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The aim of this thesis is to apply recently developed technologies for genomic variation analyses, and to ensure quality of the generated information for use in preclinical cancer research. Faster access to a patients’ full genomic sequence for a lower cost makes it possible for end users such as clinicians and physicians to gain a more complete understanding of the disease status of a patient and adjust treatment accordingly. Correct biological interpretation is important in this context, and can only be provided through fast and simple access to relevant high quality data. Therefore, we here propose and validate new bioinformatic strategies for biomarker selection for prediction of response to cancer therapy. We initially explored the use of bioinformatic tools to select interesting targets for toxicity in carboplatin and paclitaxel on a smaller scale. From our findings we then further extended the analysis to the entire exome to look for biomarkers as targets for adverse effects from carboplatin and gemcitabine. To investigate any bias introduced by the methods used for targeting the exome, we analyzed the mutation profiles in cancer patients by comparing whole genome amplified DNA to unamplified DNA. In addition, we applied RNA-seq to the same patients to further validate the variations obtained by sequencing of DNA. The understanding of the human cancer genome is growing rapidly, thanks to methodological development of analysis tools. The next step is to implement these tools as a part of a chain from diagnosis of patients to genomic research to personalized treatment. / <p>QC 20121105</p>

The objective of this thesis was to construct general models to predict pollutant fluxes in limnoplankton by incorporating characteristics of the organism and the structures of the chemical. A two-compartmental pharmacokinetic model was used to quantify the pollutant uptake, depuration and intercompartmental exchanges. The model pollutants were phosphorus and 22 organic chemicals. / The rate constants of phosphorus uptake, excretion and intercompartmental changes by algae and cladocerans decreased with cell volume or body size raised to a power close to $-$0.25, except the intercompartmental exchanges for cladocerans which showed more negative slopes. In contrast, uptake, excretion and internal exchange rates per individual increased with cell size or body weight to a power similar to 0.75 with a similar exception for the cladoceran intercompartmental exchanges, which had slopes $<$0.75. / Bioconcentration factors, rate constants and flux rates of uptake and intercompartmental exchange from metabolic pool to structural pool of 22 $ sp{14}$C-labelled organic toxicants by Chlorella pyrenoidosa and Daphnia magna were positively correlated with the octanol/water partition coefficient, molecular weight, parachor, connectivity index, boiling point and melting point, and negatively with aqueous solubility. However, those of elimination and internal transfer from structural pool to metabolic pool showed opposite changes. Comparisons of pharmacokinetic parameters between Daphnia and Chlorella demonstrated that, although all kinetic parameters displayed similar patterns, the relative magnitudes of each corresponding parameters were significantly different between two species.

<p>&nbsp / </p> <p align="left"><font face="Times New Roman">Aspalathus linearis (A. linearis), </font><font face="Times New Roman">commonly known as Rooibos tea or Red bush tea and </font><i><font face="Times New Roman">amellia sinensis (C. sinensis) </font></i><font face="Times New Roman">or Black tea are beverages that are consumed throughout the</font><font face="Times New Roman">world. These teas possess antioxidant, immunomodulating and anti-cancer actions. The aim</font> <font face="Times New Roman">of this study was to use <i>in vitro </i>assays to screen Rooibos and Black tea for endocrine</font> <font face="Times New Roman">modulation. </font><font face="Times New Roman">The immune modulating effects of Rooibos and Black tea were investigated using an <i>in vitro</i></font> <font face="Times New Roman">whole blood culture (WBC) assay. Unstimulated WBCs treated with Rooibos tea secreted</font> <font face="Times New Roman">higher levels of IL-6, IL-10 and IFN<font lang="JA">&gamma / </font>than cultures treated with DMSO control. Rooibos treatment of stimulated WBCs resulted in higher IL-6, lower IL-10 and no effect on IFN&gamma / </font><font face="Times New Roman">secretion compared to DMSO treated stimulated WBC. Black tea treatment of stimulated</font> <font face="Times New Roman">WBC resulted in decreased IL-6, IL-10 and IFN<font lang="JA">&gamma / </font>secretion compared to the DMSO