Budded virions of AcMNPV can enter a variety of non-host cells, a characteristic likely due to the presence of GP64, an envelope protein found on a small subset of baculoviruses. Results show that AcMNPV's tropism for vertebrate cells can be restricted - a prerequisite for using AcMNPV for targeted in vivo gene delivery - by replacing the gp64 gene with SeF from SeMNPV. Unlike the relatively well characterized GP64 protein, the significance and function of the F homolog (Ac23, a pathogenicity factor), is poorly understood. How Ac23 might contribute to the faster speed of kill was examined by comparing occlusion bodies and occlusion-derived virions (ODV) of Ac23null mutant viruses with control viruses at the ultrastructural level. The results show that Ac23null mutant produces a significantly higher percentage of ODVs with single or lower number of nucleocapsids than controls, suggesting Ac23 may play a role in multicapsid envelopment of ODVs. / xiii, 101 leaves : ill. (some col.) ; 28 cm. --

The molecular biology of human genetic disorders is under intensive investigation at present. In those cases where the disorder is clearly defined in terms of altered gene structure, possibilities may exist for the correction of the disorder by insertion of normal genes through the process of DNA transfection. A possible method for the transfer of genetic material is by attempting to attach DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. By this means one might be able to get DNA into cells. This thesis deals with experimental work on the chemical modification of human serum transferrin by means of water-soluble carbodiimides. The resulting N-acylurea transferrins bind DNA in a reversible manner. Characteristics and properties of the binding interactions are dealt with in detail. N-acylurea derivatives of transferrin were prepared with the water-soluble carbodiimides, N-ethyl-N' -(3-dimethylaminopropyl) carbodiimide and N-ethyl-N' -(3-trimethylpropylammonium) carbodiimide iodide. Reactions were carried out under mild conditions at room temperature for 48-72 hours. [³ H] N-ethyl-N' -(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the protein. Changes in charge properties were determined by agarose gel electrophoresis. Carbodiimide modification of proteins is thought to occur at side chain carboxyl groups of glutamic and aspartic acid residues. This was confirmed by the use of Staphylococcus aureus V8 protease, which cleaves peptide bonds at the carboxyl side of glutamic and aspartic acid residues, but not in the case of substituted side chain carboxyl groups. Through the use of puromycin as a nucleophile it has been shown that other functional groups were not activated upon reaction of transferrin with carbodiimide. The carbodiimide-modified proteins bind various types of DNA and RNA in a reversible manner. Low concentrations of N-acylurea transferrin retarded the migration of pBR322 DNA, M 13 mp 8 single-stranded DNA and Pst 1 restricted lambda DNA on agarose gel electrophoresis, while at higher concentrations the DNA was unable to enter the gel. Nitrocellulose filter binding assays showed that binding of DNA to Nacylurea transferrins was rapid, dependent on concentration of the modified transferrin and sensitive to ionic conditions. Binding was found to occur mainly through electrostatic interactions between phosphate groups of DNA and N-acylurea groups. These conclusions were based on experiments which showed that protein-DNA complexes were dissociated by increasing salt concentrations and by heparin. Non-electrostatic interactions such as hydrophobic interactions and hydrogen bonding are also involved in binding, since half dissociation of complexes, induced by chaotropic salts, KSCN and NaC10₄occurs at lower concentrations of salt than in the case of NaCl. Also RNA polynucleotides inhibit binding of DNA to Nacylurea transferrins to varying extents. The N-acyl urea transferrins have been shown to bind certain specific restriction endonuclease cleavage sites on pBR322 DNA. The N-acylurea transferrin-DNA complexes would thus be suitable for experiments in cell transfections using cells which have transferrin receptors. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

