Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1999.

S-Adenosylmethionine (SAM) synthetases isolated from both the yeast and hyphal-phase of the dimorphic fungus, C. albicans, were partially purified using DEAE cellulose ion-exchange column chromatography. Further characterization was accomplished using enzyme kinetics and specific enzyme effectors. SAM synthetase is the enzyme responsible for synthesis of SAM which is the major methyl group donor in the methylation of macromolecules. Kinetic studies on column samples, from both phases, were performed. The yeast-phase enzyme had apparent Km ranges for L-methionine and ATP of 1.06-1.42mM and 1.11-1.69mM, respectively. The hyphal-phase enzyme had apparent Km ranges for L-methionine and ATP of 1.34-2.66mM and 3.29-6.28mM, respectively. Effector studies (in vitro) indicate that 10% (v/v) dimethyl sulfoxide (DMSO) and 5mM cycloleucine inhibit SAM Synthetase from both phases, 24% and 46%, respectively. The methionine analogues DLmethionine sulfone, DL-methionine-DL-sulfoxide and L-methioninesulfoximine and sinefungin, an analog of SAM, had no effect on SAM synthetase activity. Although the data is inconclusive with respect to the existence of isozymes, the observed Km's of the yeast and hyphal-phases are different suggesting that isozymes may exist. Additionally, the yeast-phase DEAE column profile has a shoulder prior to the main peak of activity indicating that more then one form of the enzyme may be present. / Department of Biology

Bibliography: leaves 127-166. / xvi, 166, [64] leaves, [11] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study describes the isolation, sequencing and characterization of two cDNAs encoding barley (1-4)-B-xylanase isoenzymes X-I and X-II and the gene corresponding to isoenzyme X. The results of genomic Southern blot analyses indicate that the barley (1-4)-B-xylanase gene family consists of at least 3 genes which are mapped to a single locus on the long arm of chromosome 7(5H). The cDNA is used to monitor tissue-specific expression, developmental regulation and hormonal control of the (1-4)-B-xylanase genes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Bibliography: leaves 127-166. / xvi, 166, [64] leaves, [11] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study describes the isolation, sequencing and characterization of two cDNAs encoding barley (1-4)-B-xylanase isoenzymes X-I and X-II and the gene corresponding to isoenzyme X. The results of genomic Southern blot analyses indicate that the barley (1-4)-B-xylanase gene family consists of at least 3 genes which are mapped to a single locus on the long arm of chromosome 7(5H). The cDNA is used to monitor tissue-specific expression, developmental regulation and hormonal control of the (1-4)-B-xylanase genes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

Thesis (M.Sc. (Clinical Pathology))--Mahidol University, 1978.

