The correction of human genetic disorders by transfer of genetic material to cells is under intensive investigation in a number of 1aboratories. One possible way of trying to achieve the transfer of nucleic acid is by attaching DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. In order to make use of the ligand protein-receptor approach for DNA transfer, iron-loaded human serum transferrin has been modified with the water soluble carbodiimides N-ethy1-N I -(3-dilllethy1aminopropyl) carbodiimide (CDI) and its quaterary analogue (ECDI) to give modified N-acy1urea transferrins. N-Acy1urea CDI (Fe 3+) transferrin and N-acy1urea CDI (Fe ) transferrin have been found to interact with and bind DNA in a reversible manner which i! dependent on ionic strength. [1251] N-Acy1urea CDI+(Fe3+) transferrin binds to transferrin receptors on Hea cells in culture and undergoes internalization through receptor-mediated endocytosis. Binding of the modified transferrin in the presence of calf thymus DNA to transferrin receptors also takes place. However, although internalization in the presence of DNA doe! appear to take place, the results of the internalization are not fully understood. Transfection studies with N-acy1urea CDI (Fe ) transferrin and plasmid pBR322 DNA as well as plasmid ptkNEO DNA complexes in the HeLa cell system are reported. The results of a number of transfection experiments suggests that N-acy1urea transferrins are capable of transfecting DNA (ptkNEO DNA), carrying genes for resistance to the antibiotic Geneticin (G41S) in the HeLa cell system. However, further development of the transfection system is necessary in order that consistantly reproducible results may be achievd. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

Magister Scientiae - MSc (Biotechnology) / The mining industry in South Africa is of immense importance as this sector contributes largely to the countries income. In the Limpopo province, a large production of antimony (Sb) is generated per year. Antimony (Sb) is a trace element, which accumulates in the environment through anthropogenic activities, such as mining and smelting industries. Antimony is toxic to all living organisms and plants, and it is found to increase the peroxidation of membrane lipids and encourage an antioxidant response. Sb contamination in plants also accounts for DNA damage. The reduction in yield is due to the disruption of plant metabolism by reactive oxygen species (ROS). To combat abiotic stresses, plants have generated a signalling network that utilises multiple growth regulators that would offer protection against the stress. An increase in ROS is one of the responses to abiotic stresses. ROS is generated in response to the pants interaction with heavy metals, through the Harber-Weiss reaction. ROS compounds include: superoxide, hydrogen peroxide and hydroxyl radicals. Under normal conditions ROS molecules are produced as by-products, however, under stressful conditions the production of ROS molecules are increased to levels where they are detrimental to the plants. Therefore, the accumulation of ROS results in damage to proteins, lipids, carbohydrates and DNA which would lead to cellular death. ROS accumulation is thought to be a result of the disruption in the balance of ROS production and the anti-oxidation systems. The antioxidative system is thus introduced to restore the balance of ROS molecule production and to combat oxidative damage caused by the ROS molecules. The anti-oxidative system consists of various enzymes: superoxide dismutase, catalase, and ascorbate peroxidase and glutathione reductase. Each antioxidant scavenges one or two ROS molecules. / 2020-08-31

Philosophiae Doctor - PhD / The plasticity of single-stranded viral genomes permits the formation of secondary structures through complementary base-pairing of their component nucleotides. Such structures have been shown to regulate a number of biological processes during the viral life-cycle including, replication, translation, transcription, post-transcriptional editing and genome packaging. However, even randomly generated single-stranded nucleotide sequences have the capacity to form stable secondary structures and therefore, amongst the numerous secondary structures formed in large viral genomes only a few of these elements will likely be biologically relevant. While it is possible to identify functional elements through series of laboratory experiments, this is both excessively resource- and time-intensive, and therefore not always feasible. A more efficient approach involves the use of computational comparative analyses methods to study the signals of molecular evolution that are consistent with selection acting to preserve particular structural elements. In this study, I systematically deploy a collection of computationally-based molecular evolution detection methods to analyse the genomes of viruses belonging to a number of ssRNA viral families (Alphaflexiviridae, Arteriviridae, Caliciviridae, Closteroviridae, Coronavirinae, Flaviviridae, Luteoviridae, Picornaviridae, Potyviridae, Togaviridae and Virgaviridae), for evidence of selectively stabilised secondary structures. To identify potentially important structural elements the approach incorporates structure prediction data with signals of natural selection, sequence co-evolution and genetic recombination. In addition, auxiliary computational tools were used to; 1) quantitatively rank the identified structures in order of their likely biological importance, 2) plot co-ordinates of structures onto viral genome maps, and 3) visualise individual structures, overlaid with estimates from the molecular evolution analyses. I show that in many of these viruses purifying selection tends to be stronger at sites that are predicted to be base-paired within secondary structures, in addition to strong associations between base-paired sites and those that are complementarily co-evolving. Lastly, I show that in recombinant genomes breakpoint locations are weakly associated with co-ordinates of secondary structures. Collectively, these findings suggest that natural selection acting to maintain potentially functional secondary structures has been a major theme during the evolution of these ssRNA viruses.

Genetic polymorphisms can be seen as the occurrence of more than one form of a DNA- or protein sequence at a single locus in a group of organisms, where these different forms occur more frequently than can be attributed to mutation alone. The combination of genetic polymorphisms present in the genome of a particular individual is referred to as its genotype. A wide range of genotyping techniques have been developed to detect and visualize genetic polymorphisms. One such technique examines highly polymorphic repetitive DNA regions called microsatellites, also called “short tandem repeats” (STRs) and sometimes “simple sequence repeats” (SSRs) or “simple-sequence length polymorphisms” (SSLPs). A microsatellite region consists of a DNA sequence of identical units of usually 2-6 base pairs strung together to produce highly variable numbers of tandem repeats among individuals of a population. Microsatellite genotyping is a popular choice for many types of studies including individual identification, paternity testing, germplasm evaluation, genome mapping and diversity studies and can be used in many commercial, academic, social, and agricultural applications. There are, however, many obstacles in effectively managing and analysing microsatellite genotype data. Currently, researchers are struggling to effectively manage and analyse rapidly growing volumes of genotyping data. Management problems range from simply the lack of a secure, easily accessible central data repository to more complex issues like the merging and standardization of data from multiple sources into combined datasets. Due to these issues, genetic fingerprinting applications such as identity matching and relatedness studies can be challenging when data from different experiments or laboratories have to be combined into a central database. The main aim of this M.Sc study in Bioinformatics was to develop a bioinformatics resource for the management and analysis of genetic fingerprinting data from microsatellite marker genotyping studies, and to apply the software to the analysis of microsatellite marker data from ramets of Pinus patula clones with the purpose of analysing clonal identity in pine breeding programmes. The software resource developed here is called GenoSonic. It is a web application that provides users with a secure, easily accessible space where genotyping project data can be managed and analysed as a team. Users can upload and download large amounts of marker genotype data. Once uploaded to the system, DNA fingerprint data needs to be standardised before it can be used in further analyses. To do this, a two-step approach was implemented in GenoSonic. The first step is to assign standardized allele sizes to all of the input allele sizes of the microsatellite fingerprints automatically using a novel automated binning algorithm called CSMerge-1, which was designed specifically to bin data from multiple experiments. The second step is to manually verify the results from the automated binning function and add the verified data to a standardized dataset. Once the genetic fingerprints have been standardized, allele- and genotype frequencies can be viewed for any given marker. GenoSonic also provides functionalities for identity matching. One or more DNA fingerprints from unknown samples can be matched against a standardized dataset to establish identities or infer relatedness. Finally, GenoSonic implements a genetic distance tree construction function, which can be used to visualize relatedness among samples in a selected dataset. The bioinformatics resource developed in this study was applied to a microsatellite DNA fingerprinting project aimed at the re-establishment or confirmation of clonal identity of Pinus patula ramets from pine clonal seed orchards developed by a South African forestry company at one of their new agricultural estates in South Africa. The results from GenoSonic‟s automated binning function (CSMerge-1) and the results from the identity matching and tree construction exercise were compared to results obtained by human experts who have analysed the data manually. It was demonstrated that the results from GenoSonic equalled or surpassed the manual results in terms of accuracy and consistency, and far surpasses the manual effort in terms of the speed at which analyses could be completed. GenoSonic was developed with specific focus on reusability, and the ability to be modified or extended to solve future genotyping-related problems. This study not only provides a solution to current genotype data management and analysis needs of researchers, but is aimed at serving as a basic framework, or component library for future software development projects that may be required to address specific needs of researchers dealing with high-throughput genotyping data. / Dissertation (MSc)--University of Pretoria, 2011. / Biochemistry / unrestricted

<p> Human adenovirus, type 2, was utilized to investigate its effects on the host cell population. The progression of KB cells through the various phases of the cell cycle after infection was studied, with special emphasis of DNA metabolism. At different times after infection, the rate and the amount of viral DNA synthesized was determined and their efficiency of incorporation into virious was investigated.</p> / Thesis / Master of Science (MSc)

Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

Magister Scientiae - MSc (Biotechnology) / The objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population. The first step was to design the multiplex system. This was done by using inhouse SNPs. A total of seven multiplexes were designed and optimised, each consisting of two, three or four different SNPs respectively. A total of 143 saliva and buccal samples were collected from male Johannesburg Coloureds. DNA was extracted from the saliva samples using an optimised organic method. DNA was extracted from the buccal samples using an optimised salting out method. DNA was successfully extracted from 77 of the male samples. A total of 69 samples were screened using Multiplex 1; of the 69 samples 56 samples were successfully screened to infer the paternal lineage of the samples. The results show that the most frequent haplogroup of the Johannesburg male samples was haplogroup CF (39%). The second most frequent haplogroup was haplogroup DE (38%). Under further analysis of haplogroup DE it was seen that 37% of those samples were derived for the haplogroup E1b1b.

The fact that loosely bonded DNA bases could tolerate large structural fluctuations, form a dissipative environment for a charge traveling through the DNA. Nonlinear stochastic nature of structural fluctuations facilitates rich charge dynamics in DNA. We study the complex charge dynamics by solving a nonlinear, stochastic, coupled system of differential equations. Charge transfer between donor and acceptor in DNA occurs via different mechanisms depending on the distance between donor and acceptor. It changes from tunneling regime to a polaron assisted hopping regime depending on the donor-acceptor separation. Also we found that charge transport strongly depends on the feasibility of polaron formation. Hence it has complex dependence on temperature and charge-vibrations coupling strength. Mismatched base pairs, such as different conformations of the G・A mispair, cause only minor structural changes in the host DNA molecule, thereby making mispair recognition an arduous task. Electron transport in DNA that depends strongly on the hopping transfer integrals between the nearest base pairs, which in turn are affected by the presence of a mispair, might be an attractive approach in this regard. I report here on our investigations, via the I –V characteristics, of the effect of a mispair on the electrical properties of homogeneous and generic DNA molecules. The I –V characteristics of DNA were studied numerically within the double-stranded tight-binding model. The parameters of the tight-binding model, such as the transfer integrals and on-site energies, are determined from first-principles calculations. The changes in electrical current through the DNA chain due to the presence of a mispair depend on the conformation of the G・A mispair and are appreciable for DNA consisting of up to 90 base pairs. For homogeneous DNA sequences the current through DNA is suppressed and the strongest suppression is realized for the G(anti)・A(syn) conformation of the G・A mispair. For inhomogeneous (generic) DNA molecules, the mispair result can be either suppression or an enhancement of the current, depending on the type of mispairs and actual DNA sequence.

The diagnostic value of extracellular occurring DNA (eoDNA) is limited by our lack of understanding its biological function. eoDNA exists in a number of forms, namely vesicle bound DNA, histone/DNA complexes or nucleosomes and virtosomes. These forms of DNA can also be categorized under the terms circulating DNA, cell free DNA, free DNA and extracellular DNA. The DNA can be released by means of form–specific mechanisms and seem to be governed by cell cycle phases and apoptosis. Active release is supported by evidence of energy dependant release mechanisms and various immunological– and messenger functions. Sequencing has shown that eoDNA sequences present in the nucleome reflects traits and distribution of genome sequences and are regulated by ways of release and/or clearance. eoDNA enables the horizontal transfer of gene sequences from one cell to another, over various distances. The ability of eoDNA to partake in horizontal gene transfer makes it an important facet in the field of epigenetic variation. Clinical implementation of eoDNA diagnostics requires that all of the subgroups of eoDNA be properly investigated. It is suggested that eoDNA is the result of the metabolic fraction of DNA that is released by the cell. Various observations indicate that eoDNA may also be incorporated into the genome of a cell, from where it may affect cell function. Therefore horizontal gene transfer in higher organisms is a real possibility. In this study, variations and increases in eoDNA levels over time correlate with stressors that are subjected to 143B human osteosarcoma cells. It seems viable to assume that a stressor is met by a change in the molecular machinery of a cell, required to neutralise the onset of metabolic instability. This may be done by amplification of necessary cistrons, producing metabolic DNA, that may then be observed after its release as eoDNA. The presence of hydrolysing enzymes gives an updated real time picture of the state of eoDNA. The eogenics hypothesis emanating from this study, suggests that amplification and horizontal transfer of cistrons affect tissue and organ function over long periods of time, in order for an organism to evolve one or more a specialized genomes. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

The diagnostic value of extracellular occurring DNA (eoDNA) is limited by our lack of understanding its biological function. eoDNA exists in a number of forms, namely vesicle bound DNA, histone/DNA complexes or nucleosomes and virtosomes. These forms of DNA can also be categorized under the terms circulating DNA, cell free DNA, free DNA and extracellular DNA. The DNA can be released by means of form–specific mechanisms and seem to be governed by cell cycle phases and apoptosis. Active release is supported by evidence of energy dependant release mechanisms and various immunological– and messenger functions. Sequencing has shown that eoDNA sequences present in the nucleome reflects traits and distribution of genome sequences and are regulated by ways of release and/or clearance. eoDNA enables the horizontal transfer of gene sequences from one cell to another, over various distances. The ability of eoDNA to partake in horizontal gene transfer makes it an important facet in the field of epigenetic variation. Clinical implementation of eoDNA diagnostics requires that all of the subgroups of eoDNA be properly investigated. It is suggested that eoDNA is the result of the metabolic fraction of DNA that is released by the cell. Various observations indicate that eoDNA may also be incorporated into the genome of a cell, from where it may affect cell function. Therefore horizontal gene transfer in higher organisms is a real possibility. In this study, variations and increases in eoDNA levels over time correlate with stressors that are subjected to 143B human osteosarcoma cells. It seems viable to assume that a stressor is met by a change in the molecular machinery of a cell, required to neutralise the onset of metabolic instability. This may be done by amplification of necessary cistrons, producing metabolic DNA, that may then be observed after its release as eoDNA. The presence of hydrolysing enzymes gives an updated real time picture of the state of eoDNA. The eogenics hypothesis emanating from this study, suggests that amplification and horizontal transfer of cistrons affect tissue and organ function over long periods of time, in order for an organism to evolve one or more a specialized genomes. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.