Therapy of acute leukemia may be improved by a more accurate assessment of the effects of treatment on tumor burden and by anticipating relapse with greater precision. The sensitivity limit of assessing residual disease by morphology is usually 5%. Several alternative approaches are available to study minimal residual disease, defined as the presence of leukemic cells not detectable by morphology. These include studies of chromosomal abnormalities by conventional karyotyping, flow cytometry, in situ hybridization and polymerase chain reaction (PCR), investigation of gene rearrangements by Southern blotting and PCR, and immunological methods. Some of these techniques enable the detection of 1 leukemic cells among 10 000 or more normal cells. In the following, the advantages and limitations of sensitive methods for detecting small numbers of leukemic cells are reviewed. The rationale for monitoring residual disease in acute leukemia and the initial results of studies correlating minimal residual disease and clinical outcome are discussed.

Traditionally, viral particles have been primarily analyzed as a whole population according to their biochemical, genetic, and biophysical properties. Here, we describe single particle phenotypic analysis using surface markers found on Murine Leukemia Virus (MLV) by flow virometry. We used this technology to show differential incorporation of host surface markers between wild type MLV and glycosylated Gag (glycogag) deficient MLV. Moreover, we analyzed differential uptake efficiency of host proteins between two cell lines and primary lymphocytes. We hypothesize that the phenotypic profiling and quantification of antigens on the surface of individual viral particles will provide crucial information on the identity of the infected parental cells. Furthermore, we demonstrate that the MLV accessory protein glycogag is associated with the upregulation of surface antigen incorporation during assembly and release. Aside from possible evolutionary implications of glycogag, we demonstrate presence and varying antigenic composition on the surface of MLV viral particles reflective of the cell phenotype that they were released from.

MSc (Med), Dept of Haematology, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand / BACKGROUND: Human Immunodeficiency Virus (HIV) is a global epidemic with growing numbers of people on highly active anti‐retroviral therapy (HAART) programmes. Effectiveness of treatment needs to be monitored to ensure the uncompromised well being of patients. This is currently done using both Viral Load (VL) and CD4 cell counts for HAART initiation and follow‐up. Although VL is the best predictor of disease progression it is often too expensive for monitoring patients in resource‐limited settings. There is thus a need for a cheaper, more accessible alternative to monitor long term patient response to therapy. METHODS: This study evaluated the use of a recently described flow cytometric assay of CD38 expression (previously developed at the Johannesburg Flow Cytometry Reference Laboratory) in a cohort of HIV+ patients failing 1st line therapy, who were subsequently enrolled onto 2nd line HAART. CD38 and CD8 were “piggy ‐backed” onto the PLG/CD4 protocol and mean fluorescence intensity (MFI) of the CD8/38 expression was monitored longitudinally. Patterns of CD38 expression were compared to 1st line treatment observations to establish equivalence in the predictive power of CD38 expression of fluctuation in viral load on 2nd line treatment patients. In addition, the effect of sample age on assay accuracy was tested before implementation of the CD38 assay at a secondary testing site. RESULTS: The patterns observed in the cohort of 2nd line therapy patients mirrored patterns previously seen in 1st line therapy with 55% of patients showing a continuous decline in CD38 MFI that mimicked changes in VL. The remaining 33% of patients had non‐specific increases in CD38 MFI without concurrent increases in VL and one patient showed irregular VL and CD38 MFI (non‐responder). The CD38 assay showed acceptable accuracy and reproducibility up to 48 hours after venesection (%CV<5%). Implementation at the secondary testing site was successful with 98% similarity (%CV<5%) compared to the reference laboratory. CONCLUSION: CD38 monitoring of 2nd line therapy patients showed comparable patterns to observations in 1st line therapy patients. The assay proved stable over time and easy to implement at another PLG/CD4 testing facility. As such, the CD38 assay offers a cost‐effective, reliable real time supplementary test to long‐term VL monitoring of HIV infected patients on the national ART programme.

