The enzyme spermidine/spermine N1-acetyltransferase (SSAT) is a candidate biomarker for various cancers as its activity in cancerous tissues is significantly increased. An artificial molecule, amantadine, is exclusively acetylated by SSAT to acetylamantadine (AcAm), levels of which in urine can serve as a proxy biomarker for malignancy. Current method of AcAm detection is laborious, time-consuming, and lacks the possibility of transforming to a point-of-care device. In this thesis, two different approaches were applied to detect AcAm in deionized water and in human urine using optical methods. The first one was fluorescence-based indicator displacement assay using cucurbit[7]uril as the receptor molecule. The second was programmed gold nanoparticle disaggregation with cucurbit[7]uril as a molecular linker. / Graduate

Aptamers are short nucleic acid ligands generated by the process of iterative selection. Nucleic acid counterparts to protein antibodies, aptamers bind their targets with relatively high affinities by assuming characteristic shapes. Highly thermostable, open to manipulations and non-immunogenic, these olignucleotides can be readily adapted to a variety of diagnostic assays and harvested for their therapeutic potential. We have particularly focused on the unique prospects that stem from their localization patterns both in vitro and in vivo. While several assays exist for protein diagnostics, many of these are limited by the amount of target they can detect. To overcome these limitations it might prove effective to couple protein detection with nucleic acid based amplification. The Proximity Ligation Assay (PLA) is an innovative technique that facilitates protein detection on a zeptomolar range by amplifying a tiny signal via the polymerase chain reaction. PLA is based on the concept that two DNA tags when co-localized adjacent to one another on a protein surface and ligated via a connector nucleotide will form a unique amplicon that can detected using real-time PCR and in turn detect the protein. We have adapted PLA to the peptide based detection of Bacillus spores as well as the RNA aptamer based detection of cancer cells. Highly sensitive and specific, nucleic acid based PLA could serve as a promising tool in diagnostics. Aptamers have also been analyzed for their localization patterns in vivo. Using two anti-prostate specific membrane antigen RNA aptamers, we have demonstrated that there is an inherent bias for some circulating oligonucleotides over others based solely on their sequence. This phenomenon has also been explored in cancer models of mice for persistence of specific aptamers over others in tumors for therapy. An in vivo “Stealth” selection scheme has also been designed and executed to hunt for stable and robust aptamer species that are naturally chosen for survival within a mouse system. Generation of such ligands could benefit several therapeutic ventures such as targeted drug delivery past complex vasculature as in the case of the blood:brain barrier. / text

NADPH-cytochrome P450 reductase is the electron transfer partner for the cytochromes P450, heme oxygenase, and squalene monooxygenase, and is a component of the nitric oxide synthases and methionine synthase reductase. P450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine (W677A) has been shown by others to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP+ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently in vivo without inhibition by NADP+. Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (kcat/Km) with NADH. Inhibition by NADP+ was reduced by up to four orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2- to 3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P450 2E1 Ames bacterial mutagenicity assay the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay. In addition to providing a valuable tool for understanding the determinants of nucleotide cofactor specificity in this and related enzymes, these mutants might also lend themselves to creation of bioremediation schemes with increased enzymatic activity and robustness in situ, as well as cost-effective reconstitution of enzyme systems in vitro that do not require the use of expensive reducing equivalents from NADPH.

