Glyphosat ist eines der weltweit am häufigsten eingesetzten Herbizide. Sein Einsatz wird u.a. auf Grund einer möglichen karzinogenen Wirkung und eines möglichen negativen Einflusses auf die Biodiversität kritisch diskutiert. Um Aussagen über die Verbreitung von Glyphosat in der Umwelt treffen zu können, werden verlässliche Nachweissysteme benötigt. Das Ziel der vorliegenden Arbeit bestand darin, ein einfaches optisches System zum schnellen Nachweis von Glyphosat in wässrigen Proben, basierend auf einer Hydrophobin-funktionalisierten Oberfläche, die das Glyphosat Zielprotein präsentiert, zu entwickeln. Hierfür wurden verschiedene Fusionsproteine aus dem Glyphosat Zielprotein, der 5-Enolpyruvylshikimat-3-phosphatsynthase (EPSPS, hier aus dem Bakterium Escherichia coli (EcEPSPS)) und dem zur Selbstassemblierung an hydrophilen/hydrophoben Grenzflächen befähigten Hydrophobin Ccg2 aus Neurospora crassa erzeugt, welche für die Oberflächenfunktionalisierung eingesetzt wurden. Die Expression und Reinigung der Fusionsproteine und von Ccg2 in E. coli verlief erfolgreich. Nach initialen Kontaktwinkelmessungen zur Untersuchung der Funktionalität des Hydrophobins und Enzymaktivitätsmessungen für die Fusionsproteine, konnte deren Aktivität auch nach der Reinigung nachgewiesen werden. Dabei erwies sich das Fusionsprotein Ccg2_GS_EcEPSPS, aufgrund einer hohen enzymatischen Aktivität nach Immobilisierung, als am besten geeignet. Es wurden verschiedene Belegverhältnisse zwischen Hydrophobin und Fusionsprotein untersucht, um etwaige sterische Behinderungen zu minimieren. Hierbei erwies sich ein Belegverhältnis von 1 µM Ccg2_GS_EcEPSPS und 5 µM Ccg2 für die künftigen Messungen als gut geeignet. Auf Basis der so funktionalisierten Oberfläche wurden zwei Verfahren zum optischen Nachweis von Glyphosat entwickelt. Eines der Verfahren, der Malachitgrün-Assay, weist die enzymatische Aktivität der EPSPS auf der Oberfläche nach, genauer das entstehende anorganische Phosphat (Pi). Durch Glyphosathemmung entsteht weniger Pi, dies kann mittels Malachitgrün-Assay nachgewiesen werden. Unter Laborbedingungen konnte ein Detektionslimit von 50 nM erreicht werden. Des Weiteren zeigte der Assay keine nennenswerte Querempfindlichkeit und erwies sich damit als sehr spezifisch. Zusätzlich wurde der Einfluss unterschiedlicher Temperaturen und pH-Werte untersucht. Es zeigte sich, dass Schwankungen dieser Parameter den Assay beeinflussen. Auch ein Einfluss der Ionenstärke konnte festgestellt werden. Deshalb sind entsprechende Kontrollen unerlässlich. Der Einfluss nicht-reaktionsbedingten Phosphates konnte durch Vorinkubation der Oberfläche mit der Glyphosat-haltigen Analyselösung mit anschließender Entfernung der Selbigen und Durchführung des Malachitgrün-Assays minimiert werden. Das zweite Verfahren, der Hydrogelsonden (HGS)-Assay, weist direkt die Interaktion von Glyphosat und der immobilisierten EcEPSPS nach. Hierfür wurden verformbare, Glyphosat-dekorierte HGS aus Polyethylenglykol benötigt. Bei Abwesenheit von freiem Glyphosat liegen die Bindestellen der immobilisierten EPSPS frei vor, sodass sie für die Bindung des immobilisierten Glyphosats an den HGS zur Verfügung stehen. Zwischen den HGS und der Oberfläche entsteht auf diese Weise eine große Kontaktfläche, welche mittels Reflektionsinterferenzkontrastmikroksopie messbar ist. Freies Glyphosat in der Analyselösung reduziert die verfügbaren Bindestellen an der Oberfläche. Dies resultiert in einer kleineren Kontaktfläche. Auf diese Weise kann durch Ermittlung der Größe der Kontaktfläche zwischen HGS und funktionalisierter Oberfläche und der daraus berechneten Adhäsionsenergie, auf das Vorhandensein von Glyphosat in der Analyselösung geschlossen werden. Im Rahmen dieser Arbeit konnte nach Optimierung der Oberflächenbeschichtung, ein positiver Machbarkeitsbeweis für dieses Verfahren erbracht werden.

