published_or_final_version / abstract / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Pathology / Master / Master of Philosophy

No description available.

The procerebrum of terrestrial molluscs is an important processing centre for olfaction. While the physiology of the procerebrum is relatively well characterized, the procerebrum's structure and organization has not been previously investigated in detail. The goal of this thesis is to better characterize the structural organization of the procerebrum and to understand how it compares with other olfactory systems. / The morphology of the procerebral neurons in the snail Helix aspersa was investigated through intracellular injections of biocytin. The population of cells is heterogeneous, but no formal categorization of neuronal types was possible. The main difference among cells lies in the placement of the cells' neurites. Furthermore, contradicting previous results, certain neurons were found to have neurite projections outside the procerebrum, travelling as far as the contralateral cerebral ganglion. / To investigate if differences in sites of arborization represent functional differences, the distribution of synaptic contacts on labelled cells was studied using serial sections and electron microscopy. Neurons with different sites of arborization have distinct patterns of synapse distribution. Cells with arborizations in the procerebrum but not in the internal mass have large varicosities specialized for output. Cells that arborize in the internal mass or outside the procerebrum have mostly input synapses proximal to the soma and mostly output synapses in the terminal region of the neurites. These latter cells appear to transmit information from the procerebral cell body mass to other brain regions. The implications of these data are, firstly, that the procerebrum directly distributes processed information throughout the nervous system. Secondly, the procerebral neuron population may be divisible into two subgroups: intrinsically arborizing interneurons and projection neurons. / These results suggest a novel mechanism by which compartmentalization could be achieved in the procerebrum. Compartmentalization is believed to be important for processing olfactory information, is present in most olfactory centres but has not previously been described in the molluscan olfactory system. I propose that varicosities on the local interneurons generate foci of activity in the procerebrum which, in turn, activate specific subsets of output neurons, similar to what happens in other olfactory systems.

Box C/D small ribonucleoproteins (box C/D sRNPs) are responsible for the 2’-O-methylation required for the complete maturation of precursor rRNA. Archaeal box C/D sRNPs, like eucarya, are composed of four components: a guide RNA (box C/D sRNA), an RNA binding protein (L7ae), a 2’-O-methyltransferase (Fibrillarin) and a structural protein (Nop5). Here we develop several approaches for studying box C/D sRNP assembly. In particular, we have used pulldown and mobility shift assays to identify box C/D sRNP assembly intermediates (Nop5-aFib and L7ae-sR1). We have also demonstrated that isothermal titration calorimetry (ITC) can be utilized to quantitatively characterize the energetics of formation for the L7ae-sRNA assembly intermediate. / xi, 98 leaves : ill. (some col.) ; 29 cm

Several vital cellular processes are preformed by large ribonucleoprotein (RNP) complexes. In archaeal and eukaryotic cells one example of these essential RNP particles is the box C/D sRNP. In archaea, this complex is responsible for methylation of ribosomal RNA (rRNA) and transfer RNA (tRNA) during their maturation. Archaeal fibrillarin (aFib) is the 2'-O methyltransferase responsible for catalysis by this complex. In this work we have identified the ability of aFib from Sulfolobus acidocaldarius to form dimers at biologically relevant concentrations and the structural determinants essential for this association. Based on our model we have predicted the ability of aFibs to form dimers in different archaeal and eukaryotic species. The ability of aFibs and their eukaryotic homologs to potentially adopt multiple conformations provides insight into the dynamics of the box C/D sRNP complex. As observed in the study of other essential RNP particles, the ability of these complexes to be conformationally diverse is integral to efficient catalysis of their varied substrates. / viii, 74 leaves ; 29 cm.

The usefulness of RAPDs (Random Amplified Polymorphic DNA) to distinguish among different taxa of Lotus was evaluated. The following species were included: L. corniculatus, L. tenuis, L. alpinus, L. japonicus, and L. uliginosus. Several accessions for each species were studied. Following DNA extraction, amplification reactions were performed in a Hybaid DNA Thermal Cycler, and the product visualized according to a standard procedure. Twenty primers were used for each species/accession. Clear bands and several polymorphisms were obtained for all primers. A phenogram was drawn based on the genetic distance among the species. L. alpinus appears as the most distant species from L. corniculatus, followed by L. uliginosus, L. tenuis, and L. japonicus. With the exception of L. alpinus, these findings are in agreement with previous experimental studies in the L. corniculatus group. The use of a greater number of primers and increased number of species may provide a greater resolution of the systematics of these taxa.

