L’élément mariner Mos1 est un élément transposable de classe II qui code une transposase, l’enzyme permettant aux transposons de se déplacer dans les génomes. Cette transposase possède un coeur catalytique DDE similaire à celui des intégrases rétrovirales. Mon travail de thèse a consisté en l’étude de la transposase de Mos1, sous différents aspects. Mes résultats complètent le modèle de transposition précédemment établi au laboratoire et apportent une nouvelle vision de la formation du complexe synaptique qui permet l’excision de transposon du site donneur. Mes travaux ont également permis d’identifier le domaine de liaison à l’ADN de la transposase comme un domaine de type CRO. L’étude de la régulation de l’activité de la transposase, qui est phosphorylée, m’a permis d’élargir le modèle de transposition de Mos1 au contexte cellulaire eucaryote, et des études d’ingénierie de la protéine posent la question de l’impact des facteurs d’hôtes lors de la transposition de Mos1. Enfin, des inhibiteurs de la transposase de Mos1 ont été identifiés et leur mode d’action caractérisé. Ces composés présentent également une activité sur l’intégrase du VIH-1 et une autre transposase à cœur catalytique DDE. / The Mos1 mariner element is a Class II transposable element, encoding a transposase, which is the enzyme allowing them to move in the genomes. This transposase has a DDE catalytic core like the retroviral integrases. My work consisted in studying the Mos1 transposase, under different aspects. My results complete the model of transposition previously established in the laboratory and bring a new vision of the formation of synaptic complex, which allows excision of the transposon donor site. The DNA-binding of Mos1 transposase has also been identified as a CRO-like domain. Work on regulation of the activity of Mos1 transposase, which is phosphorylated, allowed me to expand the model of Mos1 transposition in a eukaryotic cell context. The engineering of the protein were also conducted and questions about the impact of host factors on Mos1 transposition. Inhibitors of Mos1 transposase have been identified and characterized. These compounds also inhibit HIV-1 integrase and an other DDE transposase.

We establish a theorem on the approximation of the solutions of delay differential equations with continuously distributed delay with solutions of delay differential equations with discrete delays. We present numerical simulations of the trajectories of discrete delay differential equations and the dependence of their behavior for various delay amounts. We further simulate continuously distributed delays by considering discrete approximation of the continuous distribution.

Master of Science / Department of Statistics / Wei-Wen Hsu / Polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (DDE) are endocrine disrupting chemicals which can imbalance the hormonal system in the human body and lead to deleterious diseases such as diabetes, irregular menstrual cycles, endometriosis, and breast cancer. These chemicals as environmental exposures still exist in the environment and food chains and can be accumulated in human fatty tissues for many years. These chemicals can also be passed from mothers to their children through placental transfer or breastfeeding; therefore, their offspring may be at increased risk of adverse health outcomes from these inherited chemicals. However, it is still unclear how the parental association with offspring health outcomes and the inter-generational phenotypic inheritance could be affected by these chemical compounds. In this study, we mainly focus on how PCBs and DDE can affect the inheritance of Body Mass Index (BMI) across generations, as BMI is the primary health outcome (or phenotype) linked to diabetes. We propose a biometrical inheritance model to investigate the effects of PCBs and DDE on the heritability of BMI over two generations. Technically, a linear mixed effects model is developed based on the decomposition of phenotypic variance and assuming the variance of the environmental effect depends on parental exposures. The proposed model is evaluated extensively by simulations and then is applied to Michigan Fisheater Cohort data for answering the research question of interest.

Stuttgart, Univ., Diplomarb., 2001.