Autoxidation, a radical chain reaction, is largely responsible for the degradation of most man-made and biological materials. These include chemical products such as lubricants, plastics and rubber; as well as biological molecules and membranes within our bodies. The development of means to hinder this process has been a major focus of the petroleum, chemical, pharmaceutical and biotechnology industry over the past century. The two most common strategies to emerge from these efforts have been the use of compounds that either prevent the initiation of autoxidation or trap the propagating radicals, so-called radical-trapping antioxidants (RTAs). Herein, we describe our efforts towards the design and development of extremely potent heterocyclic diarylamine RTAs, and their activity in a variety of applications ranging from isotropic organic solution to mammalian cells. We have elucidated the important structural motifs and mechanistic considerations necessary for the development of next-generation arylamine RTAs. Some of the substituted heterocyclic diarylamines analogs we disclose are among the best inhibitors of high temperature autoxidations described to date. Alongside, we developed novel analytical tools to facilitate the studies and acquisition of results for characterizing RTA activity in organic solutions and lipid bilayers. These fluorescent probes are highly relevant and allow for the determination of hydroperoxide and acid concentrations rapidly, as well as screen (or counter-screen) RTAs in liposomal membranes. Our methodologies address numerous drawbacks from frequently used ‘plug-and-play’ assays and we anticipate they will fill a current unmet need in both industrial and academic laboratories worldwide. Moreover, the recent characterization of ferroptosis – a novel regulated necrotic-like cell-death pathway associated with the accumulation of lipid hydroperoxides – has paved a way forward for studying oxidation induced damage in a biological context. Utilizing our expertise in lipid peroxidation and inhibition, we elucidated the prominent role of autoxidation in the execution of ferroptotic cell death. Alongside, our analytical tools and RTAs have also enabled the identification and characterization of novel ferroptosis inhibitors. Furthermore, this has prompted the development of a correlation to predict anti-ferroptosis activity of small-molecules using simple spectrophotometric assays.

Attempts were made to adapt the microtiter hemagglutination inhibition assay technique for the assay of enterotoxin A. The presence of a potent hemagglutinin in crude and partially purified preparations and the instability of sensitized erythrocytes prevented its use for routine analysis of enterotoxin from culture media and foods. A capillary tube immunological assay was developed in which 1 μ g of enterotoxin/ml was detected in less than 1 hr . Interfacial reaction of antisera and enterotoxin solutions in a 1 mm internal diameter capillary tube allowed rapid detection and serological typing of enterotoxins. Staphylococcus aureus growth and enterotoxin A development in Cheddar cheese slurry was evaluated. S. aureus growth and enterotoxin production occurred at 32 C. in 45 and 60% moisture cheese slurries following inoculation with 10 3 to 10 5 bacteria/gram. Hydrogen peroxide (0. 5%) treatment of slurry at 37 C did not inhibit S. aureus and enterotoxin A development. Heating slurry at 72 C for 30 min eliminated staphylococci but reinoculation with ripening organisms was essential. Addition of sorbic acid (0. 2 to 0. 3%) to a slurry adjusted to pH 5. 0 with lactic acid, inhibited staphylococci. in milk and slurry. Cheese flavor development was retarded due to inhibition of micrococci and lipolysis. Non-protein nitrogen increases paralleled that of sorbate-free controls. Sorbate treatment was preferred over other treatments .

