Malaria is a global pandemic that affects millions of people each year. It is a parasitic infection caused by the Plasmodium family, with Plasmodium falciparum being the most virulent strain. Malaria is transmitted to humans by the female Anopheles mosquito. The parasite undergoes two different cycles of its life cycle within the human host: the liver and intraerythrocytic life cycle. The latter consists of an asexual and sexual cycle. The intraerythrocytic cycle is perhaps the most important stage of the parasite's life cycle as it promotes the spread of the disease within and between hosts. The focus of this investigation was aimed at the invasion process of the merozoites into the erythrocytes. The Plasmodium merozoite utilises a cascade of proteins during the erythrocyte invasion process, which is a swift action that takes place in approximately 30 seconds. A number of surface proteins are expressed during merozoite development and are distributed along the merozoite surfaces to assist with attachment and invasion, the most crucial being MSP-1, AMA-1 and RON-2. MSP-1 and AMA-1 are vital targets for the development of malaria vaccines. AMA-1 is the central target protein of this investigation as it plays an essential role in the invasion process. AMA-1 commits the merozoite to invade the erythrocyte, as it assists the RON proteins in the formation of an irreversible tight-junction with the membrane of the erythrocyte. Antibodies, specific to AMA-1, bind to the protein, which prevents the formation of the tight junction and inhibits the invasion of the merozoite into the erythrocyte, therefore preventing the spread of the disease. However, before invasion, AMA-1 undergoes a number of proteolytic processes. It is synthesized as an 83 kDa (AMA-183) precursor protein in the apical organelle of the merozoite. This is then cleaved at the N-terminus to give rise to a 66 kDa (AMA-166) fragment, which is secreted onto the surface of the merozoite. The AMA-166 fragment is then cleaved into either a 48 kDa (AMA-148) or 44 kDa (AMA-144) fragment. One of these three fragments is then used by the merozoite for erythrocyte invasion. The aim of this investigation was to isolate and characterise each of the fragments of the Plasmodium falciparum AMA-1 (PfAMA-1) protein using the 3D7 lab strain of P. falciparum and to visualise the merozoite-erythrocyte invasion process, to possibly identify which of the AMA-1 fragments are involved in the invasion process. In order to achieve this large clusters of merozoites from sorbitol-synchronised cultures were isolated. Schizonts were isolated from culture by magnetic separation and incubated with E64 to prevent the release of merozoites. Merozoites that were required for the isolation of PfAMA-1 were harvested from the schizonts by saponin lysis, then homogenised, separated by SDS-PAGE and digested for LC-MS/MS analysis. Merozoites that were required for the visualisation procedures were not incubated with E64, to allow natural egression from the erythrocyte. The transmission electron microscopy results produced clear images of the merozoiteerythrocyte invasion process and the positioning of PfAMA-1 on the merozoite, before and after schizont rupture, was visualised from results obtained from confocal microscopy. Then PfAMA-1 was identified in isolated merozoite samples by LC-MS/MS analysis. However, due to its low abundance, isolation of high enough concentrations of PfAMA-1 to characterise its different fragments was not achieved. Further investigation into the development of the culturing and isolating methods could help in future projects aimed at isolating higher concentrations of merozoite proteins from synchronised cultures with a lower merozoite egression window period, in order to accomplish detailed analysis on invading proteins for the future development of treatments against malaria. / Dissertation (MSc)--University of Pretoria, 2016. / Pharmacology / MSc / Unrestricted