In vitro testing includes both cell-based and cell-free systems that can be used to detect toxicity induced by xenobiotics. In vitro methods are especially useful in rapidly gathering intelligence regarding the toxicity of compounds for which none is available such as new chemical entities developed in the pharmaceutical industry. In addition to this, in vitro investigations are invaluable in providing information concerning mechanisms of toxicity of xenobiotics. This type of toxicity testing has gained popularity among the research and development community because of a number of advantages such as scalability to high throughput screening, cost-effectiveness and predictive power. Hepatotoxicity is one of the major causes of drug attrition and the high cost associated with drug development poses a heavy burden on the development of new chemical entities. Early detection of hepatotoxic agents by in vitro methods will improve lead optimisation and decrease the cost of drug development and reduce drug-induced liver injury. Literature highlights the need for a cellbased in vitro model that is capable of assessing multiple toxicity parameters, which assesses a wider scope of toxicity and would be able to detect subtle types of hepatotoxicity. The present study was aimed at developing an in vitro procedure capable of mechanistically profiling the effects of known hepatotoxin dichlorodiphenyl trichloroethane (DDT) and its metabolites, dichlorodiphenyl dichloroethylene (DDE) and dichlorodiphenyl dichloroethane (DDD) on an established liver-derived cell line, HepG2, by evaluating several different aspects of cellular function using a number of simultaneous in vitro assays on a single 96 well microplate. Examined parameters have been suggested by the European Medicines Agency and include: cell viability, phase I metabolism, oxidative stress, mitochondrial toxicity and mode of cell death (apoptosis vs. necrosis). To further assess whether the developed method was capable of detecting hepatoprotection, the effect of the known hepatoprotectant, N-acetylcysteine, was determined. Viability decreased in a dose-dependent manner yielding IC50 values of 54 μM, 64 μM and 44 μM for DDT, DDE and DDD, respectively. Evaluation of phase I metabolism showed that cytochrome P4501A1 activity was dose-dependently induced. Test compounds decreasedlevels of reactive oxygen species, and significantly hyperpolarised the mitochondrialmembrane potential. Assessment of the mode of cell death revealed a significant elevation of caspase-3 activity, with DDD proving to be most potent. DDT alone induced dosedependent loss of membrane integrity. These results suggest that the tested compounds produce apoptotic death likely due to mitochondrial toxicity with subsequent caspase-3 activation and apoptotic cell death. The developed in vitro assay method reduces the time it would take to assess the tested parameters separately, produces results from multiple endpoints that broadens the scope of toxicity compared to single-endpoint methods. In addition to this the method provides results that are truly comparable as all of the assays utilise the same batch of cells and are conducted on the same plate under the exact same conditions, which eliminates a considerable amount of variability that would be unavoidable otherwise. The present study laid a solid foundation for further development of this method by highlighting the unforeseen shortcomings that can be adjusted to improve scalability and predictive power. / Thesis (PhD)--University of Pretoria, 2011. / Pharmacology / unrestricted

Fish and wildlife were randomly sampled in Utah from pre-selected x areas and analyzed for levels of total mercury, dieldrin, DDT, and DDE. Levels were well within consumptive tolerance or guildeline limits except for mercury levels of fish in Willard Bay Reservoir. PCB's were also identified in a large proportion of samples. None of the chukars, 6 percent of the pheasants, 4 percent of the waterfowl, and none of the fish sampled, excluding Willard Bay Reservoir, were above the 0.5 ppm guideline level for mercury established by the Food and Drug Administration. Seventy-seven percent of the fish sampled from Willard Bay Reservoir were above the 0.5 ppm guideline level. Only one duck (2 percent) was above the maximum permissible concentration for dieldrin. All of the chukars, pheasants, other waterfowl, and fish were well below human consumptive tolerance limits for dieldrin and DDT + DDE. It is recommended that studies be initiated to more closely define possible sources of mercury and the health hazard involved in consumption of these fish.