A rapid assay kit for Vibrio cholerae (Vc) was developed to detect and quantify Vc cells in oyster samples within 24 h. The kit, formulated within a two -phase (liquid and solid) 96-well plate, can detect biomarker expression of Vc when the enrichment broth and incubation temperature are optimized. The kit showed 91 % selectivity and 92 % specificity when tested with 23 inclusive Vc and 106 exclusive non-Vc strains. The kit was further optimized using 47 samples of oysters, clams, and soil. There was no significant difference in most probable number between the kit, conventional PCR and BAX PCR regardless of agar heating method (autoclaved vs. boiled). The kit’s limit of detection was below 5 cfu/g. The kit is a reliable method for the detection of V. cholerae in bivalve samples.

When released extracellularly, the purine nucleoside guanosine (Guo) can exert a wide range of physiological effects in vitro and in vivo. Guo can induce the release of neurotrophic factors such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and can initiate the differentiation, growth and proliferation of neurons and glia. While structural and pharmacological evidence support the existence of a putative Guo binding site in the rat brain, there is a paucity of information on the mechanism through which Guo exerts these effects. Through bioinformatic research, our lab has identified an orphan G-protein coupled receptor as the first Guo receptor (termed G1R). The aim of this dissertation is to determine the mechanism of action of Guo using radioligand binding assays. It is hypothesized here that G1R is a distinct purinergic receptor for Guo. Using the calcium phosphate (CaP) co-precipitation (co-i.p.) method, Drosophila Schneider 2 (S2) cells were stably and transiently transfected with G1R recombinant cDNA. A series of binding assays using tritiated Guo ([3H]-Guo) showed no difference in binding between CaP transfection groups and wild S2 controls that do not endogenously express G1R, suggesting that the [3H]-Guo may not have a high binding affinity for the G1R binding site. Preliminary experiments using the Lipofectamine® 3000 to transfect S2 cells showed higher G1R mRNA expression as well as increased binding affinity to Guo when compared to the CaP transfected groups. This suggests that the results in the CaP mediated groups may be due to low transfection efficiency. In conclusion, transfections using the CaP method resulted in too low of a transfection efficiency to see a difference in binding affinity between wild S2 and transfected S2 cells. Findings from this work can be used to further examine the binding relationship of Guo to the G1R and optimize transfections using S2 cells and radioligand binding assays using purine based compounds. / Thesis / Master of Science (MSc)

No description available.

No / Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action involves induction of CYP1A1-catalyzed biotransformation of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) to generate electrophilic species, which covalently bind to DNA, exacting lethal damage to sensitive tumor cells, in vitro and in vivo. Herein, we investigate the effects of DNA adduct formation on cellular DNA integrity and progression through cell cycle and examine whether a relevant pharmacodynamic end point may be exploited to probe the clinical mechanism of action of Phortress and predict tumor response. Single cell gel electrophoresis (SCGE) was applied to quantify DNA damage and cell cycle analyses conducted upon 5F 203 treatment of benzothiazole-sensitive MCF-7 and inherently resistant MDA-MB-435 breast carcinoma cells. Following treatment of xenograft-bearing mice and mice possessing hollow fiber implants containing MCF-7 or MDA-MB-435 cells with Phortress (20 mg/kg, i.p., 24 hours), tumor cells and xenografts were recovered for analyses by SCGE. Dose- and time-dependent DNA single and double strand breaks occurred exclusively in sensitive cells following treatment with 5F 203 in vitro (10 nmol/L¿10 ¿mol/L; 24¿72 hours). In vivo, Phortress-sensitive and Phortress-resistant tumor cells were distinct; moreover, DNA damage in xenografts, following treatment of mice with Phortress, could be determined. Interrogation of the mechanism of action of 5F 203 in silico by self-organizing map-based cluster analyses revealed modulation of phosphatases and kinases associated with cell cycle regulation, corroborating observations of selective cell cycle perturbation by 5F 203 in sensitive cells. By conducting SCGE, tumor sensitivity to Phortress, an agent currently undergoing clinical evaluation, may be determined.

