Saccharomyces cerevisiae Kre5p is important for the biosynthesis of beta-1,6-glucan, which is required for proper cell wall assembly and architecture. A functional and cell biological analysis of Kre5p was conducted to further elucidate its role in beta-1,6-glucan synthesis. Kre5p was found to be a primarily soluble N-glycoprotein of ∼200 kD that localizes to the endoplasmic reticulum. Observation of Kre5p-deficient cells reveals a severe cell wall morphological defect, and kre5Delta cells were shown to have only residual levels of beta-1,6-glucan. KRE6 was identified as a multicopy suppressor of a temperature-sensitive kre5 allele, suggesting these proteins participate in a common pathway. An analysis of truncated versions of Kre5p indicates that it may have two independent, essential activities, or that it functions in a homodimeric state. Finally, Candida albicans KRE5 was shown to partially restore growth to kre5Delta cells, suggesting it has a function similar to that of the S. cerevisiae protein.

No description available.

Human African Trypanosomiasis (HAT), also known as sleeping sickness is one of the many life threatening tropical diseases posing a serious risk to livelihoods in Africa. The disease is restricted to the rural poor across sub–Saharan Africa, where tsetse flies that transmit the disease, are endemic. Sleeping sickness is known to be caused by protozoan parasites of the genus Trypanosoma brucei, with the two sub-species: T. b. gambiense and T. b. rhodesiense, responsible for causing infection in humans. The disease develops in two stages, firstly, the infection is found in the blood and secondly, when the parasites cross the blood-brain barrier entering the nervous system. To date, no vaccines have been developed, however, there is a range of drugs and treatments available which depend on the type of infection (T. b. gambiense or T. b. rhodesiense) as well as disease stage. The trypanosome parasites have a two-host life cycle i.e. in the mammalian host as well as the tsetse fly vector. Throughout the cycle, the parasite undergoes changes, one of them being the acquisition of a variable surface glycoprotein (VSG) coat prior to entry into the human host bloodstream. Once in the host, the infection progresses and through a phenomenon known as antigenic variation, the parasite expresses a different VSG coat periodically, enabling the parasites to constantly evade the host’s immune response, facilitating their survival. The VSG genes coding for the proteins are activated by an intricate process involving the encoding of a gene which is kept silent, until activated in one of several expression sites. Despite the constant switching of VSG surface coats, there are VSG forms that are predominantly expressed in T. b. gambiense namely VSGs LiTat 1.3, LiTat 1.5 and LiTat 1.6 which are used in diagnostic tests, as antigens to detect antibodies in infected sera of HAT patients. The acquisition of these VSG antigens is, however, of high risk to staff handling the parasites, and so the first part of the study was aimed at cloning, recombinantly expressing and purifying the two VSGs known to be recognised by all gambiense HAT patients: LiTat 1.3 and LiTat 1.5, for possible use as alternative antigens in diagnostic tests. The genes encoding both VSGs, LiTat 1.3 and LiTat 1.5, were first amplified from either genomic or complementary DNA (gDNA or cDNA), cloned into a pTZ57R/T-vector and sub-cloned into pGEX or pET expression vectors prior to recombinant expression in E. coli BL21 DE3 and purification by Ni-affinity chromatography. Amplification and subsequent cloning yielded the expected 1.4 kb and 1.5 kb for the LiTat 1.3 and LiTat 1.5 genes respectively. Recombinant expression in E. coli was only successful with the constructs cloned from cDNA, i.e. the pGEX4T-1-cLiTat 1.3 and pET-28a-cLiTat 1.3 clones. Purification of the 63 kDa cLiTat 1.3His protein following solubilising and refolding did not yield pure protein and there were also signs of protein degradation. For comparison, expression was also carried out in P. pastoris and similar to the bacterial system, expression was only successful with the LiTat 1.3-SUMO construct yielding a 62.7 kDa protein. Purification of LiTat 1.3SUMO also surpassed that of cLiTat 1.3His with no degradation. The diagnostic tests based on VSGs LiTat 1.3 and LiTat 1.5 as antigens operate by binding with antibodies in infected sera, to confirm infection. These antibody detection tests have their limitations, hence an alternative would be antigen detection tests, which use antibodies to detect the respective antigens in infected sera. The second part of the study therefore involved antibody production, where chickens were immunised with the native VSGs LiTat 1.3, LiTat 1.5 as well as recombinant RhoTat 1.2 (a VSG expressed in T. evansi). Antibody production was analysed by ELISA and characterised by western blotting, prior to immunolabelling of T. b. brucei Lister 427 parasites. The chicken IgY showed a response to the immunogens, and were able to detect their respective proteins in the western blot. Interestingly, the anti-nLiTat 1.3, anti-nLiTat 1.5 and anti-rRhoTat 1.2 antibodies were able to detect their respective VSGs on the T. b. brucei trypanosome parasites in the immunofluorescence assay, thus demonstrating cross reactivity. As the antibodies showed specificity, they could potentially detect antigens in infected sera of HAT patients in an antigen detection based test. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

No description available.

T cells are believed to be important in the control of varicella zoster virus (VZV) replication but little is known of T cell epitopes and the relationships between T cell responses, viral load and clinical disease during primary infection. I initially set to investigate the immune responses to two of the main VZV glycoproteins (gE and gI) using ex vivo and cultured IFNγ ELISpot assays. I identified several novel CD4+ T cell epitopes within gE and gI and characterized the phenotype of gE DRB1*1501 tetramer specific responses in healthy immune donors. I then set out to investigate the function and phenotype of VZV specific T cells in primary infection and their relationship to viral loads and clinical disease severity by using glycoprotein E/DRB1*1501 specific MHC class II tetramers, ex vivo IFNγ ELISpot assays and quantitative real time PCR assays. I compared the frequency and phenotype of specific T cells with virological and clinical outcomes in 32 adult individuals with primary VZV infection. In healthy immune donors, the gE specific T cells showed a early intermediate stage of differentiation with evidence of recent activation. Patients with acute primary infection had higher VZV/DRB1*1501 tetramer specific T cell responses and expressed markers of activation and effector differentiation. Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001). A significant inverse correlation was seen between the viral loads and the ex vivo IFNγ ELISpot responses of the patients (p<0.05, r=-0.64). These data would be compatible with a role for gE and gl-specific T cells in the control of viral replication during both primary infection and re-activation.

A mouse testis cDNA library was screened with an oligonucleotide probe corresponding to a sequence in the 5$ sp prime$ region of mouse N-cadherin cDNA. A composite clone containing three individual cDNAs was isolated. These included a 711 bp cDNA encoding part of mouse testicular N-cadherin, an unidentified 392 bp cDNA, and a 1500 bp cDNA encoding the mouse homologue of a human breast tumor autoantigen. / The cadherins, the influenza strain A hemagglutinins, and the fibroblast growth factor receptors are three different families of integral membrane glycoproteins that harbour the amino acid motif histidine-alanine-valine (HAV) in regions involved in protein-protein interactions. In order to identify other proteins that possess the HAV motif in functionally important regions, the SwissProt database was searched using a consensus sequence derived from the cadherins, influenza strain A hemagglutinins, and fibroblast growth factor receptors. This search identified the $ alpha$ chains of the HLA class I histocompatibility antigens as a fourth family of integral membrane glycoproteins with an HAV-containing region that is involved in a protein-protein interaction. (Abstract shortened by UMI.)

Bibliography: leaves 266-303 / xviii, 303 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1985

Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 195-230). Also available via World Wide Web.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.