<p> Point Pelee National Park in Leamington, Ontario, Canada contained a large component of private farming and recreation until the 1960's. DDT was applied at the park as pest control in the orchards and recreational areas between 1948 and 1960. Recent studies have shown that the compounds DDT, DDE and DDD are highly persistent in the shallow soils of Point Pelee National Park. A laboratory treatability experiment has been effective in the removal of DDT, DDE and DDD from columns packed with soil collected from Point Pelee National Park, using hydroxypropyl-ß-cyclodextrin. Cyclodextrins are microbially produced cyclic oligosaccharides which have a unique hydrophilic shell and a hydrophobic cavity. They are able to form inclusion complexes and aid in the transport of relatively insoluble compounds. A pilotscale field remediation experiment involving the application of hydroxypropyl-ß-cyclodextrin to a Random Latin-Squares design was completed at Point Pelee National Park over the course of five months. Systematic soil sampling and analyses provided DDT, DDE and DDD concentrations throughout the remediation experiment in order to assess the removal efficiency of hydroxypropyl-ß-cyclodextrin. In-Situ volumetric moisture content was monitored throughout the study with a TDR based system. Systematic soil sampling and analyses at the conclusion of the study provided water content, organic matter content, bulk density, porosity, hydraulic conductivity as well as sampling with depth. The application of a hydroxypropyl-ß-cyclodextrin solution did result in a substantial decrease of the concentrations of DDT, DDE and DDD from the surface soil at Point Pelee National Park. By the end of the experiment, the concentration of DDD was consistently below the regulatory limits set by the Ontario Ministry of Environment and Energy for Parkland/Recreational Land-Use. In addition to the observed decrease in concentration, there was a decrease in the degree of variation between the measured concentrations, transport of the mass of DDT, DDE and DDD to depth and an observed tailing effect at late-times. There were also fundamental changes to the system due to the application of the cyclodextrin (HPCD) solution including an increase in moisture content and organic matter, a decrease in infiltration and a corresponding decrease in field saturated hydraulic conductivity. There was no appreciable benefit of the application of a high concentration solution over a low concentration solution due to an observed "clogging" -effect. The results of this study indicates that further research is necessary to determine the extent of vertical mobilization of DDT, DDE and DDD to groundwater and the role of biological matter in the observed fundamental changes responsible for the "clogging" -effect.</p> / Thesis / Master of Science (MSc)

Persistent organochlorines such as chlordecone (CD), DDT, and DDT degradation products bioaccumulate in fish and potentially impair reproduction or development via estrogenic actions. We evaluated the estrogenicity of CD, o,p'-DDT, o,p'-DDE, and p,p'-DDE injuvenile rainbow trout by assessing their potential to induce vitellogenesis; estrogen-regulated hepatic synthesis of the yolk-protein precursor, vitellogenin (Vg). In order to compare the sensitivities of various markers of estrogen stimulation, trout were injected with 17β-estradiol (0-10 mg kg-1) on days 0 and 3 and were sampled on days 3-12. Estradiol (5 mg kg-1) increased plasma Vg (2400%; 640 μg l-1), liver somatic index (200%) and hepatic cytosolic estrogen binding site levels (EBS, 300%) on day 6. These results suggested plasma Vg was the most sensitive marker of estrogen exposure. Chronic dietary exposure to CD (0.4 mg kg-1 day-1, 33 weeks) elevated plasma Vg (0.9 μg l-1), but not hepatic EBS concentrations, and resulted in relatively high hepatic CD concentrations (16 μg g-1). The in vivo estrogenicity of DDT was examined by injecting trout at 14 day intervals with single or triplicate doses of o,p'-DDT, o,p'-DDE or p,p'-DDE (0, 5, 15 or 30 mg kg-1) and monitoring vitellogenesis 14 days after the final injection. Plasma Vg and hepatic EBS concentrations were significantly elevated by o,p'-DDT and o,p'-DDE (total dose 45 and 90 mg kg-1; 23-24 μg Vg l-1) but not p,p'-DDE. Target organ doses were estimated by conducting a disposition study in which trout were injected with three doses of 14C]p,p'-DDE (30 mg kg-1), at 14 day intervals. Hepatic [14C]p,p'-DDE equivalent concentrations, 14 days after the final injection, averaged 14 μg g-1 Additionally, we evaluated the relative affinity of CD and DDT or DDE for trout hepatic EBS utilizing in vitro competitive binding assays. CD had relatively low affinity (1000-fold less than moxestrol, a synthetic estradiol) for trout hepatic EBS. o,p'-DDT and o,p'-DDE, but not p,p'-DEE also exhibited low EBS affinity (approximately 156000-fold less than moxestrol). Collectively, these results indicated that CD, o,p'-DDT and o,p'-DDE were weakly estrogenic in juvenile trout.

