In the first part of this doctoral thesis, a new analytical HPLC-MS/MS method for monitoring of concentration changes of 17β-estradiol (βE2) during in vitro mouse sperm capacitation was developed. Capacitation was performed for three initial concentrations of βE2 (200, 20 and 2 μg/L). For all the concentrations a similar trend for the total unbound βE2 was observed. In general, the βE2 concentration decreased to reach its minimum and then increased again. The position of the minimum differed for the individual tested βE2 concentrations. Experimentally obtained results were subjected to the kinetic analysis. The curves fitted through the experimentally determined points displayed an autocatalytic character. For the agreement between the curves obtained by fitting through the experimental points and the theoretical calculated curves, it is necessary to assume that the first step is adsorption of βE2 onto the surface of the sperm controlled by Langmuir isotherm. The kinetic study was also used to study the effects of fluorides and aluminium fluoride complexes on the capacitation of mouse sperm. The experimental points were in very good agreement with the shape of the theoretical curves and this fact verifies the mechanism of the mouse sperm capacitation kinetics. In the second part of this work, two...

The purpose of this study was to develop a Life Sciences programme, integrating Environmental Education, on environmental endocrine disruptors, for the Further Education and Training Phase of the Outcomes Based Educational System. This programme aims at giving learners the necessary knowledge and skills to limit their exposure to Endocrine Disrupting Chemicals (EDCs). The programme was evaluated by means of a quantitative study. Group-administered questionnaires were used to gather information before and after the programme had commenced. Lickert scales were used to establish the learners' knowledge, attitudes and values in connection with EDCs and the environment before and after the learners had followed the programme on EDCs. The results of the study indicated that the programme proved to be successful in increasing the knowledge of the target group in connection with EDCs. This study will contribute to the process of integrating Environmental Education in the Life Sciences curriculum. / Educational Studies / M.Ed.(Environmental Education)

No description available.

The disposal of waste from agricultural activities has been recognised as a source of environmental contamination by endocrine disrupting chemicals (EDCs). The New Zealand dairy industry produces a large volume of dairy farm effluent, which contains EDCs in the form of estrogens. Most of this dairy farm effluent is applied onto the land for disposal. Groundwater and soil contamination by estrogens following waste application on the land have been reported overseas, but our understanding of the processes and factors governing the fate of estrogens in the soil is poor. Therefore the main goal of the present study was to better understand the fate and transport of estrogens, in particular 17β-estradiol (E2) and estrone (E1) in soil. In order to quantify E1 and E2 in drainage water and soil samples, chemical analysis by gas-chromatography mass-spectrometry (GC-MS) was carried out. This included sample extraction, sample clean-up through silica gel and gel permeation chromatography, and sample extract derivatisation prior to analysis. In order to develop a reliable method to extract estrogens from soil, research was conducted to optimise E1 and E2 extraction conditions by adjusting the number of sonication and shaking events, as well as the volume and type of solvent. Among five solvents and solvent mixtures tested, the best recovery on spiked and aged soil was obtained using an isopropanol/water (1:1) mix. A microcosm experiment was carried out to determine the dissipation rates of E2 and E1, at 8°C and at field capacity, in the Templeton soil sampled at two different depths (5-10 cm and 30-35 cm). The dissipation rates decreased with time and half-life values of 0.6-0.8 d for E1 and 0.3-0.4 d for E2 were found for the two depths studied. A field transport experiment was also carried out in winter, over three months, by applying dairy farm effluent spiked with estrogens onto undisturbed Templeton soil lysimeters (50 cm in diameter and 70 cm deep). The hormones were applied in dairy farm effluent at 120 mg m⁻² for E2 and 137 mg m⁻² for E1. The results of the transport experiment showed that in the presence of preferential/macropore flow pathways 0.3-0.7% of E2 and 8-13% of E1 was recovered in the leachate at the bottom of the lysimeters after 3 months, and 1-7% of the recovered E2 and 3-54% of the recovered E1 was leached within 2 days of application. These results suggest that leaching of estrogens via preferential/macropore flow pathways is the greatest concern for groundwater contamination. In the absence of preferential/macropore flow pathways, a significant amount (> 99.94%) of both hormones dissipated in the top 70 cm of soil, due to sorption and rapid biodegradation. Surprisingly, in all cases, estrogen breakthrough occurred before that of an inert tracer (bromide). This could not be explained by the advection-dispersion transport of estrogens, nor by their presence as antecedent concentrations in the soil. It was therefore suggested that colloidal enhanced transport of estrogens was responsible for the earlier breakthrough of estrogens and caused the leaching of a fraction of the applied estrogens to a soil depth of 70 cm. A two-phase model, adapted from a state-space mixing cell model, was built to describe the observed estrogen transport processes under transient flow. The model takes into account 3 transport processes namely, advection-dispersion, preferential/macropore flow and colloidal enhanced transport. This model was able to successfully describe the estrogen transport observed from the lysimeters.