treated</font> <font face="Times New Roman">stimulated WBC. </font><font face="Times New Roman">Extracts of Rooibos and Black tea were assessed for phytoestrogens using quantitative </font><font face="Times New Roman">estrogen ELISAs. Both teas contain phytoestrogens. The quantitative ELISAs showed that </font><font face="Times New Roman">Rooibos tea contained significantly lower estrone (E1), estradiol (E2) and estriol (E3) levels </font><font face="Times New Roman">than Black tea. The effects of Rooibos and Black tea on proliferation of the estrogen</font> <font face="Times New Roman">dependant MCF-7 cell line was determined to further characterise the phytoestrogenic</font> <font face="Times New Roman">properties of the teas. Both Rooibos and Black tea extracts caused a significant inhibition of</font> <font face="Times New Roman">MCF-7 proliferation. </font><font face="Times New Roman">This study shows that Rooibos tea and Black tea are beverages that can either stimulate or </font><font face="Times New Roman">suppress the immune system. Also, both teas contain significant levels of phytoestrogens as </font><font face="Times New Roman">determined by quantitative ELISAs. The current study confirms previous reports showing </font><font face="Times New Roman">inhibition of growth in breast cancer cell lines by phytoestrogens. The findings extend </font><font face="Times New Roman">related observations on the anti-carcinogenic potential of the two teas.</font></p>

The Port of Durban, with its close proximity to industrial, urban and agricultural activities, receives a number of chemical pollutants that settle out and accumulate in sediments. Chemical analysis of these sediments has indicated elevated levels of chemicals that, according to sediment quality guidelines, might cause adverse biological effects. However, elevated concentrations alone do not necessarily imply that chemicals are present in bioavailable concentrations high enough to be harmful to organisms that come into contact with them. Thus, chemical tests alone cannot provide an accurate indication of the potential adverse biological effects of these chemicals. In this regard, toxicity tests of sediment porewaters have been developed using sea urchin gametes to assist in determining the bioavailability of chemicals present in porewaters. Further, procedures such as Toxicity Identification and Evaluation (TIE), which involves the manipulation and/or treatment of toxic porewater, have also been developed to assist in the isolation and identification of chemicals causing porewater toxicity. In this research, on a number of sampling occasions between July 2007 and July 2009, three replicate sediment samples were extracted from a site in the Port of Durban known to contain sediment with potentially toxic porewater. Results of initial toxicity tests, using the sea urchin fertilisation test indicated the presence of toxic porewater although, in some instances, porewater toxicity was highly variable between replicate samples. However, results from TIE procedures performed to reduce potentially toxic concentrations of metals, ammonia and organic compounds did not resolve the primary cause of porewater toxicity. Further research indicated that chemicals including hydrogen sulphide, which can occur naturally in organically enriched sediments, may have been confounding factors that masked the potential toxicity of other chemicals present in the sediment samples. Consequently, a sampling strategy and modified TIE procedure have been recommended. The sampling strategy has been designed to assist with detecting and understanding any sample variability that may occur. The modified TIE procedure, which suggests initial procedures to determine and reduce/remove the possible confounding effects of potential naturally occurring compounds such as hydrogen sulphide from the porewater, could be used in future to understand and evaluate the quality of contaminated sediments from similar environments. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Microorganisms are increasingly used in toxicological studies to determine potential environmental impacts of xenobiotic compounds. A multi-stage laboratory model was developed to facilitate the examination of environmental impacts of selected pollutants on fundamental cycling processes inherent to aquatic ecosystems, namely, the degradation of organic substances and nitrogen transformations under aerobic conditions. A microbial association representative of riverine ecosystems was enriched for, isolated and cultured within the model. Characterisation of the microbial association were undertaken. Scanning electron microscopy and bright field microscopy revealed that a diverse heterogenous community of microorganisms had