Reovirus is internalized after interaction of the outer proteins μ1, σ1 and σ3 with the host cell. Proteolysis of σ3 and cleavage of μ1 (into δ and φ) eventually leads to the formation of a more infectious subviral particle named “ISVP”. The infectious entry of viruses, but not of ISVPs, can be blocked using various entry inhibitors and therefore, suggests that there is a threshold of σ3 digestion required to allow particle to bypass entry blockers. By combining protease and detergent to the digestion of virions, data from this work showed distinct particles generated along the transition pathway. In addition, studies involving flow cytometry and specific antibodies (anti-μ1) showed that between virus and ISVP there is a gradual yet heterogeneous particle proteolysis that is directly related to the virus infectivity. The findings and approaches taken for this thesis work can possibly be extended for studying other non-enveloped viruses. Moreover, it may help to shed some light on the development of safe and effective oncolytic agents.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Malaria is currently one of the most imperative parasitic diseases in developing countries. Artesunate has a short half-life, low aqueous solubility and resultant poor and erratic absorption upon oral administration, which translate to low bioavailability. Mefloquine is eliminated slowly with a terminal elimination half-life of approximately 20 days and has neuropsychiatric side effects. Novel drug delivery systems have been utilised to optimise chemotherapy with currently available antimalarial drugs. Pheroid™ technology is a patented drug delivery system which has the ability to capture, transport and deliver pharmaceutical compounds. Pheroid™ technology may play a key role in ensuring effective delivery and enhanced bioavailability of novel antimalarial drugs. The aim of this study was to evaluate the possible efficacy and bioavailability enhancement of the selected antimalarial drugs, artesunate and mefloquine, in combination with Pheroid™ vesicles. The in vitro efficacy of artesunate and mefloquine co-formulated in the oil phase of Pheroid™ vesicles and entrapped in Pheroid™ vesicles 24 hours after manufacturing were investigated against a 3D7 chloroquine-sensitive strain of Plasmodium falciparum. Parasitemia (%) was quantified with flow cytometry after incubation periods of 48 and 72 hours. Drug sensitivity was expressed as 50% inhibitory concentration (IC50) values. An in vivo bioavailability study with artesunate and mefloquine was also conducted in combination with Pheroid™ vesicles, using a mouse model. A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to analyse the drug levels. C57 BL6 mice were used during this study. The selected antimalarial drugs were administered at a dose of 20 mg/kg with an oral gavage tube. Blood samples were collected by means of tail bleeding. The in vitro drug sensitivity assays revealed that artesunate, co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period, decreased the IC50 concentration significantly by 90%. Extending the incubation period to 72 hours decreased the IC50 concentration of artesunate, also co-formulated in the oil phase of Pheroid ™ vesicles significantly by 72%. No statistically significant differences between the reference and Pheroid™ vesicle groups were achieved when artesunate was entrapped 24 hours after manufacturing of Pheroid™ vesicles. Mefloquine co-formulated in the oil phase of Pheroid™ vesicles and evaluated after a 48 hour incubation period decreased the IC50 concentration by 36%. Extending the incubation period to 72 hours increased the efficacy of the Pheroid™ vesicles and the IC50 concentration was significantly decreased by 51%. In contrast with the results obtained with artesunate, entrapment of mefloquine in Pheroid™ vesicles 24 hours after manufacturing decreased the IC50 concentration significantly by 66%. The LC-MS/MS method was found to be sensitive, selective and accurate for the determination of artesunate and its active metabolite, dihydroartemisinin (DHA) in mouse plasma and mefloquine in mouse whole blood. Most of the artesunate plasma concentrations were below the limit of quantification in the reference group and relatively high outliers were observed in some of the samples. The mean artesunate levels of the Pheroid™ vesicle group were lower compared to the reference group, but the variation within the Pheroid™ vesicle group lessened significantly. The mean DHA concentrations of the Pheroid™ vesicle group were significantly higher. DHA obtained a higher peak plasma drug concentration with the Pheroid™ vesicle group (173.0 ng/ml) in relation to the reference group (105.0 ng/ml) and at a much faster time (10 minutes in Pheroid™ vesicles in contrast to 30 minutes of the reference group). Pharmacokinetic models could not be constructed due to blood sampling per animal limitation. The incorporation of mefloquine in Pheroid™ vesicles did not seem to have improved results in relation to the reference group. No statistical significant differences were observed in the pharmacokinetic parameters between the two groups. The relative bioavailability (%) of the Pheroid™ vesicle incorporated mefloquine was 7% less bioavailable than the reference group. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