<p>ABSTRACT</p><p>Chemotherapy is a common method used for cancer treatment. Especially when it concerns cancers that have grown invasively it seems to be the only efficient treatment due to the substances ability to reach and affect almost the entire body. One major obstacle regarding chemotherapy is that the patients often develop resistance to the cytotoxic substances used. Fluorometric microculture cytotoxicity assay (FMCA) is a method developed to measure sensitivity of tumor cells to different cytotoxic substances in vitro. The assay is based on hydrolysis of fluorescein diacetate to fluorescein by cells with intact cell membranes after incubation with drugs for 72 hours. This study investigated the impact of two methodological factors that may cause errors in the achieved results; namely the possible occurrence of drug decay during incubation and the use of an inappropriate amount of cells. These factors were tested by exposing the cytotoxic drugs to pre-incubation in absence of tumor cells for different times and to use suspensions with different concentrations of cells. The results indicated occurrence of drug decay in 3 of the 18 substances tested and that the amount of cells affected the results for most of the drugs tested but to different extent.</p>

The oxidation of lipids and antioxidants has been extensively studied in human plasma but little attention has been given to how plasma proteins are oxidised. Proteins make up the majority of biomolecules in cells and plasma and therefore are the most likely reactants with oxidants and free radicals. Previous studies in the laboratory had shown that peroxyl radicals generated by the thermolytic decay of 2-azobis (2-amdinopropane) dihydrochloride (AAPH) generated significant amounts of protein hydroperoxides, but only after a six hour lag period. In this study the existence of the six hour lag period was confirmed and shown by dialysis of the plasma to be due to the presence of low molecular weight antioxidants. The addition of both uric acid and ascorbic acid to the dialysed plasma restored the lag phase suggesting that in vivo these antioxidants act to prevent protein hydroperoxide formation. Lipid oxidation was also observed in the plasma but only after a two hour lag phase. This was the first time lipid oxidation has been observed in the absence of protein oxidation. The lipid lag phase was also abolished by dialysis of the plasma and restored by the addition of ascorbic acid and uric acid. The kinetics of tocopherol loss suggests that the tocopherol radicals act to inhibit lipid oxidation by transferring the electrons to the water-soluble ascorbate. The loss of ascorbate appears to cause the formation of a tocopherol radical mediate the lipid peroxidation process. Overall the data shows ascorbic acid scavenging the peroxyl radicals while uric acid acts to reduce the overall AAPH generated radical flux. In a separate investigation, the production of protein-bound DOPA (PB-DOPA) on albumin during X-ray radiolysis and copper mediate Fenton oxidation was investigated using a fluorescence based derivatisation method (ED-DOPA), which was compared with the more specific acid hydrolysis and HPLC analysis method. The ED-DOPA method consistently gave a much higher reading that the HPLC based methods, suggesting that the ED-DOPA method was measuring DOPA plus DOPA oxidation products. This was confirmed by oxidising X-ray radiolysis generated PB-DOPA with Cu++ to cause DOPA oxidation. The Cu++ treatment drastically increased the level of signal given by the ED-DOPA assay while HPLC analysis showed all the DOPA had been oxidised.

A quantitative competitive binding "triple sandwich" enzyme immunoassay was used to evaluate pathogen/class-specific antibody responses in Holstein-Friesian calves vaccinated against Clostridium perfringens B-toxin at various ages postpartum. Vaccination of dams at six weeks and again at two weeks prepartum increased pathogen-specific antibody levels in their colostrum and respective calf's serum. Calves initially vaccinated at three days produced both a primary and secondary pathogen-specific antibody response, whereas calves initially vaccinated at 12 and 21 days produced only secondary responses. Maternally-derived antibodies were found to suppress neonatal antibody production following primary immunization. They were also found to influence secondary humoral immune responses, although in a diminished capacity. Pathogen-specific IgG and IgM concentrations in dams' sera and colostra were found related to subsequent pathogen-specific IgG and IgM neonatal serum concentrations. Only pathogen-specific IgA in dams' colostra was correlated to neonatal levels, possibly owing to a different origin and role of this immunoglobulin class. All class-specific colostral immunoglobulin levels were related to subsequent neonatal concentrations. Based on results from this experiment, it is recommended that calves be vaccinated at three days postpartum with a booster administered at 63 days.

