La dystrophine est une protéine cytoplasmique qui lie physiquement le cytosquelette à la matrice extracellulaire par le biais du complexe dystrophine-protéines associées (DAPC), assurant ainsi la stabilité du sarcolemme. Des mutations dans le gène DMD codant pour la dystrophine, conduisant à l’absence de la protéine, sont à l’origine de la dystrophie musculaire de Duchenne qui est une maladie liée au chromosome X. Pour mes travaux de thèse, j’ai généré et caractérisé un nouveau modèle de souris transgéniques rapportrices, dénommé DmdEGFP, qui exprime une protéine dystrophine endogène fusionnée avec la protéine fluorescente EGFP. La protéine dystrophine est liée dans sa région C-terminale qui est présente dans la majorité des isoformes. Dans le modèle, une expression forte et naturelle de l’EGFP était observée dans les muscles squelettiques, lisses, le cœur, le cerveau et l’œil, ce qui suggère un étiquetage correct de tous les isoformes de la dystrophine. La fluorescence de l’EGFP co-localisait exactement avec la dystrophine dans tous les sites. Dans le muscle squelettique, la dystrophine ainsi que d’autres protéines de la DAPC étaient exprimées dans des quantités normales et dans la bonne localisation subsarcolemmale. L’architecture du tissu musculaire squelettique était normale, suggérant que la fonction de la protéine de fusion était maintenue. In vitro, l’EGFP est également exprimée dans les fibres musculaires isolées, ainsi que dans les myotubes dérivés des cellules satellites. Par conséquent, cette nouvelle souris rapportrice de la dystrophine devient un outil important pour la visualisation directe et in vivo de l’expression de la dystrophine. / Dystrophin is a rod-shaped cytoplasmic protein that physically links the cytoskeleton to the ECM through the dystrophin-associated protein complex (DAPC), thereby providing sarcolemmal stability. Mutations in the dystrophin encoding DMD gene cause the severe X-linked disorder Duchenne muscular dystrophy. In this work a novel DmdEGFP reporter mouse that expresses a fluorescently labelled endogenous dystrophin – EGFP fusion protein was generated and characterized. The protein was tagged at the C-terminus that is present in the most dystrophin isoforms. To date, no dystrophin reporter mice exist, thus imaging is only possible by indirect antibody-mediated processing ex vivo. In DmdEGFP mice strong natural EGFP expression was observed in skeletal, smooth muscles, heart, brain and the eye and EGFP fluorescence co-localized with dystrophin at all sites suggesting proper tagging of the major dystrophin isoforms. In skeletal muscle, dystrophin as well as other proteins of the DAPC were expressed in normal quantity at correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting a correct function of the fusion protein. Isolated myofibers as well as satellite-cell derived myotubes expressed EGFP in vitro. Thus, the novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression.

3D proteomics combines chemical cross-linking with mass spectrometry to study the structure of protein assemblies and protein-protein interactions both in vitro and in vivo by providing distance constraints that indicate which residues are in close spatial proximity. I addressed the main bottleneck of this technology: the reliable identification of cross-linked peptides. Reporter ion signatures for cross-linked peptides were developed, by fragmenting model compounds containing two lysine residues joined by a cross-linker backbone or a lysine residue modified with a hydrolysed cross-linker. The reporter ion signatures showed 97% specificity at 90% sensitivity and segregated cross-linked from modified and linear peptides. They decreased the false discovery rate of the identification of cross-linked peptides from 5% to 1% in a large dataset. The signatures permit data sorting during and after mass spectrometry acquisition. The advanced 3D proteomics workflow was applied to study the protein-protein interactions in Mycoplasma pneumoniae cells. In lysates of the bacterium we identified 128 protein-protein interactions (of which 24 are novel) and obtained in vivo topological data on 208 proteins, even for cases where high-resolution structures are not yet available. We showed that our data are in excellent agreement with crystal structures of proteins and complexes where available. We defined a network of ribosomal and RNA polymerase proteins that reveals an intricate link between transcription and translation in bacteria. We demonstrated that the method is suitable for identification of homomultimeric protein complexes by exploiting peptide pairs of identical amino acid sequence. The technology has the potential to provide a complete protein interaction network map after the selective enrichment of cross-lined peptides is achieved. The method was next applied to investigate the structure of condensin and cohesin complexes, which play a crucial role in stabilization of chromosome structure during mitosis. The complexes were purified, cross-linked and their linkage map created. The condensin coiled coil cross-linked on the entire length was modeled. The information was used to direct the analysis of in situ cross-linked condensin in intact chromosomes. I found two high confidence linkages between SMC2 and SMC4 coiled coils and identified H2A as a potential condensin receptor on chromosomes.

