Thesis (Ph. D.)--University of Texas at Austin, 2005. / Vita. Includes bibliographical references.

No description available.

A Dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2015 / The free radical theory of aging postulates that accumulation of oxidative damage in major cellular components is the predominant underlying cause of the aging phenotype. This damage is caused most commonly by reactive oxygen species (ROS) and antioxidant enzymes such as the superoxide dismutases (SOD) that neutralize ROS, are therefore vital. Manganese superoxide dismutase (MnSOD) is particularly critical as it is functional in the mitochondria, a major site for ROS generation. Numerous studies have demonstrated a tissue-specific decrease in the activity and mRNA levels of major antioxidants, including MnSOD, with aging, however the exact mechanism of this regulation is unclear. It was hypothesized that a general down-regulation of various antioxidant enzymes such as this may occur at the transcriptional level. In order to investigate SOD2 regulation, a comprehensively annotated rat SOD2 promoter region was established using the appropriate bioinformatics tools. Following this, SOD2 mRNA levels in tissues from young and old rat tissue were compared using quantitative PCR. The results showed increased and decreased SOD2 mRNA levels in old compared to young liver tissue and brain tissue, respectively, however these trends were not statistically significant. As MnSOD has been shown to be epigenetically downregulated in various age-related diseases it was hypothesized that the decrease in MnSOD mRNA levels seen in aging brain tissue may be a result of epigenetic regulation at the SOD2 (MnSOD gene) promoter, specifically, through DNA methylation. A methylation assay assessing the SOD2 gene promoter revealed no significant evidence of hypermethylation. Although this suggests that promoter methylation is an unlikely mechanism of SOD2 regulation in aging, further work would need to be implemented in order to prove this conclusively.

Brucella abortus is a bacterial species that infects cattle, elk, and bison herds worldwide and is a causative agent of brucellosis. B. abortus is a common form of zoonosis, as incidental spillover into the human population results in millions of infections annually. Current treatment options are limited to culling infected animals and treating humans with a rigorous antibiotic regimen, which still results in up to a 30% relapse rate. Detection of the pathogen is difficult due to the replicative niche residing within the host's immune cells, specifically macrophages and dendritic cells. Numerous small regulatory RNAs (sRNAs) were found to be expressed by B. abortus, and it was hypothesized that they may be important for virulence. One sRNA, when deleted, was shown to be linked to outer membrane stress resistance and was named MssR (membrane sensitivity sRNA). When the ΔmssR strain was tested in both macrophage and mouse models of infection, there were no virulence defects. Additionally, proteomic and transcriptomic studies of the ΔmssR strain showed very few dysregulated targets. Expression of mssR was tested under numerous biologically relevant conditions, and it was shown to be expressed significantly more during exponential phase of growth, compared to stationary phase. Initial microscopical analysis of mutant cells after treatment with sodium dodecyl sulfate (SDS_ did not reveal any morphological differences. It is unknown what contributes to the observed phenotypes and additional experiments are required to determine what is causing the perturbations in the outer membrane of the ΔmssR strain. / Master of Science / Brucella abortus is a bacterial species that causes the disease brucellosis in cattle and humans worldwide. To understand how B. abortus establishes infection, we are studying how the bacteria control the expression of genes during the process of infection. One method of bacterial gene regulation is the use of small regulatory RNAs (sRNAs). These small transcripts are similar to mRNAs but are shorter in length and typically do not encode for a protein. One such sRNA in B. abortus was shown to be linked to sensitivity to outer membrane stress and was named Membrane Sensitivity sRNA (MssR). After engineering a strain of B. abortus that does not produce MssR, there were no differences in the ability of the bacteria to infect macrophages or mice. Additionally, there were no noticable differences in the structure of the bacterial cells. When sRNAs regulate gene expression, differences can be seen at the mRNA and protein levels when the sRNAs are deleted. Very few targets were found be dysregulated at the transcript and protein level within the ΔmssR mutant. It is unknown what is causing the mutant to be more sensitive to outer membrane perturbations and additional tests are necessary to determine how MssR is linked to this phenotype.