A research report submitted to the Faculty of Science, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Master of Science by coursework and research report. Johannesburg, 13 May 2014. / The African baobab (Adansonia digitata) is a multi-purpose tree that is important among African villages as it provides food and a range of raw materials. Its fruits provide essential nutrients and are sold to generate income. As baobab fruits are important to the livelihoods of many people, it is important to understand the causes of differences in fruit production in order to maximise use and for conservation purposes. Many studies have examined fruit production to understand the causes of variation in fruit yields. In Venda, a region northern South Africa, differences in baobab fruit yield has been recorded for 8 years, thus classifying individual trees as either poor producers or producers (Venter and Witkowski, 2011). Poor producers are adult trees producing less than five fruits each year and some not producing at all. On the other hand, adult trees producing more than five fruits each year are referred as producers. Causes of this difference in fruit production have not been identified. Among other factors, the observed difference in fruit production could be related to differences in ploidy-level among baobab trees. Importantly, few or no studies to our knowledge have been carried out to confirm whether differences in fruit production among baobab trees are related to a difference in ploidy-level. The well-known and widespread mainland African baobab, Adansonia digitata, is known to be a tetraploid (four sets of chromosomes). Recently, a difference in ploidy-level has been revealed. A new diploid species, Adansonia kilima, has been identified in Africa (Pettigrew et al., 2012). Morphological characteristics (floral, pollen, and stomatal size and density), ploidy, and molecular phylogenetics suggest the presence of a new species. This new species has been reported to overlap the well-known and widespread tetraploid A. digitata’s distribution in Venda. Consequently, the presence of a diploid species that reproduces with a tetraploid species could result in triploid progeny and contribute to the observed differences in fruit production in these baobab trees. The objectives of this study were (i) to assess if there is any difference in ploidy-level between the poor producer and producer baobab trees in Venda using flow cytometry, (ii) to assess if stomatal density and size correlate to differences in ploidy-level, and (iii) to use microsatellites to estimate levels of gene flow between these baobab trees. Morphological results showed that stomatal size and density were not significantly different between poor producer and producer trees and these features may not be true indicators of difference in ploidy-level for baobabs. Gene flow results showed that there was high mean genetic heterozygosity and low population differentiation expressed in all populations. This suggests that inbreeding was not responsible for the differences in fruit production between poor producer and producer trees. Low population differentiation observed among the populations indicated that a large number of common alleles were shared among the populations. Therefore, the high gene flow observed among the populations suggests that poor producer and producer trees were sharing alleles, and what is causing the differences in fruit production remains unclear.

No description available.

Cyclosporine is a potent immunosuppressive agent used to treat a wide range of canine inflammatory diseases. Unfortunately, optimal dosing protocols for achieving immunosuppression with cyclosporine in dogs remain unclear, and standard methods that objectively monitor effectiveness of immunosuppression have not been established. We evaluated an already established panel of biomarkers of immunosuppression in vivo with two oral dosages of cyclosporine in seven normal dogs, a high dosage known to induce immunosuppression and a lower dosage used to treat atopy, with a washout period between the two dosages. The biomarker panel included the flow cytometric evaluation of T-lymphocyte cytokine expression (IL-2, IL-4, and IFN-gamma). High dosage cyclosporine resulted in significant decreases in IL-2 and INF-gamma expression, but not IL-4 expression. Low dosage cyclosporine was associated with a significant decrease in INF-gamma expression, while IL-2 expression was not affected. The results demonstrated suppression of biomarkers in a dose-dependent manner.

Crayfish exhibit innate immune responses via hemocytes and their products. There are 3 hemocyte populations: hyaline cells, granular cells, and semigranular cells. Hemocytes from laboratory housed, untreated crayfish (normal crayfish) have been quantified on the basis of cell type, cell size, and cell granularity using Flow Cytometry. These data present the first overall picture of normal hemocytes from Red Swamp Crayfish with regard to cell type, cell size, and cell granularity and will serve as a baseline for all future studies in our lab. Experiments using crayfish injected with Pseudomonas aeruginosa, Staphylococcus aureus, or crayfish saline alone showed significant and consistent changes in cell type in cells from crayfish injected with bacteria with a decrease in hyaline cells and an increase in granular cells. This effect was greater in crayfish injected with Gram - bacteria. In addition, crayfish injected with Pseudomonas aeruginosa showed a significant difference in Granular cell size with a shift to larger cells and a significant decrease in granularity in the Granular cell population. Cells from crayfish treated with Staphylococcus aureus did not show these changes.