The ubiquitous presence of anthropogenic chemicals and their transformation products in surface water represents a toxicological concern from both an ecological standpoint and a human perspective as many of these chemicals are capable of altering hormonal function. Endocrine disrupting compounds can be traced back to numerous sources and may fall under the class of pesticide, industrial chemical, pharmaceutical, personal care product, and/or heavy metals. The adrenal gland is the most common target for endocrine disruptors, although in comparison to the sex steroids, this system has received much less attention in published research. Corticosteroids play a pivotal role in many physiological processes, including immunity, cognitive function, growth, metabolism, reproduction, mineral balance, and blood pressure. A primary cell culture of rainbow trout adrenocortical cells was used to investigate the endocrine disrupting activity of two commonly detected water-borne toxicants, a personal care product, triclosan (TCS), a pesticide, dichlorophenoxyacetic acid (2,4-D), and their transformation products, methyl-triclosan (M-TCS) and dichlorophenol (DCP). Previously, it has been shown that TCS, 2,4-D, and DCP exhibit a potential for endocrine disruption, although it is currently unknown if these chemicals are capable of affecting corticosteroid balance. In this study, all four chemicals showed significant inhibitory effects on corticosteroid synthesis, even though there were considerable differences in their activity. The chemical that exhibited the highest toxicity was 2,4-D, followed by TCS, DCP, and M-TCS. Both parent-compounds proved to be more toxic than their degradation products. More research with suitable test systems is needed to determine the mechanism(s) of action of these corticosteroid disruptors and the health risk that they may present. / ix, 139 leaves : ill. ; 29 cm

The purpose of this study was to develop a Life Sciences programme, integrating Environmental Education, on environmental endocrine disruptors, for the Further Education and Training Phase of the Outcomes Based Educational System. This programme aims at giving learners the necessary knowledge and skills to limit their exposure to Endocrine Disrupting Chemicals (EDCs). The programme was evaluated by means of a quantitative study. Group-administered questionnaires were used to gather information before and after the programme had commenced. Lickert scales were used to establish the learners' knowledge, attitudes and values in connection with EDCs and the environment before and after the learners had followed the programme on EDCs. The results of the study indicated that the programme proved to be successful in increasing the knowledge of the target group in connection with EDCs. This study will contribute to the process of integrating Environmental Education in the Life Sciences curriculum. / Educational Studies / M.Ed.(Environmental Education)