A atividade biológica de aldeídos &#945;,&#914;-insaturados tem sido associada a diversos processos incluindo regulação gênica, envelhecimento Alzheimer e disfunções mitocondriais. Neste trabalho investigamos a formação de adutos do trans,trans-2,4-decadienal (DDE), um aldeído produzido endogenamente e presente como contaminante em alimentos e água, em lisina, histidina e citocromo c. Avaliamos também alterações na função de mitocôndrias de fígado de rato expostas ao aldeído. As análises por espectrometria de massas, LC-ESI/MS, indicaram a formação de diversos tipos de adutos de DDE nos aminoácidos lisina e histidina, entre eles bases de Schiff e enaminas. Os resultados obtidos por espectrometria de massas, MALDI-Tof, indicaram a formação de adutos de DDE formados via base de Schiff de maneira concentração do aldeído, tempo e pH dependentes. As análises da proteína digerida por ESI-Q-ToF, indicaram que os adutos foram formados nos resíduos H-33, K-39, 72 e 100, localizados em regiões ricas em resíduos básicos, cuja interação com membranas e citocromo e oxidase tem sido postulada. Observamos também o deslocamento da banda Soret (&#955; = 409 nm) e o desaparecimento da banda em &#955; = 695 nm, relativa a coordenação do sexto ligante do grupo heme (M-80). Este fenômeno está associado a abertura da cavidade do grupo heme e o deslocamento do ligante, indicando alterações nas estruturas secundária e terciária da proteína. Os experimentos realizados com mitocôndrias isoladas indicaram que DDE promove danos à membrana interna mitocondrial, demonstrando i pelo aumento no consumo de O2 em mitocôndrias em estado 4. Em decorrência destas lesões observamos também o intenso inchamento mitocondrial, indicado por experimentos de espalhamento de luz e microscopia eletrônica de transmissão. O inchamento não foi bloqueado por ciclosporina A, um inibidor do poro de transição mitocondrial. DDE também induziu a perda do potencial de membrana da organela, demonstrado pelo monitoramento do indicador fluorescente safranina O e aumento da peroxidação lipídica atestado pela quantificação de malondialdeido (MDA). Estes resultados indicam que DDE promove alterações estruturais no citocromo e podendo levar ao comprometimento da atividade da proteína, além de promover alterações em parâmetros mitocondriais, indicando um possível envolvimento na disfunção mitocondrial promovida por estresse oxidativo. / Lipid hydroperoxide-derived &#945;,&#946;-unsaturated aldehydes are involved in several cellular processes such as gene expression, aging, Alzheimer disease and mitochondrial dysfunction. In this work we have investigated adduct formation in lysine, histidine and cytochrome c by trans,trans-2,4-decadienal (DDE), an endogenously lipoperoxidation product. DDE is also a widespread environment aldehyde found, for example, in food and as a contaminant in water. Alterations in rat liver mitochondrial parameters such as oxygen consumption, membrane potentials, swelling and lipoperoxidation were also investigated. LC-ESI/MS experiments indicated that DDE react with aminoacids lysine and histidine producing adducts. In addition, MALDI-TOF experiments indicated increases in the molecular weight of cytochrome c consistent with the formation of DDE adducts via the Schiff base mechanism. Our data shows that the protein modification was time, pH and DDE-concentration dependent, leading to the formation of at least six adducts after 2 h incubation at pH 7.4. ESI-Q-TOF MS analysis of tryptic digests indicated that H-33, K-39, K-72 and K-100 were modified by DDE. These adducts are present in clusters of basic amino acid residues, which are believed to participate in the interaction of cytochrome c with mitochondrial membrane and cytochrome c oxidase. A blue shift in the Soret band from 409 to 406 nm was also observed, indicating heme crevice opening and displacement of heme sixth ligand (Met-80) coordination in modified protein. DDE (180 &#181;M) treatment increases the rate of mitochondrial oxygen consumption, suggesting a partial mitochondrial uncoupling. Moreover, extensive mitochondrial swelling upon treatment with DDE (900 nM-162 &#181M) was observed by light scattering and transmission electron microscopy experiments. These effects were not prevented by the mitochondrial permeability transition inhibitor cyclosporin A. A DDE concentration-dependent loss in the inner mitocondrial membrane potential, monitored by safranin O fluorescence and an increase in lipoperoxidation were also observed. All together, these results suggest that reactive aldehydes can induce inner mitochondrial membrane damage playing a role in mitochondrial dysfunction associated with oxidative stress.