The decidualized endometrium harbors abundant leukocyte populations that are proposed to regulate critical processes at the maternal fetal interface including transformation of decidual spiral arteries. The work in this thesis investigated the leukocyte subtypes in the decidua throughout the course of this vascular transformation. A particular focus was the role of the uterine Natural Killer (uNK) cells and macrophages in an in vitro model of vascular remodeling. A significant infiltration of uNK cells and macrophages, matrix metalloproteinase-2/9 activity, and evidence of apoptosis and phagocytosis were observed in remodeling arterioles. From first to second trimester, FACS analysis demonstrated dramatic changes in the decidual leukocyte subpopulations, including the decline of uNK cells and macrophages and substantial increase in T lymphocytes and neutrophils. These data demonstrate an integral role of uNK cells and macrophages in early vascular remodeling and provide evidence of unique and complex immune interactions in the decidual microenvironment during human pregnancy.

The decidualized endometrium harbors abundant leukocyte populations that are proposed to regulate critical processes at the maternal fetal interface including transformation of decidual spiral arteries. The work in this thesis investigated the leukocyte subtypes in the decidua throughout the course of this vascular transformation. A particular focus was the role of the uterine Natural Killer (uNK) cells and macrophages in an in vitro model of vascular remodeling. A significant infiltration of uNK cells and macrophages, matrix metalloproteinase-2/9 activity, and evidence of apoptosis and phagocytosis were observed in remodeling arterioles. From first to second trimester, FACS analysis demonstrated dramatic changes in the decidual leukocyte subpopulations, including the decline of uNK cells and macrophages and substantial increase in T lymphocytes and neutrophils. These data demonstrate an integral role of uNK cells and macrophages in early vascular remodeling and provide evidence of unique and complex immune interactions in the decidual microenvironment during human pregnancy.

Flocculation, the process whereby yeast cells attach in groups and sediment to the top or bottom of a fermenter, is industrially important in many fermentation batch operations. These batch operations include wine, distilled spirits, cider, bio-ethanol and production of commercial yeast metabolites. In the case of brewing yeast, it has been determined that flocculation occurs due to three forces called hydrophobic interactions, zymolectin binding and to a lesser extent, surface charge neutralization. This project sought to more closely study hydrophobic interactions and zymolectin binding. Earlier studies had shown that certain hydrophobic carboxylic acids, 3-OH oxylipins, formed in brewing yeast at flocculation onset. Therefore, these compounds showed potential as an indicator of overall cell surface hydrophobicity, and it was believed that flocculation level, cell surface hydrophobicity and oxylipin level would increase in unison in the yeast cell during brewing fermentations. During lab scale fermentations in shaker flasks and in a miniature fermentation assay setup, both flocculation level and cell surface hydrophobicity increased coincidently. However, 3-OH oxylipins could not be isolated from whole cells or cell wall isolates grown in the shaker flasks or whole cells grown in the miniature fermentation assay at detection limits approximated as 50 ng/0.5 g wet yeast. Due to their minute levels in brewing yeast cells, it was proposed that 3-OH oxylipins may mediate flocculation and aggregation via a quorum sensing mechanism instead of by increasing cell surface hydrophobicity. A disagreement exists in the literature where certain researchers believe zymolectin activity is induced, while others believe it is constitutive. The second part of this study attempted to address this by measuring zymolectin density during lab scale fermentations with a flow cytometer. Because of flow cytometry’s capacity for multiparametric analysis, large amounts of data were produced which gave information on not only zymolectin density, but also cell size and cellular complexity. Upon statistical analysis of the data, it was not possible to either refute or confirm the claim that zymolectin activity is induced or constitutive. However, the results suggested there could have been a population of cells with fewer zymolectins, and this certainly warrants further investigation. During the lab scale fermentations, cell size measured by a flow cytometer appeared to be correlated with manual measures of cell size. Furthermore, cell size tended towards uniformity during the fermentation which has also been observed in similar studies employing flow cytometry. Conversely, the cellular complexity of the yeast in this study did not change as in other studies by this may have been due either to strain differences or the methods used herein.