No / An isocratic oil-in-water microemulsion High Performance Liquid Chromatography (MELC) was developed and validated for the determination of terbutaline in urine samples. A solid phase extraction (SPE) method which used Oasis HLB cartridges was optimised to isolate terbutaline from a urine matrix followed by HPLC with fluorescence detection. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The method uses the isocratic oil-in-water micro emulsion to separate the terbutaline from the endogenous urine components. The chromatographic separation was carried out on C18-Spherisorb (250mm×4.6mm) analytical column maintained at 30 °C. The mobile phase was 94.4% aqueous orthophosphate buffer (adjusted to pH 3 with orthophosphoric acid), 0.5% ethyl acetate, 1.5% Brij35, 2.5% 1-butanol and 1.1% Octanesulfonic acid (OSA), all w/w. The terbutaline peak was detected by fluorescence detection, using excitation and emission wavelengths of 267 and 313 nm, respectively. The linearity of response was demonstrated at six different concentrations of terbutaline which were extracted from spiked urine, ranging from 60 to 1000ng/ml. The terbutaline was extracted from urine by a solid phase extraction clean-up procedure on Oasis HLB cartridges, and the relative recovery was >87.64% (n = 5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 20.21 and 61.24ng/ml, respectively. The intra-day and inter-day precisions (in term of % coefficient of variation) were <3.56% and <2.87%, respectively. In the method development the influence of the composition of the microemulsion system was also studied and the method was found to be robust with respect to changes of the microemulsion components.

Yes / The comet assay is a sensitive method used to detect DNA damage, measuring DNA breaks and alkali labile lesions in eukaryotic cells. Here, the use of whole blood in the alkaline gel electrophoresis method is described. Two hundred and seventy blood samples from individuals were examined: 120 healthy individuals, 65 suspected or pre-cancerous individuals and 85 cancer patients. Each sample was divided into two identical volumes in different falcon tubes. The blood was prepared and stored by adding the same amount of RPMI medium and 10% DMSO. Using the Student’s t-Test, the data showed a p value = 0.59 for Olive tail moment (OTM) and 0.16 for % tail DNA, and no statistically significant differences between the two methods, with or without treatment. In conclusion, using whole blood instead of isolated lymphocytes saves time, is still very sensitive and requires less than 20 µL of blood from each individual.

In the present study DNA damage was measured in peripheral blood lymphocytes from polyposis coli and colorectal cancer patients, treated with different dietary and environmental compounds and compared with lymphocytes from healthy individuals. In addition, confounding factors such as age, gender, alcohol intake and smoking habits were taken into consideration. The assays used in this study included the Comet assay, the Micronucleus assay, the Micronucleus-FISH assay and the sister chromatid exchange assay. The food mutagens, PhIP and IQ, as well as titanium dioxide nanoparticles (TiO2 NPs) induced a dose dependent increase in the DNA damage and chromosomal abnormalities in all tested groups regardless of confounding factors. Prior to experiments physicochemical characterisation of nanoparticles was conducted. In the presence of the flavonoids, quercetin and rutin that were acting in an antioxidant manner, the DNA damage resulting from the highest doses of food mutagens was significantly reduced. Thus, dietary supplementation with flavonoid-rich vegetables and fruits may prove very effective in protection against oxidative stress. The polyposis coli and colon cancer patients were more susceptible to food mutagens, PhIP and IQ, as well as TiO2 NPs, and in the majority of cases had a higher level of DNA damage in the Comet assay and higher cytogenetic damage in the Micronucleus assay. In the final project, twelve frequently encountered (NewGeneris) chemical compounds were evaluated to establish their damaging potential in lymphocytes and spermatozoa from healthy donors. The highest damage was produced by DNA reactive aldehydes, food mutagens and benzo[a]pyrene when assessed with the neutral and alkaline Comet assay with and without metabolic activation.