Ovine placental lactogen (oPL) is postulated to be involved in the repartitioning of maternal nutrients during pregnancy, through its effect on insulin metabolism. Ovine pancreatic insulin responses to exogenous glucose are depressed during pregnancy and this depression becomes more pronounced as gestation advances. In addition, under the hormonal environment of rising oPL and growth hormone (oGH) concentrations, maternal whole body glucose irreversible loss (GIL) increases. The percentage of GIL accounted for by uterine glucose uptake also increases with advancing gestation and increasing litter size. Regression analysis of oPL concentration with glucose uterine uptake as a percentage of GIL, accounts for 39% of variation. Maternal oPL concentrations which increase with gestational age, were significantly greater in multiple bearing ewes and ewes subjected to reduced metabolisable energy (ME) intakes. It is postulated that through actions on pancreatic sensitivity, oPL plays a major role as a homeorhetic control during pregnancy. Elevated oPL concentrations were strongly associated with continually depressed pancreatic insulin secretory ability. The reduction in pancreatic sensitivity to glucose was not as a result of elevation in GH or non-esterified fatty acid (NEFA) concentrations. Muscle insulin receptor number and affinity were found to increase with increasing litter size, suggesting that pregnancy associated insulin resistance occurs predominantly in adipose tissue. During ovine pregnancy there is a specific stimulation of maternal gluconeogenesis. As gestation advances, an increasingly greater proportion of this glucose is partitioned to the gravid uterus. The development of insulin resistance, together with the suppression of pancreatic activity, ensures the preferential uptake of glucose by non-insulin dependent tissues over insulin dependent tissues. These activities favour uterine glucose uptake, decrease adipose glucose uptake, and also promote adipose mobilisation and hepatic gluconeogenesis, so as to meet the increasing energy requirement of pregnancy. It is postulated that through these effects on insulin secretion and associated adipose tissue mobilisation factors, oPL plays a major role in homeorhesis during pregnancy. / Doctor of Philosophy (PhD)

Biological wastewater treatment relies on the diverse and complex metabolic activities of bacteria to remove pollutants. Its success depends on the metabolic efficiency of the bacteria. Activated sludge models use parameters that attempt to depict bacterial growth and metabolic processes. However, current methods do not separate metabolic activity from growth and maintenance. As a result, activated sludge processes are misinterpreted or over-simplified. Alternative methods for gauging bacterial activity have been proposed and include the measurements of cellular derived compounds that relate specifically to energy cycling and include Nicotinamide Adenine Dinucleotide [NADH]. To date, NADH has been largely measured within activated sludge using commercial online fluorimeters with in situ probes. However, this current method provides a measure of the 'bulk' (raw) fluorescence within the system, resulting in difficulties when interpreting fluorescence data and poor sensitivity for detecting changes in intracellular [NADH]. This study has developed a more reliable method for estimating intracellular [NADH] and thus metabolic activity within activated sludge systems. Separating extracellular from intracellular [NADH] in samples was crucial because NADH was released and accumulates in the extracellular environment at a concentration of 200 ~M immediately following bacterial death or lysis. This concentration did not decline overtime. This not only caused high background fluorescence but also reduced the sensitivity of detection for changes in intracellular [NADH]. In particular, considerably higher [NADH] values to those from the extracellular suspensions were obtained following extraction of the intracellular material, suggesting that the cell membranes were not being penetrated by the excitable light source. Of the extraction procedures examined, filtration followed by extraction of the intracellular material with a hot Tris buffer was the most efficient and was recommended for accurate estimates of intracellular [NADH] in situ. In addition, standards were used to quantify NADH (moles per cell and/or unit volume) from unknown samples. The limits of detection were found to be 1.058 - 353 uM, whereas concentrations above 353 jAM self-quenched. Sample concentrations were always within these limits of detection. Hence, the sensitivity, reliability and experimental application of the original method was improved upon and able to be used for the direct measurement of microbial metabolic activity, something that has not been demonstrated before now. This study found that bacteria have between 106~ I 08 NADH molecules per cell depending on their metabolic state. A highly metabolically active bacterial cell had between 1O6~ tO7 NADH molecules, while a less active bacterial cell had between to7 -to8 NADH molecules. These measurements of metabolic activity were simultaneously monitored alongside other measures of bacterial growth, such as the incorporation of radiolabelled thymidine into DNA as a direct measure of DNA replication (new cell synthesis), the incorporation of radiolabelled leucine into protein as a direct measure of protein synthesis, oxygen uptake rates (OUR) as a direct measure of respiration, ATP as a measure of potential energy and dissolved organic carbon (DOC) as a measure of substrate assimilation. As OUR deceased, bacterial growth (using both the thymidine and leucine assays), specific [NADH] and specific [ATP] increased. High OUR and substrate oxidation rates simultaneous with low specific [NADH] indicated high rates of electron transport and thus efficient metabolic activity. Also, low OUR and substrate oxidation rates simultaneous with high specific [NADHI indicated inefficient rates of electron transport, therefore inhibiting oxidative phosphorylation (ATP production). A lack of oxygen as the terminal electron acceptor did not efficiently reoxidise NADH to NAD and resulted in an accumulation of NADH within the cell. Thus, a measure of low specific [NADHI was linked to the efficient rate of reoxidation of NADH to NAD* and reflects high metabolic efficiency. DNA and protein syntheses were coupled following substrate enrichment (glucose or acetate), indicating that bacteria were in balanced growth. However, DNA and protein syntheses became uncoupled once substrate was depleted, indicating unbalanced growth. An average Leu:TdR ratio of 7.4 was determined for activated sludge and was comparable to values published from marine systems. This ratio increased during log growth phase and decreased during stationary growth phases. Specific growth rates determined using the [3HITdR and [3H]Leu assay yielded values ranging from 2 - 10.5 d' and from 2.5 - 6 d1, respectively and were comparable to published values. Changes in OUR, NADH, ATE', DNA replication and protein synthesis were statistically ordinated using multidimensional scaling, and changes (in magnitude and direction) in bacterial metabolic activity were observed. Such methods enable the tracing of where bacteria divert their energies, such as to growth or maintenance and thus provide a greater understanding of bacterial behaviour in activated sludge. While studying anoxic and anaerobic conditions were beyond the scope of this work, the use of such methods to monitor bacterial metabolic activity under such conditions is warranted.

La protéine FUR (Ferric Uptake Regulator) est un régulateur global ubiquitaire chez les bactéries Gram-négatives. Sa liaison au Fe2+, in vivo, entraîne la répression de l'expression des gènes qu'elle contrôle. Ce travail est une étude structurale par RMN de la forme dimérique non activée de FUR d'Escherichia coli, un dimère de 2*17 kDa contenant un ion zinc par monomère. Une forme monomérique oxydée, capable de dimériser en présence de réducteur et de zinc, a également été isolée et étudiée. Le dichroïsme circulaire et la RMN montrent que la dimérisation entraîne une structuration du domaine C-terminal lors de l'incorporation du zinc. Les structures secondaires du domaine N-terminal du monomère et du dimère sont très proches. Seuls les premiers résidus sont structurés en hélice Α dans le monomère et déstructurés dans le dimère non activé. Cette hélice, observée dans le dimère activé de FUR de P. aeruginosa, pourrait jouer un rôle dans le mécanisme de régulation. Une protéine tronquée (FUR1-82) a été construite, purifiée, cristallisée. Sa structure est superposable à celle du domaine N-terminal de FUR de P. aeruginosa et le spectre 1H-15N-HSQC est superposable aux signaux du domaine N-terminal de FUR monomère. L'étude, par anisotropie de fluorescence, de la liaison du monomère et du dimère à l'ADN montre qu'ils se lient spécifiquement à l'ADN en présence de métal, contrairement à la forme tronquée. L'affinité du monomère pour l'ADN est 5 fois plus faible que celle du dimère en excès de métal. L'ensemble de ces données nous a permis de proposer un mécanisme de dimérisation de FUR d'E. coli ainsi qu'un mécanisme d'activation mettant en jeu cette hélice Α N-terminale.

<p>Concentrations of various metals are elevated above background levels in several intertidal areas along the Tanzanian coasts. However, there is little available information concerning the toxicity of these metals and how the uptake of these metals by bioindicators are influenced by external factors, such as heavy rains and increased coastal eutrophication, which tend to fluctuate.</p><p>The present study focused on the uptake and toxicity of Cu and Zn in two common macroalgal species,<i> Padina gymnospora</i> (Phaeophyta) and<i> Ulva</i> <i>reticulata</i> (Chlorophyta). Laboratory studies were performed where metal content, growth (DGR), maximal quantum yields (Fv/Fm) and protein expression patterns (in <i>Ulva</i>) were measured as a response to exposure to Cu and Zn. The levels of metals accumulated in algal tissues correlated well to exposure concentrations and the longer the exposure time, the greater the uptake. However, an increased nutrient load (tested on <i>Padina</i>) or dilution of the seawater (tested on<i> Ulva</i>) affected both uptake of metals and their toxic effects. Here, DGR was more affected than Fv/Fm, suggesting DGR to be the more sensitive indicator of Cu and Zn toxicity. As shown by 2-D gel electrophoresis, more than ten proteins were up-regulated in <i>U. reticulata</i> after being exposed to Cu (1μg/L), while at higher concentrations (10 and 100 μg/L) of Cu numerous proteins were down-regulated.</p><p><i>P. gymnospora </i>was also used as a bioindicator to monitor long-term (1994–2005) and seasonal in-year variations in heavy metal concentrations in the Zanzibar Channel. No clear overall trends were revealed, but analysis of the combined dataset clearly pinpointed the most contaminated sites. It was concluded that seasonal and long-term variations, as well as environmental conditions need to be taken into consideration when using macroalgae as bioindicators.</p>

<p>The Arbuscular mycorrhiza (AM) is an endosymbiont of higher plant roots. Most land plants and cultivated crops are concerned to AM symbiosis. This endosymbiosis is based on the mutual exchange of nutrients between plant and fungus. Therefore, AM symbiosis leads to an increased demand for photosynthetic products. The aim of this study was to investigate the pathway used by plants during AM symbiosis to increase photosynthetic performance. Therefore, we have carried out a systematic characterization of photosynthesis in Medicago truncatula (M. truncatula), which is a model legume. We observed colonization by the fungus in roots and that AM symbiosis increases the fresh and dry plant biomass. This could be attributed to an increase in both photosynthetic efficiency and capacity in AM plants. Consistent with these observations, AM symbiosis enhanced phosphorus uptake from the soil into roots, stems and leaves, as based on analyses of phosphorus content. Based on equal chl loading, no differences were found regarding D1, Lhcb1 and Lhcb2 protein content in four plant groups. This indicates similar ratio between chl and PSII proteins. Furthermore, AM symbiosis increases the amount of chlorophyll, steady state oxygen evolution activities, maximum quantum yield (Fv/Fm), and photosynthetic electron transport rate (about 5 fold). Nevertheless, photoprotection was not affected by AM symbiosis. We observed an increase in weight of seed/fruit and weight of seed/plant in AM plants (about 2 fold). Based on these results, we propose that AM symbiosis increases both the efficiency and the capacity of photosynthetic apparatus in the M. truncatula.</p>

<p>Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response.</p><p>When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. </p><p>Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. </p><p>Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.</p>

<p>The aim of this study was to investigate the effect the plant matrix and the structure of the flavonoid (i.e. whether aglycone or glycoside) may have on the gastrointestinal uptake and metabolism of luteolin derivatives from Artemisia afra traditional plant medicine. Specifically, how these two factors influenced the intestinal uptake and disposition of luteolin derivatives in pure and in Artemisia afra plant extract forms were to be assessed by investigating the uptake and metabolism of the luteolin derivatives in human intestinal epithelial Caco-2 cells and the perfused rat intestinal loop. To realize this aim, the following were determined: (1) identification and characterization of major luteolin derivatives found in Artemisia afra, (2) the effect of the plant matrix on the uptake of luteolin derivatives in Artemisia afra aqueous-extract forms across the Caco-2 cell monolayer, (3) the effect of the plant matrix on the absorption and metabolism of luteolin derivatives in Artemisia afra aqueous-extract forms in the perfused rat small intestine, (4) the effect of gut contents on the uptake and metabolism of luteolin derivatives in intestinal loop and (5) the metabolic profiles of luteolin derivatives obtained for the pure solutions versus plant aqueous extract solutions in Caco-2 cells and the rat intestine.</p>

<p>Elevated temperatures accompanying climate warming are expected to have adverse effects on sensitive lichen species. This premise was examined by measuring the sensitivity of different lichen species to elevated temperatures in the laboratory and in the field. Laboratory studies involved the exposure of nine hydrated lichen species (Xanthoparmelia austro-africana, X. hyporhytida, Xanthoparmelia sp., Xanthomaculina hottentotta, Teloschistes capensis, Ramalina sp., Flavopuntelia caperata, Lasallia papulosa, Parmotrema austrosinensis) collected from sites of different aridity and mean annual temperature for 2 hourly intervals to temperatures ranging from 24&ordm / C to 48&ordm / C in a forced daft oven and measuring their respiration rates and maximum quantum yield of PSII. Field studies involved simultaneous hourly measurements of ground surface air temperatures and Lichen effective quantum yield of PSII of hydrated lichen species populations under ambient and artificially modified environmental conditions.</p>

Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response. When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.

Concentrations of various metals are elevated above background levels in several intertidal areas along the Tanzanian coasts. However, there is little available information concerning the toxicity of these metals and how the uptake of these metals by bioindicators are influenced by external factors, such as heavy rains and increased coastal eutrophication, which tend to fluctuate. The present study focused on the uptake and toxicity of Cu and Zn in two common macroalgal species, Padina gymnospora (Phaeophyta) and Ulva reticulata (Chlorophyta). Laboratory studies were performed where metal content, growth (DGR), maximal quantum yields (Fv/Fm) and protein expression patterns (in Ulva) were measured as a response to exposure to Cu and Zn. The levels of metals accumulated in algal tissues correlated well to exposure concentrations and the longer the exposure time, the greater the uptake. However, an increased nutrient load (tested on Padina) or dilution of the seawater (tested on Ulva) affected both uptake of metals and their toxic effects. Here, DGR was more affected than Fv/Fm, suggesting DGR to be the more sensitive indicator of Cu and Zn toxicity. As shown by 2-D gel electrophoresis, more than ten proteins were up-regulated in U. reticulata after being exposed to Cu (1μg/L), while at higher concentrations (10 and 100 μg/L) of Cu numerous proteins were down-regulated. P. gymnospora was also used as a bioindicator to monitor long-term (1994–2005) and seasonal in-year variations in heavy metal concentrations in the Zanzibar Channel. No clear overall trends were revealed, but analysis of the combined dataset clearly pinpointed the most contaminated sites. It was concluded that seasonal and long-term variations, as well as environmental conditions need to be taken into consideration when using macroalgae as bioindicators.