established within the model. Successional metabolic events, namely organic carbon catabolism, ammonification of organic nitrogen and the process of nitrification were differentiated in time and space with the microbial association integrity still being retained. The establishment of a microbial association within the model was primarily dependent on: dilution rates, specific growth rates and interactions between microorganisms and the prevailing environmental conditions. Growth-rate independent populations of microorganisms established within the model and were thought to contribute significantly to the metabolic processes within the model. Nitrifying activity was identified as a rate-limiting process within the model. Following separation of metabolic events, the ecotoxicological impacts of phenol and 2,4-dichlorophenol on the association were assessed. The biological oxidation of ammonia through to nitrate (nitrification) was found to be a sensitive indicator of perturbation. The model was found to be suitable for testing both acute and chronic intoxication by pollutant compounds as well as for biodegradation testing and the possible evaluation of ecotoxicological impacts of wastewater treatment plants. The main disadvantages of the model arose from its operational complexity, its empirical nature and its impracticality for screening large numbers of compounds. A bioassay based on the inhibition of ammonium oxidation was developed in order to fulfil the requirements for a simple and rapid test protocol for the initial screening of perturbant compounds. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Ethnopharmacological relevance: Indigenous plants such as Tulbaghia violacea(TV) and Allium sativum (garlic) are traditionally used as natural remedies to treat a variety of ailments, including cancer. This study investigated the effects of TV leaf and bulb extracts and garlic extract on a cancerous oesophageal cell line (SNO). Materials and methods: The methylthiazoltetrazolium (MTT) assay was used to determine the IC50 of TV leaf (TVL) (250μg/ml) and TV bulb extracts (TVB) (25μg/ml) and garlic (500μg/ml). Extracts were treated individually and in combination for a period of 24 hours. Oxidative damage and intracellular glutathione levels were assessed using the Thiobarbituric Acid Reactive Substances (TBARS) Assay and GSH-Glo™ Luminometry Assay, respectively. The CellTiter-Glo® Luminescent Cell Viability Assay was used to assess ATP activity. Induction of apoptosis and mitochondrial membrane potential were determined via the Caspase-Glo® 3/7 Assay, Caspase-Glo® 8 Assay, Caspase-Glo® 9 Assay and JC-1 Mitoscreen Assay, respectively. Morphological apoptotic changes were determined using the Hoechst 33342 stain. Expressions of p53, PARP and NFKB activities were determined by western blotting. Results: Bulb and leaf extracts of TV increased lipid peroxidation compared to the control (p>0.05), whilst garlic and combination of TV leaf and bulb (TVB + TVL) extracts significantly decreased lipid peroxidation relative to the control (p< 0.05). Endogenous glutathione levels significantly decreased in all TV treatments compared to the control (p<0.05).However, garlic was accompanied by insignificantly increased intracellular glutathione levels compared to the control (p> 0.05). The percentages of depolarised mitochondria in all treated cells were significantly decreased compared to untreated cells (p< 0.05). ATP levels increased significantly in garlic and combination (TVB + TVL) treated cells as compared to the control (p< 0.05), yet no significant differences were noted in TVL and TVB treatments (p> 0.05). Caspase8 and caspase 9 activities significantly increased in garlic and combination treated cells relative to the control (p<0.05). A similar trend was noted for caspase 3/7 activity in garlic and combination treatments (p< 0.05). However, initiator and executioner activities in TVL (p> 0.05) and TVB (p> 0.05) treatments did not significantly differ from the control (p> 0.05). All treatments (including garlic) resulted in increased DNA fragmentation and condensation. All treatments decreased p53 expression (p< 0.05), PARP expression (p< 0.05) and NFK B expression (p>0.05) compared to the control. Conclusions: All TV extracts and garlic induces apoptosis in the oesophageal cancerous SNO cell line through changes in oxidative stress, antioxidant systems, and nuclear chromatin condensation, as well as through induction of nuclear genes and signalling pathways. Since inhibition of apoptosis is a principal alteration in cancer, induction of apoptosis would result in a decrease in cancer cell growth. Thus, TV could be exploited as a potential anti-cancer agent. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2012.

