Spores of various strains of Bacillus cereus are the causative agents of emetic and diarrheal foodborne illnesses. Typically, spores will survive thermal treatments that destroy vegetative cells, and then go on to germinate to form the vegetative cells that are associated with toxin production. The spore has to germinate in order to develop into the vegetative cells that produce toxins, hence a thorough understanding of the proteins and molecular mechanisms that underpin spore germination are of great significance from spore control perspectives. A major objective of this thesis was to use molecular genetic and fluorescence microscopy techniques to characterise the location and function of the GerP proteins in Bacillus cereus 14579. The GerP proteins have been identified from mutagenesis studies across the Bacilli as being implicated in spore germination, most likely by impacting upon the permeability of the spore coat. Data presented in this thesis reveal that the various GerP proteins all localise to the same inner-coat vicinity within the spore, as determined via the super-resolution ellipsoid localisation microscopy technique. The study also reveals that only the GerPA protein is required for the localisation of the other GerP proteins in the developing spore. A number of other coat and or germination associated proteins in B. cereus 14579 were examined in the course of this work. These include the GerN and GerT antiporters, which are both shown to have an involvement in inosine mediated spore germination in this strain. However, hypothetical interactions between antiporter proteins and the ‘linker-like’ N-terminal domain of the GerIA inosine-responsive germinant receptor protein appear unlikely since spores engineered with a truncated GerIA receptor subunit germinate normally. The protein encoded at locus BC1245 was also examined in this work, since it too had been implicated in spore germination. Data presented in this thesis indicate that this is not the case, and that the protein is a component of the spore coat. Overall, the work conducted in this project contributes to knowledge of spore assembly, spore structure and mechanisms that underpin germination, which ultimately, should permit the development of improved methodologies for spore control.

Please read the abstract in the section 00front of this document / Dissertation (MSc)--University of Pretoria, 2005. / School of Health Systems and Public Health (SHSPH) / Unrestricted

Seven species of marine phytoplankters assigned to different taxonomic divisions were tested for toxic responses to two different molecular species of selenium known to be prevalent in seawater, selenite and selenate. Selenate proved to be more toxic than selenite, although severe toxicity was only observed at high concentrations (10⁻² and 10⁻³ M) of both selenate and selenite. At these concentrations, growth was completely or severely inhibited in most species tested. In some of the species that remained viable, both the percentage of motile cells and their swimming speed were drastically reduced. Scanning electron microscopy revealed that, under these circumstances, Dunaliella tertiolecta cells possessed much shorter flagella compared to the controls, while those that became non-motile lacked flagella altogether. Despite these striking alterations in both growth and morphology, cells of Amphidinium carterae, Dunaliella tertiolecta and Pavlova lutheri showed, after prolonged exposures, signs of adaptation to high selenium concentrations. Lower concentrations of selenium were generally non-toxic and frequently even stimulatory to growth. These observations suggest that for meaningful inferences on selenium toxicity both the concentration range and the length of the studies must be considered and the potential for adaptation to high selenium concentrations taken into consideration. The main ultrastructural and physiological changes in cells of Dunaliella tertiolecta, Pavlova lutheri and Amphidinium carterae treated with selenite or selenate involved the cell coat, mitochondria, chloroplasts as well as the respiratory and photosynthetic rates. Other changes were observed in the nucleus, lipids, vacuoles, nitrogen and carbon contents, but these showed greater variability among the microalgae studied. The major alterations suggested that energy transducing systems were severely affected by selenium toxicity. These led to significant decreases or even elimination of storage products which were indicative of severe shortage in energy and produced major reductions in growth. These occurred later upon exposure to the toxicant and coincided with the loss of cell coat material, suggesting that the shedding of cell surface material might play a major role in the detoxification and adaptation of the microalgae to toxic concentrations of selenium. / Science, Faculty of / Botany, Department of / Graduate

The biological half-life of inorganic mercury in the Dungeness crab, Cancer magister Dana, was determined experimentally to be about 25 days. Crabs were exposed to mercury solutions, sacrificed after varying periods of time, and mercury determinations of whole body homogenates made with an atomic absorption spectrophotometer. The simple and widely used negative exponential equation for calculating biological half-life was not entirely adequate for describing the mercury elimination process. A better description was obtained using a nonlinear least-squares fit of an equation describing elimination at different speeds from two types of tissues. A further model allowed for recycling of mercury that was eliminated, and gave marginally better descriptions in some cases. / Science, Faculty of / Zoology, Department of / Graduate