Metabolically active microbial communities are present in a wide range of subsurface environments. Techniques like enumeration of microbial cells, activity measurements with radiotracer assays and the analysis of porewater constituents are currently being used to explore the subsurface biosphere, alongside with molecular biological analyses. However, many of these techniques reach their detection limits due to low microbial activity and abundance. Direct measurements of microbial turnover not just face issues of insufficient sensitivity, they only provide information about a single specific process but in sediments many different process can occur simultaneously. Therefore, the development of a new technique to measure total microbial activity would be a major improvement. A new tritium-based hydrogenase-enzyme assay appeared to be a promising tool to quantify total living biomass, even in low activity subsurface environments. In this PhD project total microbial biomass and microbial activity was quantified in different subsurface sediments using established techniques (cell enumeration and pore water geochemistry) as well as a new tritium-based hydrogenase enzyme assay. By using a large database of our own cell enumeration data from equatorial Pacific and north Pacific sediments and published data it was shown that the global geographic distribution of subseafloor sedimentary microbes varies between sites by 5 to 6 orders of magnitude and correlates with the sedimentation rate and distance from land. Based on these correlations, global subseafloor biomass was estimated to be 4.1 petagram-C and ~0.6 % of Earth's total living biomass, which is significantly lower than previous estimates. Despite the massive reduction in biomass the subseafloor biosphere is still an important player in global biogeochemical cycles. To understand the relationship between microbial activity, abundance and organic matter flux into the sediment an expedition to the equatorial Pacific upwelling area and the north Pacific Gyre was carried out. Oxygen respiration rates in subseafloor sediments from the north Pacific Gyre, which are deposited at sedimentation rates of 1 mm per 1000 years, showed that microbial communities could survive for millions of years without fresh supply of organic carbon. Contrary to the north Pacific Gyre oxygen was completely depleted within the upper few millimeters to centimeters in sediments of the equatorial upwelling region due to a higher supply of organic matter and higher metabolic activity. So occurrence and variability of electron acceptors over depth and sites make the subsurface a complex environment for the quantification of total microbial activity. Recent studies showed that electron acceptor processes, which were previously thought to thermodynamically exclude each other can occur simultaneously. So in many cases a simple measure of the total microbial activity would be a better and more robust solution than assays for several specific processes, for example sulfate reduction rates or methanogenesis. Enzyme or molecular assays provide a more general approach as they target key metabolic compounds. Since hydrogenase enzymes are ubiquitous in microbes, the recently developed tritium-based hydrogenase radiotracer assay is applied to quantify hydrogenase enzyme activity as a parameter of total living cell activity. Hydrogenase enzyme activity was measured in sediments from different locations (Lake Van, Barents Sea, Equatorial Pacific and Gulf of Mexico). In sediment samples that contained nitrate, we found the lowest cell specific enzyme activity around 10^(-5) nmol H_(2) cell^(-1) d^(-1). With decreasing energy yield of the electron acceptor used, cell-specific hydrogenase activity increased and maximum values of up to 1 nmol H_(2) cell^(-1) d^(-1) were found in samples with methane concentrations of >10 ppm. Although hydrogenase activity cannot be converted directly into a turnover rate of a specific process, cell-specific activity factors can be used to identify specific metabolism and to quantify the metabolically active microbial population. In another study on sediments from the Nankai Trough microbial abundance and hydrogenase activity data show that both the habitat and the activity of subseafloor sedimentary microbial communities have been impacted by seismic activities. An increase in hydrogenase activity near the fault zone revealed that the microbial community was supplied with hydrogen as an energy source and that the microbes were specialized to hydrogen metabolism. / Mikrobielle Gesellschaften und ihre aktiven Stoffwechselprozesse treten in einer Vielzahl von Sedimenten unterschiedlichster Herkunft auf. In der Erforschung dieser tiefen Biosphäre werden derzeit Techniken wie Zellzählungen, Aktivitätsmessungen mit Radiotracer-Versuchen und Analysen der