Lycorin gilt bei vielen Amaryllidaceae als Hauptalkaloid und die Aufnahme dieser Pflanzen ist eine häufige Vergiftungsursache bei Mensch und Tier. Als Hauptsymptome infolge dieser Pflanzenvergiftungen werden Nausea und Emesis genannt, aber systematische Untersuchungen zu diesen biologischen Effekten, zum Wirkmechanismus und zur Pharmakokinetik von Lycorin, das als auslösendes Agens angenommen wird, existieren bislang nicht. In der vorliegenden Arbeit werden die Zusammenhänge zwischen verabreichter Lycorin-dosis und Lycorin-induzierter Nausea und Emesis, die Beeinflussbarkeit dieser emetischen Effekte durch etablierte Antiemetika und die Pharmakokinetik von Lycorin in einem cross-over und vehikel-kontrollierten Design in vivo untersucht. Die Studie wurde an elf Beagle-Hunden beider Geschlechter durchgeführt. Die Lycorin-induzierten emetischen Effekte wurden quantifiziert und über Videoaufzeichnungen zeitnah dokumentiert. Nausea wird hierbei mittels eines Scoring-Systems quantifiziert, während die Parameter Latenzzeit, Dauer und Anzahl der Brechakte zur Beurteilung der Emesis herangezogen werden. Die subkutane Applikation von Lycorin induziert, beginnend ab einer Dosis von 0,5 mg/kg KGW Nausea und Vomitus. Eine statistische Signifikanz ist allerdings erst ab 1,0 mg/kg und ein maximaler emetischer Effekt bei einer Dosis von 2 mg/kg (ED100) zu verzeichnen. Die Ergebnisse zeigen eine Korrelation zwischen applizierter Lycorin-Dosis und Nausea-Score sowie der Anzahl der Brechakte. Lycorin-induzierte Nausea und Emesis sind in den vorliegenden Untersuchungen selbstlimitierend und dauern maximal 2,5 Stunden an. Lycorin weist in den untersuchten Dosierungen von 0,25 mg/kg bis 2,0 mg/kg eine lineare Plasmakinetik auf. Nach subkutaner Gabe werden maximale Plasmakonzentrationen (Cmax) nach 0,5 h gemessen, die mittlere Plasma-Halbwertszeit beträgt 0,67 h nach subkutaner, respektive 0,51 h nach intravenöser Applikation. Die errechnete orale Bioverfügbarkeit beträgt ca. 40 %. Das Auftreten von Nausea und Emesis, sowie deren Verlauf decken sich weitestgehend mit dem Verlauf der Lycorinkonzentration im Plasma. In keiner der untersuchten Dosisstufen sind blutchemische oder hämatologische Abweichungen aufgetreten. Um Rückschlüsse auf die Zielstrukturen von Lycorin und somit auf den emetischen Wirkungsmechanismus der Lycorin-induzierten Emesis und Nausea zu gewinnen, wurden die Hunde jeweils mit Diphenhydramin, Maropitant, Metoclopramid, Ondansetron oder Scopolamin vorbehandelt. Diese therapeutisch etablierten Antiemetika besitzen eine selektive Rezeptoraffinität und entfalten ihre antiemetische Wirkung über einen Antagonismus an histaminergen H1- (Diphenhydramin), dopaminergen D2- (Metoclopramid), muskarinergen M1-3- (Scopolamin), serotoninergen 5-HT3- (Ondansetron) oder Neurokinin-1-Rezeptoren (NK1) (Maropitant). Durch die Bindung des jeweiligen Antiemetikums an die spezifischen Rezeptoren, soll die anschließende Bindung von Lycorin an den gleichen Rezeptoren verhindert oder reduziert werden, was sich in einer Reduktion oder Abwesenheit von Nausea und Emesis auswirkt. Die Vorbehandlung mit Ondansetron ist mit einer signifikanten Verminderung der Anzahl der Brechakte verbunden und durch die Vorbehandlung mit Maropitant kann Lycorin-induzierte Emesis komplett verhindert werden. Einzig Ondansetron reduziert darüber hinaus den Ausprägungsgrad der Nausea und verlängert die Latenzzeit bis zum Auftreten von Vomitus, was eine Beteiligung von 5-HT3 Rezeptoren bei lycorin-induzierter Nausea nahe legt. Histaminerge (H1), dopaminerge (D2) und muskarinerge (M1-3) Rezeptoren sind vermutlich nicht an Lycorin-induzierter Nausea und Emesis beteiligt. Die Befunde der vorliegenden Arbeit weisen darauf hin, dass Lycorin bei Vergiftungen mit Pflanzen oder Pflanzenteilen, die zu den Amaryllidaceae gehören, eine entscheidende Bedeutung für die klinische Symptomatik und den Verlauf von Intoxikationen hat. Nach den Ergebnissen dieser Arbeit sind eine prädominierende Beteiligung von NK1- und eine etwas geringer ausgeprägte Beteiligung von 5-HT3-Rezeptoren im emetischen Wirkmechanismus wahrscheinlich. Somit erscheint die therapeutische Anwendung von Maropitant beim Hund (und evtl. Apreptitant beim Menschen) und/oder Ondansetron zur symptomatischen Behandlung anhaltender Nausea und Emesis bei Pflanzenvergiftungen mit Amaryllidacaen bei denen die Wirkung von Lycorin dominiert, wissenschaftlich begründet und klinisch von Vorteil gegenüber anderen antiemetischen Prinzipien zu sein.

<p>Single-walled carbon nanotubes (SWNT) are highly ordered filamentous nanocarbon structures. As their commercial and industrial use becomes more widespread, it is anticipated that SWNT will enter the environment through waste streams and product degradation. Because of their highly hydrophobic nature, SWNT aggregate and settle out of aqueous environments, especially in saline environments such as estuaries. Therefore, sediments are a likely environmental sink for SWNT once released. It is important to understand how these materials will impact benthic estuarine systems since they are the probable target area for SWNT exposure in addition to containing many lower trophic level organisms whose survvial and contaminant body burdens can have a large impact on the overall ecosystem. Disruptions in lower trophic level organism survival can have negative consequences for higher trophic levels, impacting the overall health of the ecosystem. It is also important to consider contaminant bioaccumulation, trophic transfer and biomagnification. If SWNT are taken up by benthic invertebrates, there is the possibility for trophic transfer, increasing the exposure of SWNT to higher trophic level organisms that otherwise would not have been exposed. If this type of transfer occurs in environmentally important species, the potential for human exposure may increase. My research aims to determine the magnitude of the toxicity and bioaccumulation of SWNT in benthic estuarine systems, as well as determine how they interact with other contaminants in the environment. This research will contribute to the knowledge base necessary for performing environmental risk assessments by providing information on the effects of SWNT to benthic estuarine systems. </p><p> Before investigating the environmental effects of SWNT, it is imperative that a measurement method is established to detect and quantify SWNT once they enter the environment. This research utilized pristine, semiconducting SWNT to develop extraction and measurement methods to detect and quantify these specific materials in environmental media using near infrared fluorescence (NIRF) spectroscopy. Semiconducting SWNT fluoresce in the near infrared (NIR) spectrum when excited with visible&ndashNIR light. This unique optical property can be used to selectively measure SWNT in complex media. </p><p> The fate, bioavailability, bioaccumulation and toxicity of SWNT have not been extensively studied to date. Pristine SWNT are highly hydrophobic and have been shown to strongly associate with natural particulate matter in aquatic environments. In light of this, I have focused my research to examine the influence of sediment and food exposure routes on bioavailability, bioaccumulation, and toxicity of structurally diverse SWNT in several ecologically-important marine invertebrate species. No significant mortality was observed in any organism at concentrations up to 1000 mg/kg. Evidence of biouptake after ingestion was observed for pristine semiconducting SWNT using NIRF spectroscopy and for oxidized <super>14</super>C&ndashSWNT using liquid scintillation counting. After a 24 hour depuration period, the pristine semiconducting SWNT were eliminated from organisms to below the method detection limit (5 &mug/mL), and the <super>14</super>C&ndashSWNT body burden was decreased by an order of magnitude to a bioaccumulation factor (BAF) of <0.01. Neither pristine SWNT nor oxidized <super>14</super>C&ndashSWNT caused environmentally relevant toxicity or bioaccumulation in benthic invertebrates. Overall, the SWNT were not bioavailable and appear to associate with the sediment.