No description available.

Aldehyde oxidase has been partially purified from Oregon-R-C and tuh(ASU) strains of Drosophila melanogaster using an affinity technique. The two enzymes were subjected to a partial kinetic analysis and found to be very similar to one another. This indicated the problem of elevated aldehyde oxidase activity in tuh(ASU) at key developmental stages (Kuhn and Cunningham, 1976) is due to an abnormal regulation. A comparative isozyme study through the developmental stages showed no major differences between the enzymes indirectly supporting the idea of an abnormal regulation. A comparison of tuh(ASU) with four wild-type strains indicated it may be a fourth allozyme of aldehyde oxidase.

Dendritic cells (DCs) are leukocytes specialized in antigen presentation. As competent stimulators of naive T lymphocytes, they link the innate and adaptive immune responses of vertebrates. The RAG-mediated adaptive immune system appeared approximately 500 million years ago in jawed fish and a number of studies suggest that DCs exist in bony and cartilaginous fish. However, the exact role of DCs in the fish immune system is not determined and questions remain as to whether a cell type truly homologous to DCs in homeotherms does exist. My project aimed to identify potential DCs surface markers (CD209A and LAMP3) in rainbow trout (Oncorhynchus mykiss) leukocytes for evaluation of the expression patterns by qRT-PCR under different conditions and stimuli, in vitro and in vivo. Another goal was to validate and evaluate the specificity of a produced anti-trout CD209A polyclonal antibody to further characterise antigen presenting cells (APCs) in fish. The methodology was to look for up-regulation of the predicted markers together with other markers known to be expressed by DCs in mammals and to evaluate at the mRNA and protein expression level after in vitro stimulation of trout primary leukocytes with trout rIL-4/13. Trout CD209A and LAMP3 mRNA was expressed in the main lymphoid organs of fish and could be modulated with microbial mimics. Upon in vitro stimulation of trout primary leukocytes with trout rIL-4/13, trout CD209A mRNA expression was up-regulated together with both CD83 and the MHC class II chain known to be expressed by mammalian DCs. In addition, CD209A protein expression was highly induced by trout rIL-4/13. Taken together, these results suggests that the characterisation of DCs in trout with tools such as transcript evaluation of surface markers and the anti-trout CD209A antibody, could help to more precisely define these leukocyte subsets. These findings could have further impact on fish vaccine improvements and be of importance for the aquaculture industry, by optimising stimulation of adaptive immunity.