Porenwasserzusammensetzung angewendet, darüber hinaus auch molekularbiologische Analysen. Viele dieser Methoden stoßen an ihre Nachweisgrenze, wenn Sedimente mit geringer Zelldichte und mikrobieller Aktivität untersucht werden. Bei der Untersuchung von Stoffwechselprozessen mit herkömmlichen Techniken kommt dazu, dass von mehreren Prozessen, die zeitgleich ablaufen können, jeweils nur einer erfasst wird. Deswegen wäre die Entwicklung einer neuartigen Messtechnik für die gesamte mikrobielle Aktivität ein wesentlicher Fortschritt für die Erforschung der tiefen Biosphäre. Ein vielversprechender Ansatz, um die gesamte lebende Biomasse auch in Proben mit geringer Aktivität zu bestimmen, ist eine Hydrogenase-Enzym-Versuchsanordnung mit Tritium als quantifizierbarer Messgröße. In dieser Doktorarbeit wurde die gesamte mikrobielle Biomasse und Aktivität von unterschiedlichen Sedimentproben einerseits mit herkömmlichen Methoden (Zellzählungen, Analyse der Porenwasserzusammensetzung) als auch mit einer neu entwickelten Hydrogenase-Enzym-Versuchsanordnung quantifiziert. Mit einer großen Anzahl eigener Zellzählungsdaten von Sedimenten aus dem Äquatorialpazifik und dem Nordpazifik und ergänzenden publizierten Daten konnte gezeigt werden, dass Zellzahlen sich in ihrer globalen geographischen Verteilung je nach Bohrlokation um 5 bis 6 Größenordnungen unterscheiden. Dabei bestehen Korrelationen zur Sedimentationsrate und zur Entfernung zum Land, mit deren Hilfe sich die Gesamtbiomasse in Tiefseesedimenten zu 4,1 Petagramm-C abschätzen lässt. Das entspricht ~0,6 % der Gesamtbiomasse der Erde und ist damit erheblich weniger als in früheren Schätzungen angegeben. Trotz der Korrektur auf diesen Wert spielt die Biomasse der tiefen Biosphäre weiterhin eine erhebliche Rolle in biogeochemischen Kreisläufen. Um die Zusammenhänge zwischen Aktivität der Mikroben, der Häufigkeit ihres Auftretens und Zustrom von organischem Material zu verstehen, wurde eine Expedition ins Auftriebsgebiet des Äquatorialpazifiks und zum nordpazifischen Wirbel durchgeführt. Daten der Sauerstoffaufnahme in Sedimenten des nordpazifischen Wirbels, die mit Sedimentationsraten von 1 mm pro 1000 Jahren abgelagert werden, zeigen, dass mikrobielle Gesellschaften über Millionen von Jahren ohne Zufuhr von frischem organischen Kohlenstoff überleben konnten. Im Gegensatz zum nordpazifischen Wirbel wird in Sedimenten des äquatorialpazifischen Auftriebsgebiets Sauerstoff bei höherer mikrobieller Aktivität und Verfügbarkeit organischer Verbindungen oberflächennah in den ersten Milli- bis Zentimetern komplett umgesetzt. Auftreten und Variabilität von Elektronenakzeptoren nach Tiefe und Bohrlokation machen die tiefe Biosphäre zu einer komplexen Umgebung für die Quantifizierung der gesamten mikrobiellen Aktivität. Aktuelle Studien zeigen das verschiedene Elektronenakzeptorprozesse gleichzeitig ablaufen können, obwohl man bisher davon ausgegangen war, dass diese sich thermodynamisch ausschließen. In vielen Fällen wäre also eine einfache Methode zur Messung der gesamten mikrobiellen Aktivität eine bessere und verlässlichere Lösung aktueller Analyseaufgaben als Messungen mehrerer Einzelprozesse wie beispielsweise Sulfatreduktion und Methanogenese. Enzym-oder Molekular-Versuchsanordnungen sind ein prozessumfassender Ansatz, weil hier Schlüsselkomponenten der Stoffwechselprozesse untersucht werden. Das Hydrogenase-Enzym ist eine solche Schlüsselkomponente und in Mikroben allgegenwärtig. Deshalb kann die Quantifizierung seiner Aktivität mit der neu entwickelten Hydrogenase-Enzym-Versuchsanordnung als Parameter für die gesamte mikrobielle Aktivität der lebenden Zellen verwendet werden. Hydrogenase-Aktivitäten wurden in Sedimenten unterschiedlicher Lokationen (Vansee, Barentssee, Äquatorialpazifik, und Golf von Mexico) gemessen. In Sedimentproben, die Nitrat enthielten, haben wir mit ca. 10^(-5) nmol H_(2) cell^(-1) d^(-1) die geringste zellspezifische Hydrogenase-Aktivität gefunden. Mit geringerem Energiegewinn des genutzten Elektronenakzeptors steigt die zellspezifische Hydrogenase-Aktivität. Maximalwerte von bis zu 1 nmol H_(2) cell^(-1) d^(-1) wurden in Sedimentproben mit >10 ppm Methankonzentration gefunden. Auch wenn die Hydrogenase-Aktivität nicht direkt in die Umsatzrate eines spezifischen Prozesses konvertierbar ist, können zellspezifische Aktivitätsfaktoren verwendet werden, um die metabolisch aktive Mikrobenpopulation zu quantifizieren. In einer weiteren Studie mit Sedimenten des Nankai-Grabens zeigen Daten der Zelldichte und der Hydrogenase-Aktivität einen Einfluss von seismischen Ereignissen auf Lebensraum und Aktivität der mikrobiellen Gesellschaften. Ein Anstieg der Hydrogenase-Aktivität nahe der Verwerfungszone machte deutlich, dass die mikrobiellen Gesellschaften mit Wasserstoff als Energiequelle versorgt wurden und dass die Mikroben auf einen Wasserstoff-Stoffwechsel spezialisiert waren.