The project involved the study of 12 Victorian municipal wastewater treatment plant discharges. These included lagoon-based plants and those with activated sludge based processes. Permission was obtained from all the relevant water authorities to collect samples of final effluent at point of discharge to the environment, whether that was to a creek, a river, the ocean, or the land. Samples were collected in November 2003, and then again in April and June 2004, and subjected to a number of biological and chemical analyses, including toxicity tests, measurement of hormonal (estrogenic) activity using yeast-based bioassays, and the measurement of specific hormonal concentrations (17-estradiol) using enzyme-linked immunosorbent assays (ELISA). Almost all of the effluents examined showed estrogenic activity, to a greater or lesser extent (no response to 55 ng/L 17β-estradiol equivalents). On the whole, the levels of estrogenic activity observed were to the lower end of the range observed overseas in the northern hemisphere, and comparable with that recently reported in Australia and New Zealand using similar, human-estrogen receptor based assays (no response to ~ 10 ng/L 17β-estradiol equivalents). The reassuring low/no assay response is bolstered by the chemical assessment of estradiol concentrations by ELISA, which returned concentrations of these compounds for the most part in the range 2-5 ng/L. From an aquatic environmental perspective, it is difficult to say with any certainty what the potential risk to aquatic organisms in waters receiving these effluents will be. Typically, in environmental risk assessment one first looks to agreed national or international guideline or trigger values for the type of waters being assessed. In this case, there are as yet no guideline values. Without guideline values to drive the assessment, then one compares a chemical’s concentration in a sample (in this case a WWTP effluent) with data obtained from toxicological experiments in which the concentration known to elicit a specific effect has been determined. In this case, levels of 17β-estradiol were typically between the lowest reported level to induce the production of Female-indicative proteins in male fish (plasma vitellogen; 1 ng/L), and the lowest concentration of known to induce intersex in fish (8 ng/L). Consequently, such levels in a WWTP discharge are likely to be an environmental risk if there is little or no dilution of the discharge by the receiving water, i.e. discharge represents major component of stream flow. In short, to truly assess the risk (hormonal impact) of these WWTP effluents, in vivo testing needs to be undertaken, ideally with a representative native species but failing that with a ‘standard’ species such as the fathead minnow. When this programme began, the ‘watching brief’, being held in Australia on the topic of endocrine disrupting chemicals and their potential effects on aquatic wildlife was considered too passive by many. It still is, by some. Despite the assurance the results may provide (of minimal impact in most cases if there is significant dilution), there is still a need for further extensive on-ground, reassurance research to provide data for higher-level risk assessment by industry and government agencies.

Magnetic resonance imaging (MRI) reporter genes produce MRI signal in response to the molecular environment of the cells in which they are expressed. With an MRI scanner, the signal is detected and used to produce an image of the cells. We hypothesized that the magnetotactic bacterial gene mms6 has the potential to function as an MRI reporter gene. Magnetotactic bacteria produce magnetic iron oxide crystals in intracellular organelles called magnetosomes. mms6 encodes an iron-binding, magnetosome membrane protein Mms6, which plays a role in regulating the size and shape of the iron oxide crystals found within the magnetosomes. To test our hypothesis, several mammalian cell lines were transfected with mms6, and mms6-positive clones were genetically engineered. We then used MRI to image these clones in vitro. When the cells were incubated with iron-supplemented culture media, the mms6-positive clones produced more MRI image contrast than mms6-negative cells. Through a systematic process of elimination, the mms6-positive clone that generated the most in vitro MRI image contrast was identified. This clone, named 9L4S, was composed of mms6-positive rat glioma (9L) cells and was used for intracellular iron studies and in vivo imaging. The results of electron microscopy and optical emission spectrometry support the theory that mms6-positive clones enhance MRI image contrast due to an increase in intracellular iron. The main objective of this research was to assess the ability of mms6 to function as an in vivo MRI reporter gene, so a flank tumor animal model was created. Without any exogenous iron supplementation, tumors composed of mms6-positive cells produced greater negative contrast on an MRI image than mms6-negative cells. These results demonstrate that mms6 can be considered for use in studies requiring an MRI reporter gene.