The crystal (Cry) toxins from Bacillus thuringiensis (Bt) display high specificity and toxicity against relevant insect pests and the use of Bt-based products continues to contribute to insect pest management. To protect this investment, further its potential, and investigate possible unintended effects, various research questions have been proposed. One issue related to Bt usage is the evolution of pest resistance to Bt toxins. The midgut epithelium is targeted by Cry toxins killing enterocytes, facilitating invasion of the hemocoel, leading to septicemia and mortality. While resistance may emerge from alterations to these steps, most research efforts have been focused on reduced toxin binding to midgut receptors as resistance mechanism. Lepidopteran crop pest Heliothis virescens strains have been hypothesized to have enhanced midgut proliferation and differentiation of stem cell populations allowing for regeneration and resistance to diverse Cry toxins. However, the molecular mechanisms involved are not known. We developed a flow cytometry method to monitor stem cell proliferation and differentiation to compare midgut regenerative responses to Cry intoxication in larvae from susceptible and Bt-resistant strains of H. virescens. The structure of the epithelial healing response was studied in vivo using hematoxylin-eosin stained midguts derived from larvae fed Cry1Ac toxin. We detected less regenerative cells in midguts from a Bt-susceptible strain (YDK) compared to midguts from resistant (KCB and CXC) strains, and an overall increase in the total number of cells per unit surface area in KCB midguts. Using primary midgut cell cultures, the midgut regeneration response to Cry1Ac in CXC was an increase in available differentiated cells compared to YDK. In contrast, KCB exhibited an increased abundance of stem cells compared to both YDK and CXC. Using a differential proteomics approach we characterized the proteins secreted by H. virescens midgut cells in response to Cry1Ac and identified a relevant role for arylphorin in promoting midgut regeneration in response to Cry1Ac and DiPel intoxication in both susceptible and resistant H. virescens larvae. The potential fitness costs associated with altered hexamerin transcript expression were monitored using larval bioassays.

Ultrasound has been identified as a possible non-invasive drug delivery device that could avoid many of the problems found in traditional therapeutics. Studies have shown that ultrasound can deliver molecules into cells; however, the applicability of ultrasound has been limited due to uncontrollable cellular viability losses after sonication. In this study, we sought to quantify the heterogeneous bioeffects of ultrasound in order to gain more insight into how ultrasound affects cells. We were also concerned with identifying the causes of and preventing programmed cell death caused by ultrasound exposure. In order to accomplish these objectives, we used flow cytometry to group cells into quantifiable characteristic populations. This allowed us to identify the relative importance of different forms of rapid cell death. We found that up to 65% of cells (at the highest ultrasound pressure studied) can lose viability rapidly and, for the first time, quantified them among three distinct populations: (1) cells that retain normal size but lose plasma membrane integrity; (2) intact nuclei surrounded by plasma membrane remnants; (3) debris resulting from cellular lysis. Our analysis was supported by mechanical sorting of these populations and subsequent imaging using confocal microscopy. We then monitored the viable populations for 6 h after ultrasound exposure. Results indicated that up to 15% of viable cells (at the highest ultrasound pressure studied) underwent apoptosis, which we showed was associated with an influx of intracellular Ca2+; therefore, we developed a method of chelating intracellular Ca2+ after sonication in an effort to maintain viability of those cells. Using this technique, we showed for the first time that cells could be saved, and we were able to prevent apoptosis by 50%, thereby increasing the overall viability of cells exposed to ultrasound. We conclude that ultrasound is a useful method to deliver molecules into cells and that appropriate selection of sonication conditions can minimize cell death by rapid and apoptotic mechanisms.