Breast cancer is a disease that afflicts a significant proportion of women globally. 5-10% of breast cancer cases are linked to inherited polymorphisms in critical genes such as BRCA1, a tumour suppressor essential for genomic stability. A dysfunctional BRCA1 gene can increase breast cancer risk by 60-80%. Previous microarray analysis established that differential gene expression in unperturbed Epstein-Barr virus transformed lymphocyte cell lines (EBV-LCL) was able to distinguish BRCA1 mutation carriers from controls with a high degree of accuracy. A 43-gene radiation-independent classifier for BRCA1 status was constructed. We hypothesize that this differential gene expression can be observed in a subset of these genes using quantitative PCR (qPCR) in both EBV-LCL and B-lymphocytes isolated from patients with known BRCA1 mutation carrier status. The 43-gene classifier was analyzed using gene ontology analysis and 4 target genes selected based on predictive value, expression intensity and gene ontology similarity. Genes selected were CXCR3, TBX21, MX2, and IFIT1, with GusB as an endogenous reference gene. EBV-LCL established from known BRCA1 mutation carriers and from BRCA1 wildtype individuals were obtained and RT-qPCR (reverse transcriptase qPCR) performed on isolated RNA. Our results showed significant downregulation of CXCR3 and TBX21 in BRCA1 mutation carriers (p=0.018 and p=0.003, respectively), as expected from previous microarray results. IFIT1, while showing a non-significant upregulation (p=0.183), agreed with the expected trend. MX2 did not show significant differential expression. These results indicate that differential gene expression has the potential to accurately distinguish pathogenic variants, even if it may require EBV immortalization of B-lymphocytes. To determine whether the assay could be extended to fresh blood samples, B-lymphocytes were isolated from patients with known BRCA1 mutation carrier status from North York General Hospital in Toronto, ON. An optimized protocol to enrich the B-lymphocyte population using magnetic separation was developed for this purpose. RT-qPCR using RNA isolated from these lymphocytes showed no significant differential gene expression in CXCR3 and TBX21. However, a low sample size, use of non-sequenced controls and a need for further qPCR optimization may call these results into question. In addition, problems with blood sample transportation from off-site sources resulted in an unacceptable drop in RNA integrity. While this gene expression assay may be limited to screening a small number of blood samples, results indicate that may still have clinical relevance that can be explored. This would necessitate further optimization of the qPCR methodology and resolution of the issues surrounding RNA integrity and sample transport. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-09-26 13:13:50.809

Petrol attendants are exposed to petrol volatile organic compounds (VOCs) which may have genotoxic and carcinogenic effects. The single cell gel electrophoresis assay (comet assay) is a method highly sensitive to DNA damage induced by environmental and occupational exposure to carcinogenic and mutagenic agents. The aim of this study was to evaluate the level of exposure of petrol attendants to petrol VOCs and also to determine their effect on DNA damage and repair in lymphocytes of African petrol attendants. The exposed group consisted of 20 subjects, randomly selected from three petrol stations. A control group of 20 unexposed subjects was also chosen and matched for age and smoking habits with the exposed group. Sorbent tubes were used to assess personal exposure of petrol attendants. The comet assay was used to investigate the basal DNA damage and repair capacity in isolated lymphocytes of petrol attendants and control subjects. Blood samples were taken from the petrol attendants at the end of their 8 hour working shift and also from the control subjects. The petrol attendants were found to be exposed to levels of petrol VOCs lower than the occupational exposure limit (OEL) for constituent chemicals. A significant relationship was found between the volume of petrol sold during the shift and the average concentrations of benzene, toluene and the total VOCs measured. However, relative humidity had a negative correlation with the average concentrations of benzene, toluene, xylene and the total VOCs. Significantly higher basal DNA damage was observed with the exposed group compared to the control group. The period of exposure influenced the level of DNA damage and the calculated repair capacity. Smoking and age had a significant influence on the level of DNA damage. DNA repair capacity was delayed in smokers of both exposed and non-exposed group. / Thesis (M.Sc. (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2008.