POPs are a group of chemicals that have been extensively studied over the last few years. The main reason that these chemicals have received so much scientific attention is the myriad of negative effects they have on the environment and human health. The properties that cause the deleterious effects include a high molecular stability, rendering them highly persistent. Added to this is the lipophilic and hydrophobic nature of the compounds. POPs will thus tend to bio-accumulate and bio-magnify in the environment, causing a direct threat to humans and wildlife. To address this threat, the Stockholm Convention on Persistent Organic Pollutants, under the supervision of United Nations Environment programme (UNEP), was initiated and became legally binding on 17 May 2004. All countries, including South Africa, which ratified this agreement, will be expected to monitor and regulate the formation of POPs. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) are all members of the dioxin-like family of POPs. This family of chemicals pose serious health threats such as carcinogenic effects and negative effects on reproduction. These substances, with the exception of PCBs, are formed unintentionally as by-products of industrial and thermal processes. One of the main sources of dioxin-like chemicals is medical waste incinerators. In this project the area surrounding a medical waste incinerator was monitored using a bio-assay technique. The determination of dioxin concentrations is usually preformed by chemical analysis, however, bio-assays have proven themselves to be a cheaper and time-saving screening method. The Toxic Equivalency Quotient (TEQs) determined through bio-assays can support chemical analysis in determining biologically-relevant risk assessments since bio-assay data has ecotoxicological relevance. These assays represent an integrated biological response to chemical pollutants, where biological effects are accounted for which is not possible in chemical analyses. One of the bio-assays used in the determination of the dioxin-like chemical TEQ is the H411 E reporter gene bio-assay. This assay is based on the Ah-receptor mediated toxicity of dioxin-like chemicals. Using this technique the TEQs for areas surrounding an active incinerator were determined, to indicate the distribution of these substances. The TEQs for the soil samples collected ranged between nondetectable and 154 ngTEQ/kg. There was no clear distributional pattern and the total organic carbon content in the soil did not seem to play a crucial role in the distribution of dioxin-like chemicals. Although a decrease in soil tillage showed a corresponding increase in TEQ. The predominant wind direction was taken into account but no correlation could be seen. However, meteorological parameters such as the ambient temperature and low precipitation in the area may have contributed to lower TEQ values. Cytotoxicity excluded data points and the phenomenon has to be addressed. High TEQ values in a residential area where free-range chickens are raised pose a serious concern to the level of dietary dioxin-like chemical intake. Eggs in the area could theoretically contain between 2.75 and 28.75 pgTEQ/g egg fat. Further studies are needed to determine how much dioxin-like chemicals are being transferred to humans through the consumption of free-range eggs / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006.