Doctor of Philosophy / Department of Plant Pathology / James Stack / The trade of food, plant, and animal products has increased the worldwide movement and establishment of exotic pathogens with dramatic negative impacts on plant systems. Fusarium proliferatum is a broad host-range pathogen and among the most common maize pathogens globally. It is often seed-borne and symptomless in maize, making it a high risk for introduction in maize and other grains. Considering the global distribution of maize and the wide host range and production of mycotoxins by F. proliferatum, a better understanding of its life history is needed. To provide markers for tracking F. proliferatum in laboratory experiments, strains of F. proliferatum were transformed to express a green fluorescent protein (GFP). Active dispersal (at least 1.5cm at 25°C and -50mb soil matric potential) and colonization of organic matter in nonsterile field soil was demonstrated in soil microcosms. Fusarium verticillioides is commonly isolated from maize seed also colonized by F. proliferatum. A red fluorescent (mRFP) F. verticillioides transformant was developed to study competition with F. proliferatum. For quantification in host tissues, a TaqMan multiplex qPCR protocol was developed using primer and probe sets targeting fragments of the green and red fluorescence genes to detect F. proliferatum and F. verticillioides, respectively. Prior colonization of maize tissues by F. verticillioides (p=0.6749) and other seed-borne microorganisms (p=0.1910) did not affect subsequent colonization by F. proliferatum. Genotyping-by-sequencing (GBS) was used to identify genetic markers in F. proliferatum. Primer sets based GBS markers were designed to allow detection of specific isolates in field experiments. F. proliferatum populations were characterized from maize seed prior to planting and again after harvest. End-point PCR identified F. proliferatum isolates containing the GBS marker. AFLP-fingerprinting indicated that 23 of the 817 F. proliferatum isolates contained the molecular marker and were genetically related to the original isolate. Based on the subclade and percentage similarity in UPGMA phylogenetic trees, and the population grouping observed in STRUCTURE and Principal Coordinate Analysis, these isolates could have a single origin and be clonal. Understanding the life cycle of F. proliferatum is critical for learning more about the risk of introducing seed-borne exotic isolates into new environments.

The responses of invasive exotic plant species (IES) to silvicultural treatments one growing season after timber harvesting were examined in the Cumberland Plateau region of Southeastern Kentucky. Treatments included a commercial deferment harvest and unharvested control applied to five watersheds within University of Kentucky’s Robinson Forest. The effects of harvesting were compared between treatments and between preharvest and postharvest samplings. The spatial distribution and abundance of targeted IES throughout forest sites and trail systems were calculated from several sampling schemes. Additional analyses were performed to quantify forest disturbances derived from harvest activities to determine the relationships between soil, light levels, and other environmental characteristics and IES cover. Logistic and multivariate analysis techniques were used to analyze differences in IES distribution between pre-harvest and post-harvest units to relate post-harvest IES to microsite conditions. Microsite conditions within the forest and along the trail system proved important for explaining the presence and distribution of IES. Timber harvesting caused a significant increase in both Ailanthus altissima and Microstegium vimineum within harvested areas. However, many other identified IES did not initially respond to disturbances. Throughout the treatment units, species were influenced by disturbance type and intensity, as well as proximity to reclaimed surface mined land.