Orientador: Vera Nisaka Solferini / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T22:41:42Z (GMT). No. of bitstreams: 1 Andrade_SoniaCristinadaSilva_D.pdf: 14587285 bytes, checksum: 2a4ec18a37aa5d1a25489912a65439ec (MD5) Previous issue date: 2005 / Resumo: Uma das questões particularmente interessantes em Biologia é compreender o vínculo entre a ecologia e evolução das espécies. Avaliar a relação entre a capacidade de dispersão e a escala espacial na qual as populações diferem geneticamente é uma das formas de entender como esse vínculo é estabelecido. A variação espacial das freqüências alélicas em populações naturais pode ser resultado de isolamento por distância, história populacional ou seleção diversificadora. Análises populacionais em grande e pequena escala são relevantes para avaliar como essas freqüências podem variar espacial e temporalmente. O objetivo desse estudo foi investigar a distribuição da variabilidade em litorinídeos utilizando isozimas como marcador molecular. No primeiro capítulo, o padrão de desvio das proporções de Hardy- Weinberg foi analisado em três espécies de litorinídeos (Echinolittorina lineolata, Littoraria fiava e L. angulifera) em uma escala macrogeográfica ao longo da costa Brasileira (cerca de 4.000 km). Um teste de homogeneidade dentro das amostras mostrou que os valores dos FIS são, em sua maioria, heterogêneos. Este resultado exclui endogamia e efeito Wahlund como principais causas do excesso de homozigotos. Em todas as espécies, pelo menos um loco do sistema PGM apresentou valores homogêneos de desvio de Hardy- Weinberg em todas as amostras, sugerindo que essa enzima pode estar sob efeito de seleção natural ou em desequilíbrio de ligação com um loco sob seleção. No segundo capítulo, avaliamos a subdivisão em escala local em Littoraria flava a fim de testar se os desvios de Hardy-Weinberg podem ser explicados por estruturação genética em pequena escala, apesar de essa espécie possuir fase larval planctotrófica. As amostras foram coletadas em transectos horizontais no costão rochoso em três praias, três vezes em um período de cerca de um ano. Foi realizada uma análise hierárquica de 15 locos polimórficos comparando a estruturação de uma escala de 200 km em relação a uma escala de dezenas a poucas centenas de metros. Littoraria fiava apresentou maior estruturação dentro dos transectos e entre as diferentes coletas temporais do que entre as praias. Cerca de 18% dos testes de neutralidade de Ewens-Watterson apresentaram desvio significativo de neutralidade. Esses resultados sugerem um equilíbrio entre colonizações recorrentes e coeficientes seletivos variando no tempo e espaço sobre diferentes locos. No terceiro capítulo estão apresentados os resultados de uma avaliação do efeito do ambiente sobre a forma da rádula de L. fiava e L. angulifera. Além da caracterização da variação morfológica da rádula, foi realizado um experimento de transferência recíproca dos indivíduos entre o mangue e o costão. Nas duas espécies, foi observada menor variação na forma da rádula nos indivíduos coletados no mangue, indicando que cada ambiente tem um efeito diferente sobre esse caráter. No experimento de transferência, as rádulas de L. fiava apresentaram mudança de forma 40 dias após o início do experimento, apesar do tamanho da fita radular ser fortemente influenciado pelo substrato original (F6.22=17,13, p<0,001). Foram observadas mudanças na forma em diferentes intensidades, sugerindo plasticidade fenotípica da forma da rádula / Abstract: One of the main questions in biology is the link between a species ecology and its evolution. Evaluating the relationship between the geographical scale over which populations differ genetically and the species dispersal ability is a way to understand how this link is established. Spatial variation in allellic frequencies of natural populations may be explained by isolation by distance, population history or diversifying selection. Populational analyses at different scales are appropriate to evaluate how gene frequencies vary in time and space. The main goal of this study was to analyse the distribution of the genetic variability in littorinids using allozymes as molecular marker. In the first chapter, the pattern of heterozygote deficiency was evaluated in three littorinid species (Echinolittorina lineolata, Littoraria flava and L. angulifera) at a macrogeographic scale along the Brazilian coast (4,000 Km). A homogeneity test among loci showed heterogeneous FIS values in most populations. This result ruled out inbreeding and Wahlund effect as the main causes of departure of Hardy- Weinberg expectations. In the three littorinid species, at least one Pgm locus had homogeneous FIS values along all sampled populations, which suggests that this enzyme may have an important role in the fitness or may be linked to a locus under selection. In the second chapter, local-scale subdivision in Littoraria flava was investigated in order to test if Hardy- Weinberg deviations could be explained by micro-structuring, despite the planktotrophic larval phase. Samples were collected along horizontal transects in rocky shores of three different beaches, three times over a year. With this sampling design, and using 15 polymorphic allozymic loci, we searched for indications of any micro-scale or short-temporal subdivision in contrast with macrogeographic (200 Km) structuring. Littoraria flava samples presented significantly more structure within transects and along the temporal scale than at large-scale. Eighteen percent of the Ewens- Watterson neutrality test showed significant deviation of neutrality expectation. This suggested that there could be a balance among several recurrent colonizations by cohorts with different allelic frequencies, followed by a directional selection on different loci at different times and localities. In the last chapter, we assessed if environmental heterogeneity could affect radular form in L. flava and L. angulifera. We also made a reciprocal transfer experiment in natural conditions between mangrove and rocky shore locations, apart nearby 100 m. Individuals of both species from mangrove showed less variation in the shape of radula than those from rocky shores, implying in a different environmental effect in each species. In the natural transfer experiment, radulae morphology of L. flava individuals changed within 40 days, but the length of the radulae were strongly infIuenced by the original substrate (F6,22= 17 .13, p<0.00l). Changes in the shape had different intensities, suggesting that this trait could be subject to phenotypic plasticity / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

M.Sc. (Biochemistry) / Please refer to full text to view abstract

Indiana University-Purdue University Indianapolis (IUPUI) / The goal of this study was to determine the effect of a PDK4 (pyruvate dehydrogenase kinase isoenzyme 4) knock-out on mitochondrial protein expression. A 2-D gel based mass spectrometry approach was used to analyze the mitochondrial proteomes of PDK4 wild-type and knockout mice. Mitochondria were isolated from the kidneys of mice in both well-fed and starved states. Previous studies show PDK4 increases greatly in the kidney in response to starvation and diabetes suggesting its significance in glucose homeostasis. The mitochondrial fractions of the four experimental groups (PDK4+/+ fed, PDK4+/+ starved, PDK4-/- fed, and PDK4-/- starved) were separated via large- format, high resolution two-dimensional gel electrophoresis. Gels were scanned, image analyzed, and ANOVA performed followed by a pair-wise multiple comparison procedure (Holm-Sidak method) for statistical analysis. The abundance of a total of 87 unique protein spots was deemed significantly different (p<0.01). 22 spots were up- or down-regulated in the fed knockout vs. fed wild-type; 26 spots in the starved knockout vs. starved wild-type; 61 spots in the fed vs. starved wild-types; and 44 in the fed vs. starved knockouts. Altered protein spots were excised from the gel, trypsinized, and identified via tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins identified with high confidence include ATP synthase proteins, fatty acid metabolism proteins, components of the citric acid cycle and electron transport chain. Proteins of interest were analyzed with Ingenuity Pathway Analysis (IPA) to examine relationships among the proteins and analyze biological pathways, as well as ontological analysis with Generic Gene Ontology (GO) Term Mapper. IPA found a number of canonical pathways, biological functions, and functional networks associated with the 87 proteins. Oxidative phosphorylation was the pathway associated with a majority of the proteins, while the largest network of proteins involved carbohydrate metabolism and energy production. Overall, the effects of starvation were more extensive on mitochondrial protein expression than the PDK4 knockout.

No description available.