POPs are a group of chemicals that have been extensively studied over the last few years. The main reason that these chemicals have received so much scientific attention is the myriad of negative effects they have on the environment and human health. The properties that cause the deleterious effects include a high molecular stability, rendering them highly persistent. Added to this is the lipophilic and hydrophobic nature of the compounds. POPs will thus tend to bio-accumulate and bio-magnify in the environment, causing a direct threat to humans and wildlife. To address this threat, the Stockholm Convention on Persistent Organic Pollutants, under the supervision of United Nations Environment programme (UNEP), was initiated and became legally binding on 17 May 2004. All countries, including South Africa, which ratified this agreement, will be expected to monitor and regulate the formation of POPs. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) are all members of the dioxin-like family of POPs. This family of chemicals pose serious health threats such as carcinogenic effects and negative effects on reproduction. These substances, with the exception of PCBs, are formed unintentionally as by-products of industrial and thermal processes. One of the main sources of dioxin-like chemicals is medical waste incinerators. In this project the area surrounding a medical waste incinerator was monitored using a bio-assay technique. The determination of dioxin concentrations is usually preformed by chemical analysis, however, bio-assays have proven themselves to be a cheaper and time-saving screening method. The Toxic Equivalency Quotient (TEQs) determined through bio-assays can support chemical analysis in determining biologically-relevant risk assessments since bio-assay data has ecotoxicological relevance. These assays represent an integrated biological response to chemical pollutants, where biological effects are accounted for which is not possible in chemical analyses. One of the bio-assays used in the determination of the dioxin-like chemical TEQ is the H411 E reporter gene bio-assay. This assay is based on the Ah-receptor mediated toxicity of dioxin-like chemicals. Using this technique the TEQs for areas surrounding an active incinerator were determined, to indicate the distribution of these substances. The TEQs for the soil samples collected ranged between nondetectable and 154 ngTEQ/kg. There was no clear distributional pattern and the total organic carbon content in the soil did not seem to play a crucial role in the distribution of dioxin-like chemicals. Although a decrease in soil tillage showed a corresponding increase in TEQ. The predominant wind direction was taken into account but no correlation could be seen. However, meteorological parameters such as the ambient temperature and low precipitation in the area may have contributed to lower TEQ values. Cytotoxicity excluded data points and the phenomenon has to be addressed. High TEQ values in a residential area where free-range chickens are raised pose a serious concern to the level of dietary dioxin-like chemical intake. Eggs in the area could theoretically contain between 2.75 and 28.75 pgTEQ/g egg fat. Further studies are needed to determine how much dioxin-like chemicals are being transferred to humans through the consumption of free-range eggs / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2006.

A intersecção do webjornalismo com a web 2.0 faz emergir uma nova prática jornalística em rede – o webjornalismo participativo. As notícias publicadas em sites ou canais dessa natureza são produzidas por “cidadãos comuns” e, em alguns casos, passam pela mediação de jornalistas. Esta dissertação propõe-se a traçar o perfil das notícias veiculadas no canal de webjornalismo participativo vc repórter, do Portal Terra. O canal funciona com a intervenção de jornalistas que selecionam e editam o material enviado pelos internautas. Parte-se do pressuposto de que este modelo de produção jornalística acarreta novas formas de apresentação das notícias, tanto conteúdo quanto formato. Os dois meses de coleta (julho e agosto de 2007) totalizaram um corpus de 139 matérias, que foram submetidas à análise de conteúdo. Investigou-se os seguintes itens: valores-notícia; fontes consultadas; editorias mais procuradas; extensão do texto; utilização ou não de fotos e links complementares; sexo e localização geográfica dos colaboradores. / The intersection of webjournalism with Web 2.0 prompts the emergence of a new network journalistic practice – participative webjournalism. News published on sites or channels of this nature are produced by “common citizens” and, in some cases, go through the mediation of journalists. This dissertation is set to delineate the profile of the news items featured in the participative webjournalism channel vc reporter, Portal Terra. The channel functions through the intervention of journalists who select and edit the materials sent by the internet navigators. One parts from the presupposition that this journalistic production model calls for new manners to present news items, both content and format. The two months of collections (July and August) totalled a body of 139 subject matters, which were submitted to content analysis. The following items were investigated: news-values; sources consulted; most looked-after editorials; text extension; the use, or not, of photos and complementary links; gender and geographic location of the collaborators.

A intersecção do webjornalismo com a web 2.0 faz emergir uma nova prática jornalística em rede – o webjornalismo participativo. As notícias publicadas em sites ou canais dessa natureza são produzidas por “cidadãos comuns” e, em alguns casos, passam pela mediação de jornalistas. Esta dissertação propõe-se a traçar o perfil das notícias veiculadas no canal de webjornalismo participativo vc repórter, do Portal Terra. O canal funciona com a intervenção de jornalistas que selecionam e editam o material enviado pelos internautas. Parte-se do pressuposto de que este modelo de produção jornalística acarreta novas formas de apresentação das notícias, tanto conteúdo quanto formato. Os dois meses de coleta (julho e agosto de 2007) totalizaram um corpus de 139 matérias, que foram submetidas à análise de conteúdo. Investigou-se os seguintes itens: valores-notícia; fontes consultadas; editorias mais procuradas; extensão do texto; utilização ou não de fotos e links complementares; sexo e localização geográfica dos colaboradores. / The intersection of webjournalism with Web 2.0 prompts the emergence of a new network journalistic practice – participative webjournalism. News published on sites or channels of this nature are produced by “common citizens” and, in some cases, go through the mediation of journalists. This dissertation is set to delineate the profile of the news items featured in the participative webjournalism channel vc reporter, Portal Terra. The channel functions through the intervention of journalists who select and edit the materials sent by the internet navigators. One parts from the presupposition that this journalistic production model calls for new manners to present news items, both content and format. The two months of collections (July and August) totalled a body of 139 subject matters, which were submitted to content analysis. The following items were investigated: news-values; sources consulted; most looked-after editorials; text extension; the use, or not, of photos and complementary links; gender and geographic location of the collaborators.