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The fission yeast Schizosaccharomyces pombe is able to effectively degrade extracellular L-malate by means of a permease for the active transport of L-malate and a malic enzyme that catalyses the intracellular oxidative decarboxylation of L-malate to pyruvate and CO2. Sequence analysis of the S. pombe NAD-dependent malic enzyme gene, mae2, revealed an open reading frame of 1695 nucleotides, encoding a polypeptide of 565 amino acids. Mutational analyses of the mae2 promoter region revealed several putative cis-acting elements. Two of these elements have homology with binding sites for eukaryotic cAMPdependent regulatory proteins. The UAS I showed homology with the invert of the ADRI binding site, an AP-2 binding site and the TGGCA element. The other putative cAMPdependent site, UAS2, showed homology with the binding site for ATF/CREB and proved to be a strong activator sequence that is required for expression of the mae2 gene. Three negative acting elements, DRS I, DRS2 and DRS3 seem to function co-operatively to repress transcription of the mae2 gene. In this study northern and western blot analyses, as well as malic enzyme assays, showed increased levels of mae2 transcription and enzyme activity when cells were grown under fermentative conditions. The levels of mae2 expression increased approximately 4-fold in 30% glucose and 3-fold under anaerobic conditions. These increased levels of malic enzyme may provide additional pyruvate for various metabolic processes when the mitochondria are not fully functional under fermentative conditions. The regulated expression of the mae2 gene was further investigated using mae2-1acZ fusion plasmids that carried mutations in the DASI, UAS2 or the triple mutated DRSI/URS2/URS3 elements. These plasmids were transformed into S. pombe strains with mutations in the cAMP-dependent or stress-activated signal transduction pathways to determine the signal for the increased expression of the mae2 gene. The cAMP-dependent (Pkal ) and general stress activated (Styl) pathways often act in parallel to regulate the activation of transcription factors necessary for the expression of several S. pombe genes under different physiological conditions. The results presented here suggest that regulatory proteins involved in the Pka l and Styl pathways play a role in the regulation of the mae2 gene under fermentative conditions. Furthermore, some of the regulatory cis-acting elements in the mae2 promoter may interact with these trans-acting factors to regulate the transcription of the gene under different growth conditions. The mechanism of this interaction is not yet known and further research is required to identify all the transcription factors involved in the regulation of the mae2 gene. / AFRIKAANSE OPSOMMING: Die splitsingsgis S. pombe is in staat om ekstrasellulêre L-malaat effektief af te breek danksy 'n permease vir die aktiewe opname van L-malaat en 'n malaatensiem wat die intrasellulêre oksidatiewe dekarboksilering van L-malaat na pirovaat en C02 kataliseer. DNA-geen opeenvolgings van die NAD-afhanklike malaatensiemgeen, mae2, het 'n oopleesraam van 1695 nukleotiede getoon wat vir 'n polipeptied van 565 aminosure kodeer. Mutasie-analise van die mae2-promoter gebied het verskeie moontlike cis-werkende elemente getoon. Twee van die elemente toon homologie met bindingsetels vir eukariotiese cAMP-afhanklike regulatoriese proteïene. Die DAS 1 toon homologie met die omgekeerde volgorde van die ADRI bindingsetel, 'n AP-2 bindingsetel en 'n TGGCA element. Die ander moonlike cAMP afhanklike setel, DAS2, toon homologie met die bindingsetel vir ATF/CREB en is 'n sterk aktiveringselement wat vir die uitdrukking van die mae2-geen benodig word. Drie onderdrukker-tipe elemente, DRSI, DRS2 en DRS3, funksioneer moontlik gesamentlik om die transkripsie van die mae2-geen te onderdruk. In hierdie studie het northern en western klad analise, sowel as malaatensiem aktiwiteitstoetse verhoogde vlakke van mae2-transkripsie en ensiemaktiwiteit getoon wanneer die kulture onder fermentatiewe toestande gegroei het. Die uitdrukking van die mae2-geen het ongeveer 4-voudig toegeneem in 30% glukose en 3-voudig onder anaërobiese toestande. Hierdie verhoogde uitdrukking van die malaatensiem mag addisionele pirovaat vir verskeie metaboliese behoeftes voorsien wanneer die mitochondria onder fermentatiewe toestande nie volkome funksioneer nie. Die uitdrukking van die mae2-geen is verder onder fermentatiewe toestande bestudeer deur gebruik te maak van mae2-lacZ-fusie plasmiede wat mutasies in die moontlike DASI, DAS2, of die drievoudig-gemuteerde DRS I/URS2/URS3 setels bevat. Hierdie plasmiede is in S. pombe rasse met mutasies in die cAMP-afhanklike of stres-geaktiveerde seintransduksie paaie getransformeer om die sein vir die verhoogde mae2-geen uitdrukking te bepaal. Die cAMP-afhanklike (Pkal) en algemene stres-aktiverings (Styl) pad werk soms in parallel om die aktivering van transkripsiefaktore betrokke in die uitdrukking van verskeie S. pombe gene onder verskillende fisiologiese toestande to bewerkstellig. Ons resultate dui daarop dat die regulatoriese proteïene van die Pkal en die Styl paaie 'n rol in die regulering van die mae2- geen onder fermentatiewe toestande speel. Daar is ook aanduidings dat sommige van die regulatoriese cis-werkende elemente in die mae2-promoter wisselwerking met die transwerkende faktore toon om die transkripsie van die geen onder verskillende groeitoestande te reguleer. Die meganisme van hierdie interaksie is nog nie bekend nie en verdere navorsing is nodig om al die transkripsiefaktore wat by die regulering van die mae2-geen betrokke is, te identifiseer.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy