M.Sc. (Zoology) / Sediment contamination occurs as a result of various anthropogenic activities; mainly through mining-, agricultural- and industrial practices. Many of the contaminants arising from these activities enter the aquatic system and precipitate from the surrounding water, binding to sediment particles. In the sediment compartment, these contaminants reach concentrations much higher than in solution with the overlying water. Even though the quality of the overlying water may prove acceptable in accordance to water quality uidelines, an aquatic system may still be at risk from the contaminated sediment. If the contaminated sediment were to be disturbed through flooding, bioturbation or changes in the water chemistry, these contaminants will desorb into the water column and as a result be detrimental to life forms in contact and dependent on that water source. Monitoring sediment has been a widespread initiative internationally and has led to the development of various sediment toxicity test methods, including different bioassays. This study focused on sediment bioassays such as the Phytotoxkit-F and Ostracodtoxkit-F, and the Diptera bioassay to assess the sediment quality of the Tweelopiespruit-Rietspruit-Bloubankspruit (TRB) river system in Gauteng, South Africa. This river is known to be impacted by acid mine drainage (AMD) since late August 2002. Exposure of the bioassays to river sediment from preselected sampling sites (Site 1, closest to the mine, to Site 6, furthest from the mine, and Site 7, the reference site) provided an eco-toxicological estimation of the acute toxicity emanating from contaminants in the sediment. Physico-chemical analyses revealed high concentrations of metals and other contaminants in the water and sediment. A general linear decrease in contaminant concentrations was observed from Site 1 to Site 6. The results from the bioassays displayed a similar trend, since there were greater sensitivities (mortalities and growth inhibition) to sediments sampled closer to the mine. Due to high levels of contamination in sediments, compared to the overlying water, and the potential impact on aquatic organisms, sediment toxicity monitoring should be a compulsory requirement for environmental studies in South Africa

Often predictor variables in regression models are measured with errors. This is known as an errors-in-variables (EIV) problem. The statistical analysis of the data ignoring the EIV is called naive analysis. As a result, the variance of the errors is underestimated. This affects any statistical inference that may subsequently be made about the model parameter estimates or the response prediction. In some cases (e.g. quadratic polynomial models) the parameter estimates and the model prediction is biased. The errors can occur in different ways. These errors are mainly classified into classical (i.e. occur in observational studies) or Berkson type (i.e. occur in designed experiments). This thesis addresses the problem of the Berkson EIV and their effect on the statistical analysis of data fitted using linear and nonlinear models. In particular, the case when the errors are dependent and have heterogeneous variance is studied. Both analytical and empirical tools have been used to develop new approaches for dealing with this type of errors. Two different scenarios are considered: mixture experiments where the model to be estimated is linear in the parameters and the EIV are correlated; and bioassay dose-response studies where the model to be estimated is nonlinear. EIV following Gaussian distribution, as well as the much less investigated non-Gaussian distribution are examined. When the errors occur in mixture experiments both analytical and empirical results showed that the naive analysis produces biased and inefficient estimators for the model parameters. The magnitude of the bias depends on the variances of the EIV for the mixture components, the model and its parameters. First and second Scheffé polynomials are used to fit the response. To adjust for the EIV, four different approaches of corrections are proposed. The statistical properties of the estimators are investigated, and compared with the naive analysis estimators. Analytical and empirical weighted regression calibration methods are found to give the most accurate and efficient results. The approaches require the error variance to be known prior to the analysis. The robustness of the adjusted approaches for misspecified variance was also examined. Different error scenarios of EIV in the settings of concentrations in bioassay dose-response studies are studied (i.e. dependent and independent errors). The scenarios are motivated by real-life examples. Comparisons between the effects of the errors are illustrated using the 4-prameter Hill model. The results show that when the errors are non-Gaussian, the nonlinear least squares approach produces biased and inefficient estimators. An extension of the well-known simulation-extrapolation (SIMEX) method is developed for the case when the EIV lead to biased model parameters estimators, and is called Berkson simulation-extrapolation (BSIMEX). BSIMEX requires the error variance to be known. The robustness of the adjusted approach for misspecified variance is examined. Moreover, it is shown that BSIMEX performs better than the regression calibration methods when the EIV are dependent, while the regression calibration methods are preferable when the EIV are independent.

Alcohol abuse is a very common practice (just like in many other parts of the world) in Nkonkobe Municipality, Eastern Cape Province, South Africa. This is associated with liver disease. An ethnobotanical survey of plants used for the treatment of alcohol-induced liver damage in Nkonkobe Municipality was conducted. During the survey and also from information gathered in the literature, Pelargonium reniforme Curtis, was prominently mentioned, among other plants, as the species used generally for the treatment of alcohol-induced liver damage. This project was designed to evaluate the effects of the plant on alcohol-induced liver damage, including its antioxidant and antimicrobial properties. It also involves safety evaluation studies to determine if the plant is safe for consumption. Studies using rats of the Wistar strain were carried out to determine the protective and curative effects of P. reniforme on alcohol-induced liver damage. Results obtained showed that the plant extract can protect the liver cells as well as enhance recovery from tissue damage. The plant also showed good antimicrobial and antioxidant activity and this further validates its use in the treatment of liver diseases. Safety evaluation studies of the extract were carried out by investigating the effects of the oral administration on some haematological and biochemical parameters in male Wistar rats. The results obtained from the study suggest that the plant extract is not toxic at the doses used and is therefore safe for medicinal uses. The results of the various bioassays carried out in this project have justified the traditional uses of P. reniforme for the treatment of alcohol-induced liver damage.

L’objectif de cette thèse, dans le cadre de l'ANR TRICETOX (CESA, 2013-2017), était de développer des méthodes alternatives aux méthodes d’analyse chimiques classiques pour la détection spécifique des herbicides de la famille des β-tricétones. Ces nouveaux outils analytiques doiventt être faciles à utiliser, rapides, peu coûteux et devant répondre aux exigences imposées pour la détection de ces herbicides (valeur critique de 0,1 µg.L-1). Les β-tricétones ont pour cible spécifique l'enzyme p-HydroxyPhénylPyruvate Dioxygénase (HPPD), impliquée dans la biosynthèse des caroténoïdes, son inhibition entraîne un blanchiment des feuilles des plantes traitées. Deux stratégies ont été envisagées, basées sur l’inhibition de l’HPPD : un bio-essai colorimétrique à cellules entières utilisant un clone recombinant d'Escherichia coli exprimant l’HPPD d’Arabidopsis thaliana, et un biocapteur enzymatique à transduction électrochimique. Le principe du bio-essai repose sur la capacité du clone recombinant à produire un pigment brun à partir de la voie de catabolisme de la tyrosine. L’addition de β-tricétones entraîne une diminution de la pigmentation dans le milieu. Après optimisation des conditions opératoires, les LODs obtenues pour la mésotrione, la tembotrione, la sulcotrione et la leptospermone sont de 0,069, 0,051, 0,038 et 20 μM, respectivement. En format biocapteur, l'enzyme HPPD purifiée a été immobilisé sur la surface d’une électrode de graphite sérigraphiée. Le biocapteur à enzyme libre a permis l’obtention de limites de détection de 1,36-10 M, 1,25.10-11 M et 1,08.10-11 M pour la sulcotrione, la tembotrione et la mésotrione, respectivement. A ce jour, des échantillons réels ont été analysés sur le bio-essai et les résultats ont été validés par HPLC. / The goal of this thesis, in the frame of the ANR TRICETOX (CESA, 2013-2017), was to develop some alternative methods to conventional chromatographic methods for the monitoring of the β-triketone herbicides. These new analytical tools have to be easy to handle, fast, low-cost, and to respond to the requirements for the detection of these herbicides (critical value of 0.1 μg.L-1). The β-triketones specifically target the 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme, involved in the carotenoids biosynthesis in plants ; its inhibition results in foliage bleaching of the treated plant. Two strategies have been considered, both based on the HPPD inhibition : a whole-cell colorimetric bioassay involving a recombinant E. coli expressing the HPPD from A. thaliana, and an electrochemical enzymatic biosensor. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble brown pigment from tyrosine catabolism. The addition of β-triketone induces the decrease of the pigment production in the medium. After optimization of the operational conditions, the LODs obtained for mesotrione, tembotrione, sulcotrione, and leptospermone were 0.069, 0.051, 0.038, and 20 μM, respectively. In the biosensor format, the purified HPPD enzyme was immobilized on the surface of a screen-printed graphite electrode. Using the free enzyme biosensor, LODs as low as 1.36-10 M, 1.25.10-11 M and 1.08.10-11 M were obtained for sulcotrione, mesotrione and tembotrione, respectively. Up to date, real samples have been analyzed using the bioassay and the results were validated by HPLC.

A esquistossomose é incidente em países tropicais e subtropicais e o uso de moluscicidas é adequado para prevenir a infecção de pessoas. O objetivo deste estudo foi buscar compostos em Piper ativos em Biomphalaria glabrata e avaliar a atividade esquistossomicida e toxicidade do composto mais ativo. O fracionamento biomonitorado de Piper diospyrifolium resultou no isolamento da flavocavaína A e ácido 4-hidroxi-3-[3,7,11-trimetildodeca-2,6,10-trienil]benzoico. A busca de compostos também foi realizada em amidas e chalconas e dos oito compostos avaliados, quatro foram ativos, sendo a piplartina mais ativa. A piplartina não foi letal a miracídios e cercárias de Schistosoma mansoni e foi tóxica em Daphnia similis e Danio rerio, contudo ainda foi menos tóxica que a niclosamida. Neste estudo, os compostos moluscicidas foram obtidos pelo fracionamento biomonitorado de P. diospyrifolium e pela avaliação da atividade de amidas e chalconas. Ambas as metodologias foram adequadas e a análise de componente principal mostrou ser uma ferramenta viável para a busca de compostos. / Schistosomiasis is a parasitic disease and the use of molluscicides has been considered an appropriate method to prevent human infection. The aim of this study was to search for compounds in Piper active in B. glabrata and evaluate schistosomicidal activity and toxicity of the most active compound. The P. diospyrifolium bioguided fractionation resulted in flavokavain A and 4-hydroxy-3-[3,7,trimetildodeca-11-2,6,10-trienil]benzoic acid isolation. The search for active compounds was carried out in amides and chalcones. Eight compounds were evaluated, four were active and piplartine was the most active. There was no mortality of miracidia and cercariae exposed to piplartine. It was classified as toxic to D. similis and D. rerio; nevertheless was less toxic than niclosamide. In this study, molluscicidal compounds were obtained from bioguided fractionation of P.diospyrifolium and by evaluating the activity of amides and chalcones. Both methods were suitable to obtain active compounds and principal component analysis also proved to be a viable tool for obtain compounds.

New experimental strategies and algorithmic approaches were devised and tested to improve the analysis of pharmaceutically relevant materials. These new methods were developed to address key bottlenecks in the design of amorphous solid dispersions for the delivery of low-solubility active pharmaceutical ingredients in the final dosage forms exhibiting high bioavailability. <br>

Although seasonal influenza epidemics represent a significant threat to public health, their treatment options remain limited. With deaths from the 1918 influenza pandemic estimated at >50,000,000 worldwide and future pandemics predicted, the need for a potent broad-spectrum influenza therapy is critical. In this thesis, I describe the use of a structurally modified zanamivir, an influenza neuraminidase inhibitor that blocks the release of nascent virus, to deliver attached therapeutic agents specifically to the surfaces of viruses and virus-infected cells, leading to simultaneous inhibition of virus release and immune-mediated destruction of both free virus and virus-infected cells. Chapter 1 describes the major characteristics of the influenza virus, the morbidity and mortality associated with annual infections by current strains of the virus, and the treatments available to reduce the disease burden associated with these infections. Chapter 2 describes the design, synthesis, and evaluation of a zanamivir-related targeting ligand and its conjugation to two orthogonal imaging agents which are then used to characterize the binding specificity and biodistribution of the targeting ligand in influenza virus-infected cells and in infected mice. Chapter 3 describes the development of an influenza virus-targeted immunotherapy, where a zanamivir-targeted hapten is exploited to redirect the immune system to destroy influenza virus and virus-infected cells. When tested in vivo, this immunotherapy is shown to be significantly superior to zanamivir in protecting mice from lethal influenza virus infections. Finally, both a zanamivir-targeted chemotherapy and a CAR-T cell therapy with different mechanisms of cytotoxicity against neuraminidase expressing cells are introduced in Chapter 4.

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and their presence in water bodies is documented. They discharge into surface water (SW) unmonitored, posing a threat to both aquatic and terrestrial lives. This is a challenge as not all populations have access to treated drinking water (TDW). The EDC contaminated serves as a route of exposure, together with ineffective treatment plants. Given the complexity of the endocrine system, EDCs may mimic or antagonise natural hormones or disrupt their synthesis, metabolism and excretion. The associated health effects include testicular dysgenesis syndrome, metabolic disorders and cancers. Policy and internationally standardised test methods are however sti ll limited. This study therefore aimed to assess the suitability of two assays used for screening estrogenic activity and one for androgenic activity in different water sources. The study consisted of two phases. In phase 1, water sample (tap, surface and treated wastewater) were collected from a catchment area in Pretoria. The samples and a spiked MilliQ laboratory water sample were extracted with solid phase extraction (SPE) and sent to Germany for distribution to participating laboratories. Samples (n=24) from six different countries were received to test for androgenic activity in the MDA-kb2 reporter gene assay. In phase 2, SW and TDW samples were collected from April 2015 until March 2016. The samples were filtered, extracted using SPE and assayed with the YES assay, T47D-KBluc reporter gene assay for estrogenic activity and MDA-kb2 reporter gene assay for androgenic activity. In phase 1, androgenic activity was detected in 4 out of 24 (21%) samples and ranged from 0.23 ± 0.040 ng/L to 0.008 ± 0.001 ng/L DHTEqs. In phase 2, estrogenic activity was detected in 16 out of 24 (67%) SW samples in the T47DKBluc reporter gene assay and ranged from 0.31 ± 0.05 pg/L to 10.51 ± 5.74 pg/L EEqs. It was below the detection limit (dl) in the YES assay. Androgenic activity was detected in 4 out of 24 (17%) SW samples, ranging from 0.0033 ± 0.0050 ng/L to 0.090 ± 0.040 ng/L DHTEqs. Androgenic and estrogenic activity was higher i n pretreatment samples compared to post-treatment in both treatment plants. In phase 1, the MDA-kb2 reporter gene assay was successfully applied to water samples from different sources. Androgenic activity was highest in treated wastewater. In phase 2, treatment plants proved to be effective in removing estrogens detected in the SW samples, as the TDW samples were below the dl. Estrogenic activity is within the ranges reported in other studies. Positive samples were below the 0.7 ng/L proposed trigger value for health risk assessments. Detected androgenic activity was lower in TDW samples compared to the SW samples supplying the two treatment plants indicating that they were both effective in removing the androgenic activity detected. Few studies have reported androgenic activity in tap water. This study strengthens the argument for using a battery of assays when monitoring endocrine activity as EDCs occur at low concentrations in mixtures. / Dissertation (MSc)--University of Pretoria, 2017. / School of Health Systems and Public Health (SHSPH) / MSc / Unrestricted

Es wird eine Bioassay-Analyse von 168 Testsubstanzen gegenüber Tetrahymena pyriformis beschrieben. Mithilfe dieses In-vitro-Ansatzes konnte eine erste prognostische Aussage über die toxische Wirkung unbekannter Stoffe getroffen werden. Aus den Konzentrations-Wirkungs-Beziehungen erfolgte eine Bestimmung der Toxizitätserhöhung gegenüber der Narkoselevel-Toxizität. Dadurch war eine mechanistische Interpretation der Wirkstärke der Fremdstoffe möglich. Die Wirkung der Fremdstoffe basiert auf direkten toxikologisch relevanten Reaktionsmechanismen mit nukleophilen Biomolekülen oder nach entsprechender enzymatischer Aktivierung und ermöglichte die Aufstellung von Strukturalarmen. Aus jeder Stoffklasse waren Vertreter direkt oder nach enzymatischer Biotransformation erhöht toxisch. Somit ist mithilfe der Ciliaten eine Vorhersage der direkten reaktiven Toxizität möglich. Zudem enthält es für eine metabolische Aktivierung bedeutsame Enzymsysteme. Damit ist es für eine prognostische Vorhersage der Toxizität unbekannter Stoffe anwendbar.

Reynoutria spp. rank among highly invasive taxa in the Czech Republic. Therefore, a considerable effort is focused on its eradication. This is usually done by application of non- selective herbicide based on glyphosate. However, only little is known about the spontaneous recovery of the native vegetation after the eradication, as well as whether the restoration can be facilitated by additional management. In my master thesis I (i) describe the process of restoration after Reynoutria spp. eradication, (ii) evaluate the influence of factors which could potentially inhibit the succession (such as glyphosate, allelopathy, changes in soil conditions due to Reynoutria spp. invasion), and (iii) evaluate the influence of native-species seed addition on establishment of target plant communities. The process of vegetation recovery was followed on 17 sites in a forest and in ruderal forest-free area from 2007 to 2010. Over the three years of the study, the sites in the forest- free area were covered by diverse vegetation, secondary succession on sites in a forest progressed more slowly. Any soil changes were detected. The process of succession was limited probably because a dead biomass was left on the sites after the eradication. The seed addition was also not crucial for the restoration of plant communitites.