Acid phosphatases (apase) are important hydrolytic enzymes that function in the acquisition, production; transport and recycling of inorganic phosphate (Pi), thus making a significant contribution towards nutrients dynamic of many ecological niches. The aim of this study was to characterize the apase enzyme found in the lichen Cladonia portentosa at the molecular level. The initial experiment entailed cloning the apase gene by PCR using degenerate primers designed from close relatives of C. portentosa from the Ascomycete family. The isolation of apase gene from Cladonia portentosa using PCR was not successful. Attempts were then made to purify the secreted apase and to determine its biochemical and molecular properties and to allow comparison with already characterized secreted phosphatases from other fungal sources existing in the NCBI database. It was anticipated that the partial sequence of the purified enzymes would provide a corresponding apase gene. The acid phosphatase enzyme was partial purified to 45 fold by a gel filtration with a yield of 18%. It gave a single, broad glycoprotein band on native PAGE and SDS-PAGE corresponding in size to 250 and 148 kDa, respectively. Under reducing conditions, the purified enzyme migrated as two bands of 116 and 32 kDa, indicating the heterodimer nature of this enzyme. Only one distinct band, (pI 6.4) was observed after electrofocusing. The optimum temperature for the enzyme was 65 °C where an optimal pH was detected at 2.5. The enzyme was inhibited by known acid phosphatase inhibitors (fluoride, molybdate, orthovanadate and tartrate) and the metals (Cu²⁺ and Zn²⁺). The purified enzyme demonstrated broad substrates selectivity and had a KM of 31.2±0.25 μM for phytic acid. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF indicated the presence of two apase proteins. The amino sequences of purified apase/s from Cladonia portentosa were FLAETNPAPFGH, AVGLGYVEELLAR and AQGLGYVQEVLAR. Comparing the amino acids of the sequenced protein with that of already known proteins confirmed the enzyme to be a secreted histidine acid phosphatase, resembling other acid phosphatases and phytase from several filamentous fungi with respect to amino acid composition. To investigate the effect of phosphorus on C. portentosa apase, the mycelium was grown under different concentrations of Pi [0.05, 1.0, 3.0, 10 and 100 mM (KH₂PO₄)]. The aim was to localize the apase enzyme and to screen for the occurrence of the gene coding for the acid phosphatase enzyme. A treatment of 3.0 mM Pi induced high levels of apase compared to all other treatments. In addition, cultures of C. portentosa were grown in axenic cultures to study the effect of pH and Pi versus menadione on the production of acid phosphatase and mycelia growth. A culture media of pH 4.8 and 6.0 resulted in higher apase secretion than when compared with pH 2.5 medium. The presence of 2.0 μM menadione marginally increased levels of the apase compared to the control treatment. Apase was further localized cytochemically using fluorescent substrate-enzyme-labelled fluorescence (ELF-97) which forms a fluorescent crystalline precipitate at the site of phosphate activity. Fluorescent microscope revealed that the enzyme was present in all treatments, irrespective of Pi concentration, however, the fluorescence signals were intense in low Pi concentrations (0.05 and 1.0 and 3.0 mM Pi). Ultrastructure localization using live mycelium under confocal microscopy using Vector blue III substrate revealed that the enzyme was localized in the cytoplasm, cell membrane, vacuole and small organelles, presumed to be endosomes. Co-staining with FM4-64, confirmed the punctuate structure to be secretory vesicles or a vacuolar network. To investigate the effect of P starvation on C. portentosa at a molecular level, the effect of Pi on the gene expression profile was examined. The generation of a cDNA library from axenic grown mycelium treated with P provided a foundation for the identification and characterization of genes expressed in the P treated mycelium through expressed sequence tags (ESTs). Several genes were identified whose transcriptional profiles have been significantly changed by phosphorus treatment and menadione. They include genes required for signal transduction and vesicular transport, cell biosynthesis and protein metabolism and stress response. In conclusion, this study constitutes the first step towards understanding the molecular mechanism governing acid phosphatase in C. portentosa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Bloodstream forms of African trypanosomes, which endocytose macromolecules exclusively through their flagellar pockets, contain an acid phosphatase (AcP) activity in this organelle. In the present thesis, AcP activity was demonstrated cytochemically in some intracellular vesicles and on the surface of Trypanosoma congolense as well as in the flagellar pocket. Unlike other trypanosomatids such as Leishmania spp. and Trichomonas spp., these trypanosomes, while viable, did not release this enzyme into the surrounding medium. In contrast to mammalian cells, the AcP in T. congolense was shown by cell fractionation to be a non-lysosomal enzyme. The enzyme was mostly recovered in the microsomal and cytosolic fractions which had 52.7% and 44.4% of the total activity, respectively. Further separation of the microsomal fraction showed an association of AcP activity with vesicles derived from the plasma membrane, Golgi apparatus and endoplasmic reticulum. After ammonium sulfate precipitation and chromatography on a succession of columns containing Sephacryl S-300, DEAE-cellulose and Sephadex G-75, two acid phosphatases (AcPi and ACP2) were produced from the cytosolic fraction. A membrane-bound acid phosphatase (ACP3) was isolated from the microsomal pellets extracted with Triton X-l 14 and subjected to the above chromatographic procedures. The molecular mass of AcP 1 was higher than 700 kDa. It had an isoelectric point of 4.7. AcP2 (pi 5.3) and AcP3 (pi 6.5) had molecular masses of 33 and 320 kDa, respectively. AcPi and ACP3 were strongly inhibited by vanadate while ACP2 was strongly inhibited by p-chloromercuribenzoate. None of the enzymes was inhibited by tartrate but all were inhibited by NaF. The Km values for each of the various substrates differed widely between the three AcPs indicating that the binding site of each enzyme was distinct. The best of all the substrates tested was para-nitrophenyl phosphate. On non-denaturing gels the enzymes exhibited very high molecular masses but on denaturing SDS-PAGE, two similar bands of activity, localised at 62 and 65 kDa, were observed in all three AcP preparations. Thus the three isolated enzymes may be derived from the same base 62 and 65 kDa units. Differences between enzymes may be derived from differential processing of the isoenzymes for different functions at different locations. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.