Pesticide poisoning is a global health concern with approximately three million cases being reported on an annual basis. The latter includes both intentional and unintentional poisonings. Organophosphorus and carbamate insecticides are frequently found to be ‘responsible’ for pesticide poisoning in developing countries. In South Africa, aldicarb is the most potent carbamate pesticide and is sold in the informal markets as Temik. It is colloquially known as “Two step” or “Galephirimi” resulting in numerous cases of acute poisoning, especially in urban areas. Underreporting of suspected or confirmed pesticide poisoning cases has been a problem encountered in the national notification systems. Although a number of carbamate poisonings have been identified at the Pretoria Medico-Legal Laboratory, the presence of carbamates in post-mortem samples is rarely confirmed analytically. This may be ascribed to insufficient sample preparation, analytical methods not being sensitive enough or storage conditions not being optimal or too long before analysis takes place. It is well documented that most analytical errors occur during the pre-analytical phase, leading to a high prevalence of inconclusive results being attained. This may possibly be due to pre-analytical degradation, binding to biological matrix or the analytical method not been sensitive enough for detection in collected samples. Post-mortem redistribution factors such as physicochemical properties of the xenobiotic compounds (pH, volume of distribution, protein binding affinity, bacterial biotransformation and lipophilicity), characteristics of the matrix, specimen collection procedure and the use of preservatives may also influence the carbamate stability. The primary aim of the study was to optimise the sample preparation and analysis of biological matrices for select carbamates using LC-MS/MS method. Additionally, to analyse pesticide samples sold by street vendors as well as post-mortem samples collected from suspected cases of carbamate intoxication to determine whether the developed method can detect carbamates in real samples. Assessment of the aldicarb decay kinetics was done by spiking biological samples (whole blood, plasma, urine) collected from consenting healthy volunteers. Post-mortem samples (blood, urine, stomach content) of suspected carbamate poisoning cases, were screened for possible carbamate compounds and their metabolites or breakdown products. Optimisation and validation of the method was performed using a high-performance liquid chromatography (HPLC) system coupled to a triple quadrupole mass spectrometer following different extraction methods. The system was operated in positive electrospray ionisation (ESI+) mode. Different columns, mobile phase buffers and cartridges were used to compare the chromatographic separation of the carbamate compounds. Validation according to ICH guidelines was done for aldicarb. A set of matrix-matched standard calibration curves, was constructed using Analyst (version 1.5.2) software. Initial sample preparation of carbamate pesticides using three different SPE cartridges proved to be unreproducible with poor recoveries of specific compounds due to the wide range of carbamate pesticide polarities, so this was abandoned for the stability testing and forensic samples tested. About 85% reduction of the concentration of aldicarb was seen in whole blood only at ambient temperature but was stable at lower temperatures. Stability proved to be better in plasma compared to whole blood, for aldicarb and its oxidation products. Aldicarb was stable in urine stored with boric acid preservative. The ideal storage temperature for biological samples containing these carbamate compounds was found to be -80°C. During analysis of forensic samples, unknown peaks were consistently detected which are believed to correspond to adulterants and diluents which are added to “backstreet” pesticides. A possible match of an organophosphate, terbufos, found in some “backstreet” pesticide products was detected in some of the post-mortem samples. Considering their different physicochemical properties and that several factors can influence the biodegradation of carbamate compounds, no extrapolation of results from one carbamate compound to another can be formulated. The development and validation of an analytical method to quantify aldicarb and its oxidation products (aldicarb sulfoxide and sulfone) in whole blood, plasma and urine, using the protein precipitation method and LC-MS/MS was successful. Method validation to quantify ten standard carbamate pesticides using SPE and UPLC-q-TOF/MS was unsuccessful. The LC-MS/MS technique was found to be a suitable tool for the quantitation of aldicarb and its oxidation products in typical post-mortem sample matrices. / Dissertation (MSc (Medical Criminalistics))--University of Pretoria, 2021. / Faculty of Health Sciences Research Committee / Department of Pharmacology / University of Pretoria Masters research and research award grant / Forensic Medicine / MSc (Medical Criminalistics) / Unrestricted