L'adaptation d'une bactérie aux changements de son environnement est contrôlée par un réseau de régulation large et complexe, faisant intervenir de nombreux acteurs et modules différents. Dans ce travail, nous avons étudiés un module de régulation spécifique, contrôlant l'adaptation de la bactérie Escherichia coli à un changement de sources de carbone. Dans un milieu contenant du glucose et de l'acétate, la croissance est divisée en deux phases : les bactéries utilisent préférentiellement le glucose et commencent à métaboliser l'acétate qu'après l'épuisement du glucose. En effet, la présence du glucose réprime la transcription d'un gène nécessaire à la croissance sur acétate, le gène acs (codant pour l'acétyl-CoA synthétase). Le mécanisme régulateur fait intervenir le facteur de transcription Crp-AMPc et le système de transfert de phosphate (PTS), qui permet l'import du glucose. Plusieurs modèles décrivent en détail la cascade de réactions moléculaires à l'origine de cette « répression catabolique ». Cependant, certaines de nos observations expérimentales ne sont pas correctement prédites par les modèles actuels. Ces modèles doivent être révisés ou complétés. L'outil majeur que nous employons pour les expériences est la fusion transcriptionnelle : une région promotrice fusionnée en amont d'un gène rapporteur (GFP, luciferase). Avec ces constructions, nous mesurons la dynamique de l'expression génique dans différentes souches (mutants) et différentes conditions environnementales. Les observations à l'échelle de la population sont corroborées par des mesures similaires à l'échelle de la cellule unique. Nous utilisons cette même technologie pour construire de petits systèmes synthétiques qui sondent davantage le phénomène de répression catabolique. Nous avons ainsi créé un interrupteur génétique dont le fonctionnement est contrôlé par le flux glycolytique et nous avons construit un petit système de communication intercellulaire basé sur la molécule AMPc. Enfin, nous proposons une manière originale de mesurer l'état métabolique des cellules en utilisant la dépendance énergétique de la luciferase. / The adaptation of bacteria to changes in their environment is controlled by a large and complex regulatory network involving many different actors and modules. In this work, we have studied a specific module controlling the adaptation of Escherichia coli to a change in carbon sources. In a medium containing glucose and acetate, growth is divided into two phases : the bacteria preferentially use glucose and start to metabolize acetate only after glucose exhaustion. Indeed, the presence of glucose represses the transcription of a gene needed for growth on acetate : the acs gene (coding for acetyl-CoA synthetase). The regulatory mechanism involves the Crp-cAMP regulator and the phosphate transfer system (PTS), which is responsible for glucose import. Several models describe the cascade of molecular reactions responsible for this « catabolite repression ». However, our work shows that many of our experimental observations are incorrectly predicted by current models. These models have to be amended.We use transcriptional fusion, i.e., the fusion of a promoter region upstream of a reporter gene (GFP, luciferase), to measure the dynamics of gene expression in different genetic backgrounds and environmental conditions. Observations at the population level are corroborated by similar measurements at the single cell level. We use this same technology to construct small synthetic systems that probe further aspects of the phenomenon of catabolite repression. We have thus created a genetic toggle switch controlled by the glycolytic flux and we have built an inter-cellular communication system mediated by cAMP. Finally, we propose a novel way to measure the metabolic state of cells by using the energy dependence of the luciferase enzyme.

No description available.