Abstract Between October 1999 and October 2003, 30 adult and 48 young (< 1 year) raccoon dogs (Nyctereutes procyonoides) were monitored using radio-telemetry in an area of Germany which has been occupied by this invasive alien species since the early 1990s. Additionally, three pairs of raccoon dogs were observed by continuous radio-tracking during the first six weeks after parturition in 2003. Furthermore 136 raccoon dog pubs were ear-tagged between June 1999 and August 2006. No adult animals dispersed from the area during the study period and home ranges tended to be used for several years, probably for life. The average annual home range size, calculated using 95% fixed kernel, was 382.2 ha ± 297.4 SD for females (n = 30 seasonal home ranges) and 352.4 ha ± 313.3 SD for males (n = 32 seasonal home ranges). Paired raccoon dogs had home ranges of similar size, with pair sharing the same area all year round. Raccoon dogs occupied large core areas (85% kernel) covering 81.2% of their home ranges. The home ranges were at their smallest during the mating season. The slightly larger size of home ranges in winter suggests that, due to the temperate climate, raccoon dogs do not hibernate in Germany. Males and females formed a long-term (probably lifelong) pair bond. Same-sex neighbours ignored each other and even adjacent males/females showed neither preference nor avoidance. Thus, it can be assumed that the raccoon dog in Central Europe is monogamous without exclusive territories, based on the results of home range overlap analysis and interaction estimations. Habitat composition within home ranges and within the whole study area was almost equal. Although, percentage shares of farmland and meadow was 16.35% smaller and 12.06% higher within the home ranges, respectively. All nine habitat types (farmland, forest, settlement, water, meadows, maize fields, small woods, reeds and hedges) were used opportunistically by raccoon dogs. No significant, recognisable difference for habitat preferences between seasons was detected. Male and female raccoon dog showed equal habitat preference pattern. A comparison of active and inactive locations in different habitats found no remarkable differences. Habitat composition of individual home ranges was used to classify animals. If the percentage of forest within a home range exceeded 50% the individual was classified as a ‘forest type’ raccoon dog. If the percentage of forest habitats within a home range was less than 5%, the share of pastureland was mean 81.82% ± 16.92 SD. Consequently the individual was classified as a ‘agrarian type’ raccoon dog. Neither habitat preference nor habitat selection process differed between the two ‘types’. Habitat use and preference is discussed with relation to the ability of the raccoon dog to expand its range towards Western Europe. Males spent noticeably more time (40.5% of the time ±11.7 SD) alone with the pups than females (16.4% of the time ±8.5 SD). Females had noticeably larger 95% kernel home ranges (98.24 ha ±51.71 SD) than males (14.73 ha ±8.16 SD) and moved much longer daily distances (7,368 m ±2,015 SD) than males (4,094 m ±2,886 SD) in six weeks postpartum. The raccoon dogs being studied left the breeding den in the 6th week after the birth of the pups. In situ video observation showed that the male carried prey to the den to provide the female and the litter with food. A clear division of labour took place among parents during the period in which the pups were nursed: males guarded the litter in the den or in close vicinity of it, while the females foraged to satisfy their increased energy requirements. There were relocations of 59 (43.4%) ear-tagged young racoon dogs and mean distance from marking point was 13.5 km ±20.1 SD. Dispersal mortality rate was 69.5% among young raccoon dogs. Most animals (55.9%) were recovered nearer than 5 km from the marking point, whereas only 8.5% relocations were recorded further than 50 km from the marking point. There was no difference in the distances of relocations between sexes. Most (53.7%) relocations of ear-tagged young raccoon dogs were in August and September and, only 34.1% were recorded from October to April. Hunting (55 %) and traffic (27 %) were the major mortality factors. Radio-collared young raccoon dogs generally dispersed between July and September. The mean natal home range size (MCP 100%) with and without excursions was 502.6 ha ±66.4 SD (n = 9) and 92.1 ha ±66.4 SD (n = 17), respectively. There were no differences between sexes in the month of dispersal. The direction of travel for dispersing animals appeared to be random, with distances from 0.5 km to 91.2 km. A highly flexible dispersing behaviour is certainly one of the reasons which contribute to the high expansion success of the species.

