Thesis (M.Tech.: Clinical Technology)- Dept. of Clinical Technology, Durban University of Technology, 2008. xviii, 160 leaves.

No description available.

Many proteins with special properties have been identified in aquatic organisms. Due to their peculiarity, some of these biomolecules could be used advantageously for some industrial and health care applications. However, the preparation of these biochemicals from its original source is highly impractical and generally unfeasible for commercial purposes. Nevertheless, the advances in DNA manipulation open the possibility of producing the recombinant proteins in different organisms to allow a viable production and extraction on a commercial scale. Among such particular proteins, the cunner-fish trypsin (CFT) is an enzyme with a high potential for exploitation by the food processing industry. The CFT is a cold adapted protease that has a proven ability to inactivate undesirable endogenous enzymes during the processing of some foodstuffs. Thus, the CFT gene was characterized and cloned in E. coli and Pichia pastoris for over-expression. A cDNA library from pancreatic mRNA was screened using a salmon trypsin cDNA probe. Positive clones harboring a cDNA insert of the predicted size were sequenced and a full-length cDNA was obtained that contained an ORF encoding the zymogen and a signal peptide. Multiple alignment of the gene sequence showed 85 to 90 per cent identities with other fish trypsins and the deduced amino acid sequence for the mature enzyme exhibited the entire characteristic features observed in trypsins. E. coli expressed the recombinant CFT (rCFT) at levels higher than 20% in the form of insoluble aggregates, while P. pastoris showed expression levels below 1%. Despite the low expression, the yeast rCFT appeared to be correctly folded and could be partially purified by immobilized nickel affinity chromatography. Trypsin-specific activity was confirmed in the purified rCFT after digestion with bovine enterokinase.

No description available.

RNA m5C methyltransferases are a group of enzymes that catalyze the transfer of a methyl group to a cytosine nucleotide of RNA. Only two of these enzymes have been well characterized: Fmu from E. coli and Trm4p from S. cerevisiae. YNLO22c is one of three ORFs identified in S. cerevisiae that have homology with both known and putative RNA m5C methyltransferases, but its encoded protein, YNLO22p, has not been confirmed to have enzyme activity. Verifying that YNLO22c encodes an RNA m5C methyltransferase will require adequate amounts of soluble YNLO22p for enzyme assays. A bacterial expression plasmid for YNLO22c was developed, but the result was insoluble protein. Therefore, several methods known to improve protein solubility were tested to develop a system in which a sufficient amount of soluble YNLO22p could be produced. Results of this study found that coexpression of YNLO22c with chaperone proteins can provide sufficient quantities of soluble YNLO22p. / Department of Biology

Transfusion medicine is an important part of modern health care and the provision of reliably phenotyped red blood cells (RBC) is essential for safe and effective blood transfusions. For identification of many RBC antigens, monoclonal antibodies of either murine or human origin are available for use in agglutination assays, in which they perform as well as or better than the human polyclonal antibody preparations which they have replaced. However, the detection of some blood groups is still reliant on the use of human polyclonal antisera, which is a less reliable reagent source with respect to availability, batch to batch variation and bio-safety. The use of recombinant antibody and phage display technology for the discovery of new monoclonal antibodies with specificity for some of these RBC antigens has the potential to deliver an economical, unlimited supply of specific antibody reagents suitable for use in RBC phenotyping. Samples of human B cells from donors producing useful phenotyping antibodies were identified and transformed using Epstein Barr virus into lymphocyte cell lines. Antibody genes were obtained from the cell lines in the form ofRNA which was reverse transcribed, amplified by PCR and cloned into a phagemid vector system to generate several combinatorial antibody libraries. These antibody libraries were displayed on the surface of phage particles and subjected to antigen-driven selection by several rounds of phage display biopanning using soluble and cell based RBC antigens. In addition a large naIve library was biopanned against the same antigens in an attempt to isolate a wide range of antibodies suitable for blood typing. Several high quality combinatorial antibody libraries with respect to size (> 107 clones) and diversity were generated. Biopanning of recombinant libraries resulted in enrichment of phage antibodies specific for RBC antigens, and several clones were isolated which were shown to be specific for Duffy a antigen. The isolated antibodies would be ideal candidates for re-engineering into multivalent antibody molecules capable of direct agglutination of RBC and as such, have the potential to replace human polyclonal sera in the identification of Duffy a RBC antigen phenotyping.

Two leaves of amendments in pocket on front end paper. / Bibliography: leaves 177-185. / xix, 194 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes selective and nonselective procedures to extract recombinant protein from the cytoplasm of Escherichia coli. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 1997

The mouse parvovirus designated MPV can infect laboratory mice and affect the humoral and cellular immune response of infected mice, reducing their value for biomedical and medical research. The development and maintenance of MPV-free mouse colonies for biomedical research is therefore essential and requires routine monitoring of the infection status of mice, using serological surveillance procedures. Recent experience in the Animal Resources Centre (ARC), a major supplier of mice to the medical research community in Australia, was that MPV infection was present but was not detectable with the serological tests that were then in routine use. This thesis reports the development of a polymerase chain reaction (PCR) assay for the detection of the MPV in the ARC mouse colonies, the genetic characteristics of the strain of MPV detected, the development of a recombinant virus protein that provided a suitable antigen for enzyme-linked immunosorbent assay (ELISA) and a Western immunoblot (WIB) assay for the detection of MPV antibodies, and use of these various assays to determine aspects of the epidemiology and pathogenicity of the infection that were critical to the eradication of virus infection and future immunological surveillance to ensure the absence of infection. The recombinant protein produced as an antigen was a biotinylated fusion protein, a truncated capsid protein of the strain of MPV detected in the ARC, and was produced using the PinpointTM vector and with expression in Escherichia coli. The protein was produced as an insoluble intracellular product within inclusion bodies and was solubilised using urea and purified. The purified protein was utilised as an antigen for ELISA and the WIB assays to detect virus antibody in infected mice. The outbreak of MPV infection in the ARC was used as an unique opportunity for assessment of the seroprevalence of MPV-1 infection in a large laboratory mouse colony and to utilise this data to determine the sampling size needed to reliably detect MPV-1 infection within such large laboratory mouse colonies. An overall seroprevalence of 16.5% was detected using the developed serological tests, but considerable variation in prevalence was detected in different mouse strains. The response to MPV infection of 4 different but common strains of mice was determined as a basis for developing appropriate surveillance procedures and the selection of appropriate sentinel animals. The effect of infection of these strains at different ages was also investigated. Virus replication was detected in tissues of all the mice strains infected (outbred ARC(s) and inbred C57BL/6JArc, BALB/c and BALB/c-Foxn1nu/Arc) as juveniles and adults, with the exception of C57BL/6JArc inoculated as adults. However, while seroconversion in mice inoculated as juveniles and adults was detected in ARC(s) and C57BL/6JArc mice, it was not detected in BALB/c mice. The high rate of seroconversion to MPV, the early and prolonged development of an immune response, and the lack of age differences in their susceptibility indicated that ARC(s) mice would provide reliable sentinels for the detection of MPV. The genomic nucleotide sequence of the ARC strain, excluding the terminal palindromic regions and the predicted amino acid sequences of the non-structural and structural proteins was determined. This strain was very similar (98-99% nucleotide identity) to the previously described MPV strains MPV-1a, MPV-1b and MPV -1c. The similarity suggested there were unlikely to be significant antigenic differences in the proteins of the ARC strain and those strains of MPV reported previously.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 106-117

Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 2000? / Bibliography: leaves 182-198.