A atividade biológica de aldeídos &#945;,&#914;-insaturados tem sido associada a diversos processos incluindo regulação gênica, envelhecimento Alzheimer e disfunções mitocondriais. Neste trabalho investigamos a formação de adutos do trans,trans-2,4-decadienal (DDE), um aldeído produzido endogenamente e presente como contaminante em alimentos e água, em lisina, histidina e citocromo c. Avaliamos também alterações na função de mitocôndrias de fígado de rato expostas ao aldeído. As análises por espectrometria de massas, LC-ESI/MS, indicaram a formação de diversos tipos de adutos de DDE nos aminoácidos lisina e histidina, entre eles bases de Schiff e enaminas. Os resultados obtidos por espectrometria de massas, MALDI-Tof, indicaram a formação de adutos de DDE formados via base de Schiff de maneira concentração do aldeído, tempo e pH dependentes. As análises da proteína digerida por ESI-Q-ToF, indicaram que os adutos foram formados nos resíduos H-33, K-39, 72 e 100, localizados em regiões ricas em resíduos básicos, cuja interação com membranas e citocromo e oxidase tem sido postulada. Observamos também o deslocamento da banda Soret (&#955; = 409 nm) e o desaparecimento da banda em &#955; = 695 nm, relativa a coordenação do sexto ligante do grupo heme (M-80). Este fenômeno está associado a abertura da cavidade do grupo heme e o deslocamento do ligante, indicando alterações nas estruturas secundária e terciária da proteína. Os experimentos realizados com mitocôndrias isoladas indicaram que DDE promove danos à membrana interna mitocondrial, demonstrando i pelo aumento no consumo de O2 em mitocôndrias em estado 4. Em decorrência destas lesões observamos também o intenso inchamento mitocondrial, indicado por experimentos de espalhamento de luz e microscopia eletrônica de transmissão. O inchamento não foi bloqueado por ciclosporina A, um inibidor do poro de transição mitocondrial. DDE também induziu a perda do potencial de membrana da organela, demonstrado pelo monitoramento do indicador fluorescente safranina O e aumento da peroxidação lipídica atestado pela quantificação de malondialdeido (MDA). Estes resultados indicam que DDE promove alterações estruturais no citocromo e podendo levar ao comprometimento da atividade da proteína, além de promover alterações em parâmetros mitocondriais, indicando um possível envolvimento na disfunção mitocondrial promovida por estresse oxidativo. / Lipid hydroperoxide-derived &#945;,&#946;-unsaturated aldehydes are involved in several cellular processes such as gene expression, aging, Alzheimer disease and mitochondrial dysfunction. In this work we have investigated adduct formation in lysine, histidine and cytochrome c by trans,trans-2,4-decadienal (DDE), an endogenously lipoperoxidation product. DDE is also a widespread environment aldehyde found, for example, in food and as a contaminant in water. Alterations in rat liver mitochondrial parameters such as oxygen consumption, membrane potentials, swelling and lipoperoxidation were also investigated. LC-ESI/MS experiments indicated that DDE react with aminoacids lysine and histidine producing adducts. In addition, MALDI-TOF experiments indicated increases in the molecular weight of cytochrome c consistent with the formation of DDE adducts via the Schiff base mechanism. Our data shows that the protein modification was time, pH and DDE-concentration dependent, leading to the formation of at least six adducts after 2 h incubation at pH 7.4. ESI-Q-TOF MS analysis of tryptic digests indicated that H-33, K-39, K-72 and K-100 were modified by DDE. These adducts are present in clusters of basic amino acid residues, which are believed to participate in the interaction of cytochrome c with mitochondrial membrane and cytochrome c oxidase. A blue shift in the Soret band from 409 to 406 nm was also observed, indicating heme crevice opening and displacement of heme sixth ligand (Met-80) coordination in modified protein. DDE (180 &#181;M) treatment increases the rate of mitochondrial oxygen consumption, suggesting a partial mitochondrial uncoupling. Moreover, extensive mitochondrial swelling upon treatment with DDE (900 nM-162 &#181M) was observed by light scattering and transmission electron microscopy experiments. These effects were not prevented by the mitochondrial permeability transition inhibitor cyclosporin A. A DDE concentration-dependent loss in the inner mitocondrial membrane potential, monitored by safranin O fluorescence and an increase in lipoperoxidation were also observed. All together, these results suggest that reactive aldehydes can induce inner mitochondrial membrane damage playing a role in mitochondrial dysfunction associated with oxidative stress.