Recent proceedings in biotechnology have enabled completely new avenues in life science research to be explored. By allowing increased parallelization an ever-increasing complexity of cell samples or experiments can be investigated in shorter time and at a lower cost. This facilitates for example large-scale efforts to study cell heterogeneity at the single cell level, by analyzing cells in parallel that also can include global genomic analyses. The work presented in this thesis focuses on massively parallel analysis of cells or nucleic acid samples, demonstrating technology developments in the field as well as use of the technology in life sciences. In stem cell research issues such as cell morphology, cell differentiation and effects of reprogramming factors are frequently studied, and to obtain information on cell heterogeneity these experiments are preferably carried out on single cells. In paper I we used a high-density microwell device in silicon and glass for culturing and screening of stem cells. Maintained pluripotency in stem cells from human and mouse was demonstrated in a screening assay by antibody staining and the chip was furthermore used for studying neural differentiation. The chip format allows for low sample volumes and rapid high-throughput analysis of single cells, and is compatible with Fluorescence Activated Cell Sorting (FACS) for precise cell selection. Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences by constantly producing increasing amounts of data from one sequencing run. However, the reagent costs and labor requirements in current massively parallel sequencing protocols are still substantial. In paper II-IV we have focused on flow-sorting techniques for improved sample preparation in bead-based massive sequencing platforms, with the aim of increasing the amount of quality data output, as demonstrated on the Roche/454 platform. In paper II we demonstrate a rapid alternative to the existing shotgun sample titration protocol and also use flow-sorting to enrich for beads that carry amplified template DNA after emulsion PCR, thus obtaining pure samples and with no downstream sacrifice of DNA sequencing quality. This should be seen in comparison to the standard 454-enrichment protocol, which gives rise to varying degrees of sample purity, thus affecting the sequence data output of the sequencing run. Massively parallel sequencing is also useful for deep sequencing of specific PCR-amplified targets in parallel. However, unspecific product formation is a common problem in amplicon sequencing and since these shorter products may be difficult to fully remove by standard procedures such as gel purification, and their presence inevitably reduces the number of target sequence reads that can be obtained in each sequencing run. In paper III a gene-specific fluorescent probe was used for target-specific FACS enrichment to specifically enrich for beads with an amplified target gene on the surface. Through this procedure a nearly three-fold increase in fraction of informative sequences was obtained and with no sequence bias introduced. Barcode labeling of different DNA libraries prior to pooling and emulsion PCR is standard procedure to maximize the number of experiments that can be run in one sequencing lane, while also decreasing the impact of technical noise. However, variation between libraries in quality and GC content affects amplification efficiency, which may result in biased fractions of the different libraries in the sequencing data. In paper IV barcode specific labeling and flow-sorting for normalization of beads with different barcodes on the surface was used in order to weigh the proportion of data obtained from different samples, while also removing mixed beads, and beads with no or poorly amplified product on the surface, hence also resulting in an increased sequence quality. In paper V, cell heterogeneity within a human being is being investigated by low-coverage whole genome sequencing of single cell material. By focusing on the most variable portion of the human genome, polyguanine nucleotide repeat regions, variability between different cells is investigated and highly variable polyguanine repeat loci are identified. By selectively amplifying and sequencing polyguanine nucleotide repeats from single cells for which the phylogenetic relationship is known, we demonstrate that massively parallel sequencing can be used to study cell-cell variation in length of these repeats, based on which a phylogenetic tree can be drawn. / QC 20111031