Between October 1999 and October 2003, 30 adult and 48 young (< 1 year) raccoon dogs (Nyctereutes procyonoides) were monitored using radio-telemetry in an area of North-East Germany which has been occupied by this invasive alien species since the early 1990s. Additionally, three pairs of raccoon dogs were observed by continuous radio-tracking during the first six weeks after parturition in 2003. Furthermore 136 raccoon dog pubs were ear-tagged between June 1999 and August 2006. No adult animals dispersed from the area during the study period and home ranges tended to be used for several years, probably for life. The average annual home range size, calculated using 95% fixed kernel, was 382.2 ha ± 297.4 SD for females (n = 30 seasonal home ranges) and 352.4 ha ± 313.3 SD for males (n = 32 seasonal home ranges). Paired raccoon dogs had home ranges of similar size, with pair mates sharing the same area all year round. Raccoon dogs occupied large core areas (85% kernel) covering 81.2% of their home ranges. The home ranges were at their smallest during the mating season. The slightly larger size of home ranges in winter suggests that, due to the temperate climate, raccoon dogs do not hibernate in Germany. Males and females formed a long-term (probably lifelong) pair bond. Same-sex neighbours ignored each other and even adjacent males/females showed neither preference nor avoidance. Thus, it can be assumed that the raccoon dog in Central Europe is monogamous without exclusive territories, based on the results of home range overlap analysis and interaction estimations. Habitat composition within home ranges and within the whole study area was almost equal. Although, percentage shares of farmland and meadow was 16.35% smaller and 12.06% higher within the home ranges, respectively. All nine habitat types (farmland, forest, settlement, water, meadows, maize fields, small woods, reeds and hedges) were used opportunistically by raccoon dogs. No significant, recognisable difference for habitat preferences between seasons was detected. Male and female raccoon dog showed equal habitat preference pattern. A comparison of active and inactive locations in different habitats found no remarkable differences. Habitat composition of individual home ranges was used to classify animals. If the percentage of forest within a home range exceeded 50% the individual was classified as a ‘forest type’ raccoon dog. If the percentage of forest habitats within a home range was less than 5%, the share of pastureland was mean 81.82% ± 16.92 SD. Consequently the individual was classified as a ‘agrarian type’ raccoon dog. Neither habitat preference nor habitat selection process differed between the two ‘types’. Habitat use and preference is discussed with relation to the ability of the raccoon dog to expand its range towards Western Europe. Males spent noticeably more time (40.5% of the time ±11.7 SD) alone with the pups than females (16.4% of the time ±8.5 SD). Females had noticeably larger 95% kernel home ranges (98.24 ha ±51.71 SD) than males (14.73 ha ±8.16 SD) and moved much longer daily distances (7,368 m ±2,015 SD) than males (4,094 m ±2,886 SD) in six weeks postpartum. The raccoon dogs being studied left the breeding den in the 6th week after the birth of the pups. In situ video observation showed that the male carried prey to the den to provide the female and the litter with food. A clear division of labour took place among parents during the period in which the pups were nursed: males guarded the litter in the den or in close vicinity of it, while the females foraged to satisfy their increased energy requirements. There were relocations of 59 (43.4%) ear-tagged young raccoon dogs and mean distance from marking point was 13.5 km ±20.1 SD. Dispersal mortality rate was 69.5% among young raccoon dogs. Most animals (55.9%) were recovered nearer than 5 km from the marking point, whereas only 8.5% relocations were recorded further than 50 km from the marking point. There was no difference in the distances of relocations between sexes. Most (53.7%) relocations of ear-tagged young raccoon dogs were in August and September and, only 34.1% were recorded from October to April. Hunting (55 %) and traffic (27 %) were the major mortality factors. Radiocollared young raccoon dogs generally dispersed between July and September. The mean natal home range size (MCP 100%) with and without excursions was 502.6 ha ±66.4 SD (n = 9) and 92.1 ha ±66.4 SD (n = 17), respectively. There were no differences between sexes in the month of dispersal. The direction of travel for dispersing animals appeared to be random, with distances from 0.5 km to 91.2 km. A highly flexible dispersing behaviour is certainly one of the reasons which contribute to the high expansion success of the species.