MSc (Zoology) / Department of Zoology / Many chemicals released into the environment are believed to disrupt normal endocrine functions in humans and animals. These endocrine disrupting chemicals (EDCs) affect reproductive health and development. A major group of EDCs that could be responsible for reproductive effects are those that mimic natural oestrogens, known as xeno-oestrogens. A number of in vivo and in vitro screening strategies are being developed to identify and classify xeno-oestrogens, in order to determine whether they pose a health risk to humans and animals. It is also important to be able to apply the assays to environmental samples for monitoring purposes. Oestrogens and androgens mediate their activity via intracellular receptors – directly in muscular tissue as well as indirectly via stimulation of growth hormones from the pituitary glands and other growth factors from liver plus several other organs. Mancozeb is a metal ethylenebisdithiocarbamate (EBDC) fungicide used to protect many fruits and vegetables and field crops against pathogenic fungal. It causes a variety of defects on the female reproductive system in experimental animals and is therefore considered a suspected EDC. This fungicide can also induce toxic effects in cells of the immune system and other non-immune cells leading to genotoxicity and apoptosis. The mechanisms of EDCs involve divergent pathways including (but not limited to) oestrogenic, antiandrogenic, thyroid receptors; that are highly conserved in wildlife and humans, and which can be modelled in laboratory in vitro and in vivo models. The endocrine disrupting properties of Mancozeb are not known as of yet and therefore the T47D-KBluc reporter gene assay, GH3.TRE-Luc and MDA-kb2 reporter gene assay were used determine the possible endocrine disrupting activity/potential there-of. No activity was detected in any of the assays and no mancozeb was detected in any of the dams either. Oestrogenic activity was detected in Albasini Dam, Nandoni Dam and Xikundu weir but all values were below 0.7 ng/ℓ trigger value for oestrogenic activity in drinking water. / NRF

No description available.

PFOS (pluorooctane sulfonic acid) and PCB 180 (2,2′,3,4,4′,5,5′-heptachlorobiphenyl) are endocrine-disrupting chemicals (EDCs) ubiquitously found throughout the environment, given their persistence and extreme long biological half-life. PFOS and PCB 180 are predicted to disrupt retinoic acid receptor (Rar) signaling, interfering with important events of brain development, including neural differentiation and proliferation. Despite accumulating reports on the adversities of these EDCs, studies on the underlying mechanism continue to be largely unknown. The aim of this study was to validate transcriptional markers predictive of Rar disruption and to assess whether the same effects are induced by PFOS and PCB 180 exposure. Murine neural progenitor C17.2 cells were employed to mimic the developing brain. The cells were exposed to increasing nanomolar concentrations (nM) of Rar (ant)agonist, PFOS, and PCB 180. Interestingly, of all the transcriptional markers investigated, Ccn2 (cellular communication network factor 2), Il18 (Interleukin -18), and Ntn1(Netrin 1) were significantly altered by the Rar agonist (P< 0.05). Likewise, the expression of Il18 and Ntn1 was also altered by developmental exposure to PFOS and PCB 180. Altogether, these findings indicate that Il18 and Ntn1 may be promising markers for studying developmental neurotoxicity induced by disruption of the retinoic acid pathway.

Genome-wide association studies have greatly improved our understanding of the contribution of common variants to the genetic architecture of complex traits. However, two major limitations have been highlighted. First, common variant associations typically do not identify the causal variant and/or the gene that it is exerting its effect on to influence a trait. Second, common variant associations usually consist of variants with small effects. As a consequence, it is more challenging to harness their translational impact. Association studies of rare variants and complex traits may be able to help address these limitations. Empirical population genetic data shows that deleterious variants are rare. More specifically, there is a very strong depletion of common protein truncating variants (PTVs, commonly referred to as loss-of-function variants) in the genome, a group of variants that have been shown to have large effect on gene function, are enriched for severe disease-causing mutations, but in other instances may actually be protective against disease. This thesis is divided into three parts dedicated to the study of protein truncating variants, their medical relevance, and their functional consequences. First, I present statistical, bioinformatic, and computational methods developed for the study of protein truncating variants and their association to complex traits, and their functional consequences. Second, I present application of the methods to a number of case-control and quantitative trait studies discovering new variants and genes associated to breast and ovarian cancer, type 1 diabetes, lipids, and metabolic traits measured with NMR spectroscopy. Third, I present work on improving annotation of protein truncating variants by studying their functional consequences. Taken together, these results highlight the utility of interrogating protein truncating variants in medical and functional genomic studies.