The thioredoxin system consisting of thioredoxin (Trx), thioredoxin reductase and NADPH plays a significant role in a large number of redox-dependent processes such as DNA synthesis and anti-oxidant defense. Elevated levels of this system have been associated with a number of diseases including cancer and HIV. Understanding the regulation of this network from a systems perspective is therefore essential. However, contradictory descriptions of thioredoxin as both an enzyme and redox couple have stifled the adoption of systems biology approaches within the field. Using kinetic modeling, this discrepancy was resolved by proposing that saturation of Trx activity could be due to the saturation of the Trx redox cycle which consequently allowed development of the first computational models of the thioredoxin system in Jurkat T-cells and Escherichia coli. While these models successfully described the network properties of the thioredoxin system in these organisms, further confirmatory studies were required before this modeling approach could be generally accepted. The aim of this study was to utilize computational and molecular methods to confirm or reject this proposed mechanism for thioredoxin activity. To determine if there is any difference in the kinetic models obtained when thioredoxin was modeled as an enzyme or as a redox couple, representative core models were developed. The data showed that when modeling Trx as a redox couple, the system was able to achieve steady state, there was a re-distribution of Trx into its oxidized form and, thioredoxin reductase affected the rates within the system. On the other hand, when Trx was modeled as an enzyme, the system could not reach a steady state, Trx remained in the reduced form and thioredoxin reductase concentration had no effect on the rates within the system. As these properties could be directly tested invitro, we sought to directly confirm which model was correct. The thioredoxin system from Saccharomyces cerevisiae was cloned, expressed and purified and substrate saturation curves were generated using insulin as a model substrate. The data showed that the system reached steady state and with increasing concentrations of insulin, the system saturated with a progressive re-distribution of the thioredoxin moiety into its oxidized form. Further, increasing the thioredoxin reductase concentration increased the flux through the system. Collectively, the results obtained through invitro analyses provided unambiguous support for the thioredoxin redox couple model. These results will enable the construction of a complete computational model of the yeast thioredoxin system and provide a basis for the analysis of this network in a number of pathologies. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Alcanivorax species are gram negative bacteria that usually require aliphatic hydrocarbon as the sole carbon source for growth. The ability to use petroleum in polluted environments as energy source makes Alcanivorax species biotechnologically relevant in bioremediation. Universal stress proteins confer ability to respond to unfavourable environments, thus the present study was done to analyse the genomic context of genes for universal stress proteins in Alcanivorax genomes. A combination of bioinformatics and visual analytics approaches were used to analyze genome-enabled data including sequences and gene expression datasets. On the basis of transcription unit and adjacent genes, two types of Alcanivorax USP genes observed were (i) adjacent to cyclic nucleotide-binding and oxygen sensing functions; and (ii) adjacent to sulfate transporter function. Both types of genes encode two universal stress protein domains (pfam00582) also referred to as tandem-type universal stress proteins. The sequence and structural characteristics of each of the four USP domains in Alcanivorax needs to be further investigated. This dissertation research evaluated data from Alcanivorax borkumensis cells (grown on either pyruvate or hexadecane as carbon source) that were stressed with 1-octanol and data collected at 15 min, 30 min, 60 min and 90 min after 1-octanol addition. The two genes for Alcanivorax borkumensis SK2 universal stress proteins, ABO_1340 and ABO_1511, had the same direction of expression for adjacent genes. A limitation of this research was that findings based on bioinformatics and visual analytics methods may need confirmation by molecular methods. The differences observed may also reflect the quality of the annotations provided for genes. The sequence and structural characteristics of each of the four USP domains in Alcanivorax needs to be further investigated. Further research is needed on the relationship between number, length and order of genes in operons that include genes for universal stress proteins. Additionally, in vitro studies to confirm the functional prediction made from the genomic context of the universal stress protein in Alcanivorax genome. The knowledge discovered from this genome context analytics research could contribute to improving the performance of Alcanivorax species in bioremediation of environments polluted with petroleum / College of Agriculture and Environmental Sciences / M. Sc. (Environmental Science)

Pichia pastoris is a methylotrophic yeast that is commonly used for its ability to express and secrete heterologous proteins. However, some proteins are not readily secreted in P pastoris and so adjustments in the secretion pathway must be made in order to achieve secretion. The Lin-Cereghino lab previously developed mutant strains using restriction enzyme-mediated integration that enabled P pastoris to secrete Pgalactosidase at higher levels than the wild type strain. This study focuses on characterizing the random pREMI-Z mutations in the genomic DNA and examining their secretory phenotype, in hopes of creating a super secretor strain. The ah3 mutant was specifically chosen and characterized for its ability to secrete HRP and SLPI proteins and the effect of the pREMI-Z mutation on the morphology of the cells using transmission electron microscopy. An examination into the AH3 protein yielded a comparative B-galactosidase secretion study between the ah3 mutant and ah3 mutant cells transformed with the pKANB-AH3 rescue construct. Lastly, a cell localization experiment was done to examine where the AH3 protein may be found. These experiments help to increase the current understanding of the secretion pathway in Pichia pastoris and serves as an outline of how to characterize other pREMI -Z mutant strains.