No description available.

Investigations of potato (Solanum tuberosum) have been hampered by its complicated genetics and high genetic load. This dissertation applies genome reduction techniques to investigate a broad swath of genomic and physiological phenomena. It begins with the presentation and evaluation of a protocol to characterize endoreduplication within potato tubers, demonstrating substantial variation between tissue types and among wild species which may facilitate research into the genesis and growth of these starchy underground stems. Next, we transitioned to explore the distribution and consequences of a specific class of transposable element, Miniature Inverted Transposable Elements (MITEs), showing that they comprise approximately 5% of the potato genome, occur more frequently in genes with stress-related functions, and may be associated with changes, especially decreases, in gene expression. We then combined homology and sparsity based approaches to predict recent MITE activity, identifying five families as especially active. Finally, we expose the gene underlying the potato flower color locus, a homolog of AN2, while showing the effects it exerts on the flavonoid biosynthesis and fruit ripening pathways. This region was shown to be particularly dynamic, replete with MITEs and structural variants which we hypothesize to be the ultimate cause of differences in AN2 expression within the germplasm we examined. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the utilization of genomic reduction approaches to acquire homozygous genotypes for study and currently available genomic resources. / Ph. D. / Despite their humble appearance and routine consumption, potatoes have a complex genetic structure and a life cycle capable of both sexual reproduction through flowers, fruit and seed, and asexual reproduction through the tubers which also comprise the edible product. From an agronomic perspective, one of the most important qualities of a potato tuber is size, a feature influenced by genetics and environment. Cell-to-cell variation for the amount of DNA per cell, one component that influences tuber size, is known to occur, yet our ability to measure DNA content in starchy tuber cells has been obscured by debris generated through routine preparation techniques. We present and evaluate a new method for measuring the DNA content of potato tuber cells, which provides reliable results across a range of different potato varieties and species. ‘Jumping genes’ also known as transposons, first reported in maize but now known to occur in most advanced plant and animal species, have been found to comprise ~5% of the recently sequenced potato genome. We show that a particular class of transposons is more likely to occur adjacent or actually in certain types of genes, such as those which confer resistance to disease, where they may have meaningful effects on how those genes operate. We then proceed to predict the current activity of the various families of these jumping genes to understand how they continue to alter the genetic landscape of potato. Finally we identify a particular gene which dictates flower color in potato (purple vs. white). We demonstrate that several transposons occur in some forms of the flower color gene. Originally we hypothesized that transposons were associated with the turning off of the purple flower color form; however, on closer examination, we could express the white flower form in transgenic plants that were originally white-flowered and convert them to have purple flowers, demonstrating that even the white flower form was functional. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the availability of the special strains of potatoes with reduced chromosome number and the publication of the potato genome.

Yes / To develop an efficient nanotechnology fluorescence-based method to track cell proliferation to avoid the limitations of current cell-labeling dyes. Material & methods: Synthesis, PEGylation, bifunctionalization and labeling with a fluorophore (Cy5) of 200 nm polystyrene nanoparticles (NPs) were performed. These NPs were characterized and assessed for in vitro long-term monitoring of cell proliferation. Results: The optimization and validation of this method to track long-term cell proliferation assays have been achieved with high reproducibility, without cell cycle disruption. This method has been successfully applied in several adherent and suspension cells including hard-to-transfect cells and isolated human primary lymphocytes. Conclusion: A novel approach to track efficiently cellular proliferation by flow cytometry using fluorescence labeled NPs has been successfully developed.