</p><p> In addition to investigating the toxicity and bioaccumulation of SWNT as an independent toxicant, it is important to consider how they will interact with other contaminants in the environment (i.e., increase or decrease toxicity and bioaccumulation of co&ndashcontaminants, alter the environmental transport of co&ndashcontaminants, induce degradation of co&ndashcontaminants, etc.). I wanted to investigate the effects of SWNT on a complex mixture of contaminants already present in a natural system. New Bedford Harbor (NBH) sediment, which is contaminated with polychlorinated biphenyls (PCBs), was amended with pristine SWNT to determine if the presence of SWNT would mitigate the toxicity and bioaccumulation of the PCBs in deposit-feeding invertebrates. A dilution series of the NBH sediment was created using uncontaminated Long Island Sound (LIS) sediment to test 25% NBH sediment, 50% NBH sediment, 75% NBH sediment, and 100% NBH sediment. The results of this work showed increased organism survival and decreased bioaccumulation of PCBs in treatments amended with SWNT, with the greatest reduction observed in the 25% NBH sediment treatment group amended with 10 mg SWNT/g dry sediment. Polyethylene (PE) passive samplers indicated a reduction of interstitial water (ITW) PCB concentration of greater than 90% in the 25% NBH sediment + 10 mg SWNT/g dry sediment amendment. The ITW concentration was reduced because PCBs were not desorbing from the SWNT. Lower bioavailability leads to reduced potential for toxic effects, supporting the observation of increased survival and decreased bioaccumulation. Once in the sediment, not only are SWNT not bioavailable, they act as a highly sorptive phase, such as black carbon (BC), into which hydrophobic organic contaminants (HOCs), such as PCBS and polycyclic aromatic hydrocarbons (PAHs), can partition, thereby reducing the toxicity and bioavailability of co-occurring HOCs.</p><p> To more fully understand the impact of SWNT in this environment, their biodegradability also needs to be investigated. Biodegradation of SWNT could lead to release and/or transformation of sorbed HOCs as well as a change in the inherent transport, toxicity, and bioaccumulation of SWNT in the estuarine environment. Because the persistence of SWNT will be a primary determinant of the fate of these materials in the environment, I conducted experiments to determine if the fungus <italic>Trametes versicolor</italic>, the natural bacterial communities present in NBH sediment, and municipal wastewater treatment plant sludge could degrade or mineralize oxidized <super>14</super>C&ndashSWNT. Over a six month time period, no significant degradation or mineralization was observed. In all treatments, approximately 99% of the 14C-SWNT remained associated with the solid phase, with only approximately 0.8% of added <super>14</super>C present as dissolved species and only 0.1% present as <super>14</super>CO₂. These small pools of non-SWNT <super>14</super>C were likely due to trace impurities, as no differences in production were observed between treatments and abiotic (killed) controls.</p> / Dissertation

Two studies were conducted to evaluate the effectiveness of using copper sulfate (CuSO4) as a drench in Hampshire ewes to control stomach worms (Haemonchus contortus). A study was conducted to assess the effectiveness of CuSO4 to control gastrointestinal nematodes (GIN) over a three year period. Ewes were FAMACHA scored, hematocrit evaluated for packed cell volume (PCV), and fecal egg counts (FEC) were determined from 2007 through 2009. Ewes received only CuSO4 to control GIN. Ewes with FEC exceeding 6,000 eggs/g feces were drenched. A separate study during the summer of 2008 assessed the potential of CuSO4 drench to cause copper toxicity in Hampshire ewes. Eighty-four ewes were blocked to one of two treatments according to parity and balanced for FEC. One group received CuSO4 (D) and the other was not drenched (ND). Jugular blood samples were collected at pre-determined intervals after CuSO4 was administered to D ewes. Serum was analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK). Elevated serum levels indicate copper toxicity. Results suggest CuSO4 has the potential to control stomach worms in Hampshire ewes without causing copper toxicity.