Proteins of the cytoplasmic membrane of Mycobacterium avium were investigated to identify those which were: (1)intrinsic or extrinsic, (2) attached to the cell wall, (3)surface accessible and (4) excreted. In addition sera containing anti-cytoplasmic membrane proteins were obtained and preliminary purification of the cytoplasmic membrane protein was attempted. The predominating cytoplasmic membrane protein of 31,000 daltons (MCMP) was found to be intrinsic, attached to the cell wall and possibly surface accessible. The MCMP was not excreted, even in media in which the MCMP is not found in the cytoplasmic membrane. Other cytoplasmic membrane proteins were also found to be intrinsic; a few were likely to be extrinsic based upon their separation from the membrane in sucrose gradients. Cytoplasmic membrane proteins of 66, 000, 115, 000 and 129 dalton were surface accessible as judged by I 125-Iodobead labeling. Antisera against the HCMP and other cytoplasmic membrane proteins was obtained and will be useful in further cytoplasmic membrane protein characterization. Acetone precipitation of a cytoplasmic membrane preparation was performed to partially purify the MCMP. The data from this study can be used for the development of serodiagnostic reagents for detecting mycobacterial infection. / Master of Science

The concentrated protein fractions of soyabeans, known as soya protein isolate, was produced by three different methods from the same raw material namely defatted soya flakes. Extraction of the soluble fraction of the raw material is common to all three methods. A study was therefore undertaken to optimise the extraction process conditions in terms of time, temperature, pH, extraction time, extraction volume and raw material particle size, thereby maximising yields of soluble material. The three different methods, namely isoelectric precipitation, ultrafiltration and swollen gel technology were then used to separate the soluble and non-soluble protein fractions. Both the isoelectric and ultrafiltration methods gave good yields of finished product, with the ultrafiltration process giving the better overall yield, but the swollen gel method gave disappointing results and was not feasible in practice. Functional properties of the products from the isoelectric and ultrafiltration methods were compared and found to be broadly similar although different in certain respects from those of commercial soya isolates. Levels of the anti-nutritional factors trypsin inhibitor and phytate in products from the three processes were determined and the substantial differences observed in trypsin inhibitor levels were further investigated. Determination of lysinoalanine levels was also attempted but the results obtained were unsatisfactory. Amino acid composition and polyacrylamide gel electrophoresis were used to compare the chemical composition of products from the three processes. The comparative economics of the isoelectric and ultrafiltration processes for large scale production of soya protein isolates were evaluated, taking into account the comparative efficiencies of the two processes as determined during the study. It was established that, while the isoelectric process initially appears more economical, it may be possible to modify the ultrafiltration process in such a manner as to make it more economical than the isoelectric process. Overall figures however indicate that the manufacture of soya protein isolate in South Africa is not currently a viable economic proposition, due to high raw material costs. / Thesis (M.Sc.)-University of Natal, 1995.

Imidazolium trans-[tetrachloro (dimethyl sulfoxide)(imidazole)ruthenate(III)], NAMI-A, is an experimental metastasis inhibitor whose specific mechanism of activation and action remains to be elucidated. In the nucleophilic and reducing physiological environment; it is anticipated that the most relevant and available reductants upon introduction of NAMI-A as a therapeutic agent will be the biologically-relevant free thiols. The kinetics and mechanisms of interaction of NAMI-A with biologically-active thiols cysteamine, glutathione, cysteine and a popular chemoprotectant, 2-mercaptoethane sulfonate (MESNA) have been studied spectrophotometrically under physiologically-relevant conditions. The reactions are characterized by initial reduction of NAMI-A with simultaneous formation of dimeric thiol and subsequent ligand exchange with water to various degrees as evidenced by Electospray Ionization Mass Spectrometry. Stoichiometry of reactions shows that one molecule of NAMI-A reacted with one mole of thiol to form corresponding disulfide cystamine, dimeric MESNA, oxidized glutathione and cystine. Observed rate constants, ko, for the reaction of NAMI-A with cysteamine, MESNA, GSH and cysteine were deduced to be 6.85 + 0.3 x 10-1, 9.4 + 0.5 x 10-2 , 7.42 + 0.4 x 10-3 and 3.63 + 0.3 x 10-2 s-1 respectively. Activation parameters determined from Arrhenius plots are indicative of formation of associative intermediates prior to formation of products. A negative correlation was obtained from the Brønsted plot derived from observed rate constants and the pKa of the different thiols demonstrating significant contribution of thiolate species towards the rate. In conclusion, interactions of NAMI-A with biologically-active thiols are kinetically and thermodynamically favored and should play significant roles in in vivo metabolism of NAMI-A.