Abstract Between October 1999 and October 2003, 30 adult and 48 young (&amp;lt; 1 year) raccoon dogs (Nyctereutes procyonoides) were monitored using radio-telemetry in an area of Germany which has been occupied by this invasive alien species since the early 1990s. Additionally, three pairs of raccoon dogs were observed by continuous radio-tracking during the first six weeks after parturition in 2003. Furthermore 136 raccoon dog pubs were ear-tagged between June 1999 and August 2006. No adult animals dispersed from the area during the study period and home ranges tended to be used for several years, probably for life. The average annual home range size, calculated using 95% fixed kernel, was 382.2 ha ± 297.4 SD for females (n = 30 seasonal home ranges) and 352.4 ha ± 313.3 SD for males (n = 32 seasonal home ranges). Paired raccoon dogs had home ranges of similar size, with pair sharing the same area all year round. Raccoon dogs occupied large core areas (85% kernel) covering 81.2% of their home ranges. The home ranges were at their smallest during the mating season. The slightly larger size of home ranges in winter suggests that, due to the temperate climate, raccoon dogs do not hibernate in Germany. Males and females formed a long-term (probably lifelong) pair bond. Same-sex neighbours ignored each other and even adjacent males/females showed neither preference nor avoidance. Thus, it can be assumed that the raccoon dog in Central Europe is monogamous without exclusive territories, based on the results of home range overlap analysis and interaction estimations. Habitat composition within home ranges and within the whole study area was almost equal. Although, percentage shares of farmland and meadow was 16.35% smaller and 12.06% higher within the home ranges, respectively. All nine habitat types (farmland, forest, settlement, water, meadows, maize fields, small woods, reeds and hedges) were used opportunistically by raccoon dogs. No significant, recognisable difference for habitat preferences between seasons was detected. Male and female raccoon dog showed equal habitat preference pattern. A comparison of active and inactive locations in different habitats found no remarkable differences. Habitat composition of individual home ranges was used to classify animals. If the percentage of forest within a home range exceeded 50% the individual was classified as a ‘forest type’ raccoon dog. If the percentage of forest habitats within a home range was less than 5%, the share of pastureland was mean 81.82% ± 16.92 SD. Consequently the individual was classified as a ‘agrarian type’ raccoon dog. Neither habitat preference nor habitat selection process differed between the two ‘types’. Habitat use and preference is discussed with relation to the ability of the raccoon dog to expand its range towards Western Europe. Males spent noticeably more time (40.5% of the time ±11.7 SD) alone with the pups than females (16.4% of the time ±8.5 SD). Females had noticeably larger 95% kernel home ranges (98.24 ha ±51.71 SD) than males (14.73 ha ±8.16 SD) and moved much longer daily distances (7,368 m ±2,015 SD) than males (4,094 m ±2,886 SD) in six weeks postpartum. The raccoon dogs being studied left the breeding den in the 6th week after the birth of the pups. In situ video observation showed that the male carried prey to the den to provide the female and the litter with food. A clear division of labour took place among parents during the period in which the pups were nursed: males guarded the litter in the den or in close vicinity of it, while the females foraged to satisfy their increased energy requirements. There were relocations of 59 (43.4%) ear-tagged young racoon dogs and mean distance from marking point was 13.5 km ±20.1 SD. Dispersal mortality rate was 69.5% among young raccoon dogs. Most animals (55.9%) were recovered nearer than 5 km from the marking point, whereas only 8.5% relocations were recorded further than 50 km from the marking point. There was no difference in the distances of relocations between sexes. Most (53.7%) relocations of ear-tagged young raccoon dogs were in August and September and, only 34.1% were recorded from October to April. Hunting (55 %) and traffic (27 %) were the major mortality factors. Radio-collared young raccoon dogs generally dispersed between July and September. The mean natal home range size (MCP 100%) with and without excursions was 502.6 ha ±66.4 SD (n = 9) and 92.1 ha ±66.4 SD (n = 17), respectively. There were no differences between sexes in the month of dispersal. The direction of travel for dispersing animals appeared to be random, with distances from 0.5 km to 91.2 km. A highly flexible dispersing behaviour is certainly one of the reasons which contribute to the high expansion success of the species.