Coronary artery disease is a leading cause of morbidity and mortality in developed countries. Percutaneous transluminal coronary angioplasty (PTCA) is an important treatment option when intervention is required; namely for patients with relatively severe occlusions. However, adverse events including recurrence of angina pectoris and restenosis of the treated artery limit patient prognosis, with subsequent re-vascularisation often necessary. Platelet activation accompanies PTCA, with platelet adhesion and aggregation involved in thrombus formation during restenosis. During platelet activation, highly coagulant platelet-derived microparticles (PMPs) are formed, and it is likely that these PMPs will also be produced during PTCA. While platelet aggregation inhibitors used during PTCA limit platelet aggregation and decrease the incidence of restenosis, they do not prevent PMPs being formed. PMPs are capable of adhesion and aggregation, and adhere to PTCA treated arteries in an animal model. Therefore, in order to understand the phenomenon of restenosis and its improved limitation, it is necessary to investigate PMP formation during PTCA. The field of PMP study is in its infancy, with conflicting results from the substantial inequities in methods of PMP measurement, which may be exacerbated by PMP heterogeneity. The current literature on this topic is reviewed in Chapter 2, where the PMP surface and possible functions are considered, and the PMP size and morphology examined. To conclude, the relationship between PMPs and PTCA is explored, with a focus on the potential role of PMPs in restenosis. The knowledge deficiencies in this field are highlighted at the conclusion of this chapter. Very little is known regarding the production of PMPs with PTCA. The level of PMPs during PTCA was monitored in paired arterial blood samples obtained from seventy-five patients undergoing the procedure (Chapter 3). A significant increase in PMPs from baseline to completion of PTCA was clearly demonstrated for the first time. This indicated that procoagulant PMPs are produced during PTCA and may contribute to subsequent restenosis. Furthermore, administration of the platelet aggregation inhibitor abciximab to a group of thirty-eight high risk patients limited PMP formation; given that abciximab patients required more rigorous PTCA, the protective benefit of this medication for PMP production is underlined. Although few patients in this study experienced restenosis, it is interesting to note that of those treated with abciximab, all patients suffering subsequent restenosis were revascularised using PTCA. This demonstrates that their occlusions were comparatively mild as the need for coronary artery bypass grafting was avoided, and suggests that minimisation of PMP levels may assist in limiting the progression of severe restenosis. The increased peripheral level of PMPs predicated investigation of the coronary circulation to determine local events. Although the level of PMPs increased significantly within the coronary arteries of PTCA patients, there was no corresponding increase in the coronary sinus (Chapter 4). This important finding indicated that significant levels of PMPs remained within the coronary circulation, where their ability to adhere, aggregate and accelerate haemostasis may allow them to contribute directly to restenosis. During the time when increased levels of PMPs were being formed, there was no evidence of platelet lysis, which refuted the hypothesis that PMPs are merely membrane fragments of lysed platelets. A wide variation in reported PMP sizes has contributed to the hypothesis that PMPs are heterogeneous. As morphological information can assist in understanding physiology, the final study was designed to investigate platelet morphology from PTCA patients (Chapter 5). Most platelets were activated prior to and following PTCA, with a slight decrease in body size occurring due to PTCA, presumably due to loss of cytoplasm in PMPs being shed as reported in the previous chapter. Importantly, platelet distal pseudopod buds were observed, and these did not alter significantly with PTCA. These buds were probably unformed PMPs, although the exact mechanism of PMP formation remains undetermined. The platelet pseudopods were longer and significantly thinner distally with PTCA, which may be due to movement of cytoplasm into these terminal swellings. In addition, buds or swellings directly on the platelet body were smaller following PTCA, and it is likely these may also be PMPs prior to detachment from the parent platelet. This work has contributed substantially to knowledge of PMPs produced during PTCA. The level of PMPs increased significantly in peripheral arterial samples, with the platelet aggregation inhibitor abciximab preventing this occurrence. This may indicate that functional aggregation receptors are an essential requirement for PMP formation under these conditions. However, it is possible for PMPs to be formed when aggregation is inhibited, and therefore the molecular mechanisms of PMP formation remain unconfirmed. The examination of PMPs from the coronary circulation provided valuable data indicating that PMPs are produced during PTCA but remain within the coronary circulation. As PMPs are capable of adhesion and aggregation, this strongly suggests that PMPs within the coronary circulation would contribute directly to pathogenesis of restenosis, although further investigation on PMPs with PTCA is necessary to confirm this association. The examination of platelet morphology during PTCA indicated that platelets possessed terminal pseudopod swellings, and cell surface swellings. Importantly, the terminal swellings, which are likely to be unformed PMPs, were observed for the first time during PTCA.