The 70 kDa heat shock proteins expressed by Plasmodium falciparum (PfHsp70s) are believed to be essential to both the survival and virulence of the malaria parasite. A total of six Hsp70 genes have been identified in the genome of P. falciparum. However, only four of these encode canonical Hsp70s, which are believed to localise predominantly in the cytosol (PfHsp70-1 and PfHsp70-x), the endoplasmic reticulum (PfHsp70-2) and mitochondria (PfHsp70-3) of the parasite. These proteins bind and release peptide substrates in an ATP-dependent manner, with the aid of a J-domain protein cochaperone and a nucleotide exchange factor (NEF). The aim of this study was to identify the residues involved in the interaction of these PfHsp70s with their peptide substrates, their J-domain cochaperones and potential NEFs. These residues were then mapped to three-dimensional (3D) structures of the proteins, modelled in three different conformations; each representing a different stage in the ATPase cycle. Additionally, these proteins were compared to different types of Hsp70s from a variety of different organisms and sequence features found to be specific to each PfHsp70 were mapped to their 3D structures. Finally, a novel modelling method was suggested, in which the structures of templates were remodelled to improve their quality before they were used in the homology modelling process. Based on the analysis of residues involved in interactions with other proteins, it was revealed that each PfHsp70 displayed features that were specific to its cellular localisation and each type of Hsp70 was predicted to interact with a different set of NEFs. The study of conserved features in each PfHsp70 revealed that PfHsp70-x displayed various sequence features atypical of both Plasmodium cytosolic Hsp70s and cytosolic Hsp70s in general. Additionally, residues conserved specifically in Hsp70s of Apicomplexa, Plasmodium and P. falciparum were identified and mapped to the each PfHsp70 model. Although these residues were too numerous to reveal any information of specific value, these models may be useful for the purposes of aiding the design of drug compounds against each PfHsp70. Finally, the novel modelling approach did show some promise. Half of the models produced using the modified templates were of a higher quality than their counterparts modelled using the original templates. This approach does still require a lot of validation work and statistical evaluation. It is hoped that it could prove to be a useful approach to homology modelling when the only templates available are poor quality structures.

Parasitic diseases remain a health burden affecting more than 500 million people worldwide with malaria having the highest mortality rate. The parasites can be transferred to the human bodies either through the mouth by ingestion of contaminated food and water or through the skin by bug bites or direct contact to environments harbouring them. Epidemiological control seems to be impossible since there is failure to control the insect vectors as well as practice of hygiene. Therefore, this has led to the development of a number of vaccines, chemotherapy and disease control programs. However, parasites have increasingly developed resistance to traditionally used anti-parasitic drugs and due to that fact there is need for alternative medication for parasitic diseases. Heat shock protein 90 (Hsp90) facilitates the folding of proteins in all living cells and their role is more important to parasites because of their environmental changes, from vector to host. Hsp90s play a major role; therefore this justifies the need for a deeper analysis of the parasitic Hsp90s. Recent studies have revealed that, the Plasmodium sp. Hsp90 has an extended linker region which increases the protein’s affinity for ATP and its inhibitors. Therefore we hypothesize that there are also significant features in other parasitic Hsp90s which would lead to Hsp90 being defined as potential drug targets. In the present study an attempt was made to gain more insight into the differences in primary structure of human and parasitic Hsp90s. The sequences were retrieved from the NCBI database and analysis was done in three groups basing on the localization of the Hsp90. The physicochemical properties were calculated and in every group, the protozoan Hsp90s showed significant differences when compared to the human orthologs. Multiple sequence alignments (MSA) showed that endoplasmic reticulum Hsp90s have an extended region in the middle domain indicating their ability to bind to a unique subset of client proteins. Sequence identities between the human and parasites showed that the protozoan Hsp90s are less related to the human Hsp90s as compared to the other parasites. Likewise, motif analysis showed the trypanosomatids and apicomplexan groups have their own unique set of motifs and they were grouped together in the phylogenetic analysis. Phylogenetic analysis also showed that, the protozoan Hsp90s forms their own clades in each group while the helminths did not form in endoplasmic reticulum group. In this study, we concluded that, Hsp90 can be a potential drug target for the protozoan species and more specifically those from the apicomplexan and trypanosomatids groups.