Between October 1999 and October 2003, 30 adult and 48 young (< 1 year) raccoon dogs (Nyctereutes procyonoides) were monitored using radio-telemetry in an area of North-East Germany which has been occupied by this invasive alien species since the early 1990s. Additionally, three pairs of raccoon dogs were observed by continuous radio-tracking during the first six weeks after parturition in 2003. Furthermore 136 raccoon dog pubs were ear-tagged between June 1999 and August 2006. No adult animals dispersed from the area during the study period and home ranges tended to be used for several years, probably for life. The average annual home range size, calculated using 95% fixed kernel, was 382.2 ha ± 297.4 SD for females (n = 30 seasonal home ranges) and 352.4 ha ± 313.3 SD for males (n = 32 seasonal home ranges). Paired raccoon dogs had home ranges of similar size, with pair mates sharing the same area all year round. Raccoon dogs occupied large core areas (85% kernel) covering 81.2% of their home ranges. The home ranges were at their smallest during the mating season. The slightly larger size of home ranges in winter suggests that, due to the temperate climate, raccoon dogs do not hibernate in Germany. Males and females formed a long-term (probably lifelong) pair bond. Same-sex neighbours ignored each other and even adjacent males/females showed neither preference nor avoidance. Thus, it can be assumed that the raccoon dog in Central Europe is monogamous without exclusive territories, based on the results of home range overlap analysis and interaction estimations. Habitat composition within home ranges and within the whole study area was almost equal. Although, percentage shares of farmland and meadow was 16.35% smaller and 12.06% higher within the home ranges, respectively. All nine habitat types (farmland, forest, settlement, water, meadows, maize fields, small woods, reeds and hedges) were used opportunistically by raccoon dogs. No significant, recognisable difference for habitat preferences between seasons was detected. Male and female raccoon dog showed equal habitat preference pattern. A comparison of active and inactive locations in different habitats found no remarkable differences. Habitat composition of individual home ranges was used to classify animals. If the percentage of forest within a home range exceeded 50% the individual was classified as a ‘forest type’ raccoon dog. If the percentage of forest habitats within a home range was less than 5%, the share of pastureland was mean 81.82% ± 16.92 SD. Consequently the individual was classified as a ‘agrarian type’ raccoon dog. Neither habitat preference nor habitat selection process differed between the two ‘types’. Habitat use and preference is discussed with relation to the ability of the raccoon dog to expand its range towards Western Europe. Males spent noticeably more time (40.5% of the time ±11.7 SD) alone with the pups than females (16.4% of the time ±8.5 SD). Females had noticeably larger 95% kernel home ranges (98.24 ha ±51.71 SD) than males (14.73 ha ±8.16 SD) and moved much longer daily distances (7,368 m ±2,015 SD) than males (4,094 m ±2,886 SD) in six weeks postpartum. The raccoon dogs being studied left the breeding den in the 6th week after the birth of the pups. In situ video observation showed that the male carried prey to the den to provide the female and the litter with food. A clear division of labour took place among parents during the period in which the pups were nursed: males guarded the litter in the den or in close vicinity of it, while the females foraged to satisfy their increased energy requirements. There were relocations of 59 (43.4%) ear-tagged young raccoon dogs and mean distance from marking point was 13.5 km ±20.1 SD. Dispersal mortality rate was 69.5% among young raccoon dogs. Most animals (55.9%) were recovered nearer than 5 km from the marking point, whereas only 8.5% relocations were recorded further than 50 km from the marking point. There was no difference in the distances of relocations between sexes. Most (53.7%) relocations of ear-tagged young raccoon dogs were in August and September and, only 34.1% were recorded from October to April. Hunting (55 %) and traffic (27 %) were the major mortality factors. Radiocollared young raccoon dogs generally dispersed between July and September. The mean natal home range size (MCP 100%) with and without excursions was 502.6 ha ±66.4 SD (n = 9) and 92.1 ha ±66.4 SD (n = 17), respectively. There were no differences between sexes in the month of dispersal. The direction of travel for dispersing animals appeared to be random, with distances from 0.5 km to 91.2 km. A highly flexible dispersing behaviour is certainly one of the reasons which contribute to the high expansion success of the species.