Software-defined networking (SDN) is an emerging architecture with a great potentialto foster the development of modern networks. By separating the controlplane from the network devices and centralizing it at a software-based controller,SDN provides network-wide visibility and flexible programmability to networkadministrators. However, the security aspects of SDN are not yet fully understood.For example, while SDN is resistant to some topology poisoning attacks inwhich the attacker misleads the routing algorithm about the network structure,similar attacks by compromised hosts and switches are still known to be possible.The goal of this thesis is to thoroughly analyze the topology poisoning attacksinitiated by compromised switches and to identify whether they are a threat toSDN. We identify three base cases of the topology poisoning attack, in which theattack that requires a single compromised switch is a new variant of topologypoisoning. We develop proof-of-concept implementations for these attacks inemulated networks based on OpenFlow, the most popular framework for SDN.We also evaluate the attacks in simulated networks by measuring how muchadditional traffic the attacker can divert to the compromised switches. A widerange of network topologies and routing algorithms are used in the simulations.The simulation results show that the discovered attacks are severe in many cases.Furthermore, the seriousness of the attacks increases according to the number oftunnels that the attacker can fabricate and also depends on the distance betweenthe tunnel endpoints. The simulations indicate that network design can help tomitigate the attacks by, for example, shortening the paths between switches in thenetwork, randomizing regular network structure, or increasing the load-balancingcapability of the routing strategy.

Two groups of parents from different socio-economic levels were questioned about the behavioral characteristics of their children before 18 months and subsequent incidences of accidental poisoning. The data collected showed proportional numbers of accidental poisonings regardless of parent education, race or income. Unusual activity in the child before 18 months was the best predictor of accidental poisoning during childhood.

Thirty years since the removal of lead from gasoline, lead still poses a health risk. Children are most at-risk for adverse health outcomes caused by lead toxicity due to both behavioural (e.g., hand-to-mouth behaviour) and physiological differences (i.e., increased intake of lead by body weight, higher uptake rate and a higher vulnerability to the effects of lead) compared to adults (Yeoh et al., 2009). As a result, governments must identify children that may be at-risk of lead poisoning and develop practical methods to mitigate lead exposure. Before a government can develop a policy to help mitigate exposure of lead for children, we need to understand the spatial distribution of lead within the city. A popular spatial model used within air pollution research may allow more accurate, and more localized predictions than the most common interpolation method, kriging. Land use regression (LUR) is a technique leveraging multiple predictor variables to help estimate the spatial distribution of the dependent variable. By using historical sources of lead, LUR can be used to model soil lead levels (SLL) with localized variation. Unfortunately, spurious relationships can be the basis of a LUR model, which may lead to an overfitted spatial model resulting in a model with little generalizability and questionable ability to estimate the dependent variable at unobserved locations. Ultimately, Empirical Bayesian Kriging may be the best option for soil contamination research due to its ability to provide a smoothed prediction surface and its dependence on the spatial structure of the data to provide estimations. The benefit to society and the return on investment (ROI) is often the justification for lead remediation. Gould (2009) estimates a $17 to $221 ROI for every dollar spent on lead hazard control. One of the main components of this estimate of ROI comes from the decrease in intelligence quotient (IQ) that a child may experience as a consequence of lead toxicity. There are three main ways that a decrease in IQ can negatively impact the economy, (i) lower potential lifetime earnings, (ii) reduced tax revenues, and (iii) higher spending on special education (Gould, 2009). Since IQ has such a significant role in the ROI estimates, chapter 3 seeks to achieve a greater understanding of the relationship between blood lead levels (BLLs) and IQ. The loss of IQ points for an increase in blood lead concentration proposed by Lanphear et al. (2005) and referenced by Gould (2009) is significantly higher than what we found in our meta-analysis. Thus, the projected ROI proposed by Gould (2009) may be much lower than previously calculated. In the final chapter, the cost associated with permanent lead abatement is investigated based on ROI projections as a case study in Hamilton, Ontario. We show that, in most cases, permanent lead remediation is far too expensive for a municipal government. Furthermore, the capital initially invested may not be distributed back into the local economy, as the ROI suggests. We suggest that municipal governments make decisions based on need, rather than basing remediation decisions on ROI projections. Furthermore, we recommend the use of hazard quotient maps to help justify lead remediation as a more accurate representation of potential lead toxicity, instead of only looking at SLL exceedances across the city. / Dissertation / Doctor of Philosophy (PhD)

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