been developed for in vivo investigation of simultaneous protein-protein and protein:DNA interactions. The traditional yeast one-hybrid assay (Y1H) permits examination of one expressed protein targeting one DNA site, whereas our MY1H allows coexpression of two different proteins and examination of their activity at the DNA target. This single-plasmid based MY1H was validated by use of the DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. The MY1H system could be used to examine proteins that contribute inhibitory, repressive, coactivational or bridging functions to the protein under investigation, as well as potential extension toward library screening for identification of novel accessory proteins. After development and validation of the MY1H with the p53/LTAg/53BP2 system, we applied the MY1H system to investigate the DNA binding activities of heterodimeric proteins, the bHLH/PAS domains of AhR and Arnt that target the xenobiotic response element (XRE). The AhR/Arnt:XRE interaction, which served as our positive control for heterodimeric protein binding of the XRE DNA site, showed negative signals in initial MY1H experiments. These false negative observations were turned into true positives by increasing the number of DNA target sites upstream of the reporter genes and increasing the number of activator domains fused to the two monomers. This methodology may help trouble-shooting false negatives stemming from unproductive transcription in yeast genetic assays, which can be a common problem. In the study of XRE-binding proteins, two bHLHZ-like hybrid proteins, AhRJunD and ArntFos were designed and coexpressed in the MY1H and yeast two-hybrid (Y2H) systems; these proteins comprise the bHLH domains of AhR and Arnt fused to the leucine zipper (LZ) elements from bZIP proteins JunD and Fos, respectively. The in vivo assays revealed that in the absence of the XRE DNA site, heterodimers and homodimers formed, but in the presence of the nonpalindromic XRE, only heterodimers bound to the XRE and activated reporter transcription. The present results provide valuable information on DNA-mediated protein heterodimerization and specific DNA binding, as well as the relationship between protein structure and DNA-binding function.

The electronegativity of thiolate ligation in the hemeprotein nitric oxide synthase (NOS) proteins has been identified as an influence on autoinhibition in this enzyme. The mutation of a conserved tryptophan residue, which hydrogen bonds to the coordinating thiolate ligand and therefore influences its electronegativity, to either phenylalanine or tyrosine has had various effects including heme loss and dimer disruption in the inducible isoforms, while hyperactivity occurs in the neuronal isoforms. I have performed the analogous mutations in W180 of eNOSoxy, the endothelial isoform. UV/visible and resonance Raman spectroscopy have demonstrated that the mutants experienced increased basicity of the thiolate due to loss of the hydrogen bond between the mutated residue in the absence of the cofactor (6R)5,6,7,8-tetrahydrobiopterin (H4B). The mutants also displayed relative rates of NO2- production that were comparable to the nNOSoxy mutants, which is consistent with the nNOSoxy results. The presence of H4B alters porphyrin planarity, which enabled hydrogen bonding to occur in W180Y, thus restoring thiolate basicity to that of wild-type eNOSoxy. Reduced overall activities by the proteins suggest that H4B stabilizes the heme. The analogous W56 mutants of saNOS, a NOS oxygenase domain-like protein from Staphylococcus aureus (saNOS), have been previously characterized using resonance Raman spectroscopy. These mutants also exhibit increased thiolate electronegativity over wild-type. As the homodimers had already been investigated, saNOS was an ideal system in which to explore heterodimers. Heterodimers were generated through the co-expression of one wild-type and one mutated subunit, enabling the examination of each subunit individually through resonance Raman spectroscopy. The subunits of the resulting proteins were shown to have heme environments that resembled those of their corresponding homodimers. The activity of saNOS did not vary significantly for the various W56 mutants, suggesting that saNOS catalysis may be unaffected by thiolate electronegativity. / Thesis (Master, Chemistry) -- Queen's University, 2008-09-04 11:37:38.688