Regular physical activity (PA) leads to an increased quality of life by means of certain physiological adaptations. Regular PA is beneficial to the human body and its functionality, including the physiological, biochemical and even psychological modalities. During PA an increased burden is placed on all physiological mechanisms due to the increased energy demand, resulting in an adaptation of the physiological systems. Currently the biochemical mechanisms by which these adaptations occur are not well understood or defined. During the flow of electrons through the electron transport chain in the mitochondria free radicals and reactive oxygen species (ROS) are produced. PA results in increased ROS production. The relationship of different exercise intensities and ROS production with resulting DNA damage is unclear. These free radicals and ROS disturb the pro-oxidant anti-oxidant balance resulting in oxidative stress. When this balance is disturbed oxidative stress could lead to potential oxidative damage, Oxidative damage occurs in lipid, protein and nucleic acid macromolecules. ROS can attack DNA bases or deoxyribose residues to produce damaged bases and/or single and double strand breaks. When the DNA is regarded and the damages are replicated it could cause mutations or apoptosis, affecting the cell function and physiology. The purpose of this study was to investigate the influence of different aerobic intensities on oxidative DNA damage and repair in middle aged men by means of the Comet assay. Five PA males and five physically inactive males were assigned to an experimental and control group respectively. The subjects did not differ significantly at baseline. The VO2-max of each subject was determined at baseline. Subjects were then randomly assigned to 60, 70, 80 and 90% of individual baseline VO2-max intensities for an acute exercise intervention of 30 minutes on a bicycle ergometer. Blood sampling was done at baseline, post-exercise and 24 hours post-exercise for oxygen radical absorbance capacity (ORAC) and hydroperoxide analysis (dROM). Peripheral blood was obtained for DNA damage testing by means of Comet analysis at baseline, post-exercise, 5, 15, 30 minutes, and also 6, 12, 24, 48 and 72 hours after exercise. The results obtained indicated that subjects who regularly participate in PA had an increased baseline reading of ORAC and dROM values. ORAC levels after each acute exercise session increased, with the highest increase in the control group, with a decrease in the direction of baseline readings 24 hours post exercise. A biphasic damage-repair cycle over the 72 hour period was observed with the Comet analysis. The most damaged cells occur directly after acute exercise. The highest incidence of DNA damage over a 72 hour period was observed at 70% VO2-max, with the least amount of damage after 90% VO2-max. In conclusion the study indicates stress proteins or other kinds of physiological reaction to minimize the damaging effect of oxidative stress, is in place to restore the cell's homeostasis. Thus PA results in the development of oxidative DNA damage. To minimize DNA damage the optimal intensity for acute physical exercise is between 70-80% VO2-max. At higher intensities the release of stress proteins are initiated to buffer the damaging effect of oxidative stress and to restore homeostasis. / Thesis (M.Sc. (Human Movement Science))--North-West University, Potchefstroom Campus, 2007.