La phytoremediation consiste à utiliser les plantes pour nettoyer des sols contaminés. Jusqu’ici, des plantes naturellement capables de tolérer et d’accumuler les polluants ont été utilisées pour cette approche. Cependant, l’utilisation de plantes transgéniques doit être considérée pour optimiser l’efficacité de la phytoremédiation. Le peuplier est une espèce adaptée pour la phytoremédiation et peut être utilisé pour des approches transgéniques. Néanmoins, son efficacité de phytoextraction est limitée par une forte accumulation de métaux dans les feuilles qui retournent au sol lors de leur chute. L’ingénierie génétique pourrait être utilisée pour résoudre ce problème, en modifiant l’expression de transporteurs de métaux soit pour limiter l’accumulation de métaux dans les feuilles, soit pour stimuler leur accumulation dans le bois.Dans le cadre de cette thèse, trois transporteurs potentiellement impliqués dans la tolérance et l’accumulation de métaux ont été caractérisés : PtIREG1, PtNRAMP3.1 et PtNRAMP3.2. L’expression de PtIREG1 chez la levure et chez Arabidopsis thaliana a montré que ce transporteur contribue à la tolérance au nickel. Des peupliers transgéniques chez lesquels l’expression de PtIREG1 est globalement augmentée ou ciblée dans le bois ont été générés. Des peupliers transgéniques chez lesquels l’expression de PtNRAMP3.1 ou PtNRAMP3.2 est modifiée ont également été générés au cours de cette thèse. Cela a permis de montrer que ces protéines fortement homologues ont des localisations subcellulaires distinctes : la membrane vacuolaire pour PtNRAMP3.2 et un compartiment connecté à l’appareil de Golgi pour PtNRAMP3.1. Des mesures de concentrations en métaux dans les feuilles des peupliers transgéniques ptNRAMP3.1 et PtNRAMP3.2 ont montré des différences avec le type sauvage non transformé, pour le cuivre, le manganèse, le cadmium et le zinc. Les résultats obtenus contribueront à l’élaboration de stratégies biotechnologiques pour réhabiliter les sols pollués / Phytoremediation is the use of plants to clean up polluted soils. Previous approaches have mostly used native plants able to tolerate, degrade and accumulate environmental pollutants such as toxic metals, but transgenic plants may also be considered for phytoremediation in the future. Poplar is well adapted for phytoremediation and suitable for molecular genetic studies. However, high metal accumulation in poplar leaves limits phytoextraction due to toxic metal return to the soil after leaf abscission. In order to circumvent this problem, genetic engineering can be used to limit metal accumulation in leaves or direct metal accumulation in poplar trunks using relevant metal transporter genes under the control of tissue-specific promoters. This thesis focuses on the characterization of 3 candidate metal transporters potentially involved in metal tolerance and accumulation in poplar: PtIREG1, PtNRAMP3.1 and PtNRAMP3.2. Expression of PtIREG1 in yeast and in Arabidopsis thaliana indicated that it contributes to nickel tolerance. Transgenic poplars were generated in which PtIREG1is either ectopically overexpressed or expressed specifically in wood tissues. PtNRAMP3.1 and PtNRAMP3.2 transgenic plants were also generated during this thesis. Despite their high similarity, PtNRAMP3.1 and PtNRAMP3.2 displayed distinct localizations in poplar: PtNRAMP3.2 is targeted to the vacuolar membrane whereas PtNRAMP3.1 localizes in a compartment connected with the Golgi apparatus. Metal concentrations were modified in leaves of transgenic plants grown on metal-contaminated or non-contaminated soil. The results obtained will contribute to develop a biotechnological approach using transgenic plants for the rehabilitation in metal polluted soils.

The Drosophila mutant fumble has a defect in mitochondrially targeted pantothenate kinase (PANK) and exhibits a movement disorder in the females. The human disease pantothenate kinase associated neurodegeneration (PKAN) has the same genetic defect and a neurodegenerative phenotype as well as iron accumulation in the brain. We have found that fumble females accumulate almost 2 fold more iron in the heads than wildtype. Dietary iron supplementation increases the iron accumulation in the heads further. The small isoform of malvolio (MVL), a homologue of mammalian NRAMP iron transporters, is expressed in the heads of flies. Its expression is upregulated in the fumble females, as well as in dietary iron supplemented wildtype flies. Unlike in the wildtype, dietary iron supplementation leads to a downregulation of MVL in the fumble flies. Although iron levels were elevated in fumble, ferritin expression was relatively unchanged and remained unchanged in the heads of fumble and wildtype with dietary iron supplementation. <p>The Drosophila mutant malvolio was used to determine how iron metabolism is affected when the MVL gene is defective. Iron levels were unchanged in malvolio relative to its parental strain (w1118) with or without dietary iron supplementation. Despite similar iron levels, a small decrease in ferritin expression was found in malvolio relative to w1118, and dietary iron increased ferritin expression in malvolio. However ferritin expression decreased in the parental strain of malvolio after iron supplementation. <p>Most of the iron in the Drosophila heads was in the form of goethite and ferrihydrite. The presence of iron oxides implies that this iron is in a mineralized storage form, likely ferritin. Dietary iron supplementation induced the appearance of ferric phosphates in fumble, malvolio, and wildtype. The subcellular location of this iron is unknown. It may be non-transferrin bound iron in the hemolymph, or a cytosolic intermediate in the labile iron pool. Also of note was the presence of transferrin-bound iron in wildtype heads on normal diet that was not seen after iron supplementation or in the heads of the fumble mutant. The presence in fumble of the kind of ferrihydrite characteristic of the mitochondrial protein frataxin may indicate that iron is accumulating in mitochondria.<p>The upregulation of MVL in the fumble mutant is of significant interest because it is the first protein involved in iron metabolism found to be altered with mitochondrial PANK deficiency. A disruption in MVL could be relevant to the brain iron accumulation in fumble and could be a treatment target for human PKAN.

The Drosophila mutant fumble has a defect in mitochondrially targeted pantothenate kinase (PANK) and exhibits a movement disorder in the females. The human disease pantothenate kinase associated neurodegeneration (PKAN) has the same genetic defect and a neurodegenerative phenotype as well as iron accumulation in the brain. We have found that fumble females accumulate almost 2 fold more iron in the heads than wildtype. Dietary iron supplementation increases the iron accumulation in the heads further. The small isoform of malvolio (MVL), a homologue of mammalian NRAMP iron transporters, is expressed in the heads of flies. Its expression is upregulated in the fumble females, as well as in dietary iron supplemented wildtype flies. Unlike in the wildtype, dietary iron supplementation leads to a downregulation of MVL in the fumble flies. Although iron levels were elevated in fumble, ferritin expression was relatively unchanged and remained unchanged in the heads of fumble and wildtype with dietary iron supplementation. <p>The Drosophila mutant malvolio was used to determine how iron metabolism is affected when the MVL gene is defective. Iron levels were unchanged in malvolio relative to its parental strain (w1118) with or without dietary iron supplementation. Despite similar iron levels, a small decrease in ferritin expression was found in malvolio relative to w1118, and dietary iron increased ferritin expression in malvolio. However ferritin expression decreased in the parental strain of malvolio after iron supplementation. <p>Most of the iron in the Drosophila heads was in the form of goethite and ferrihydrite. The presence of iron oxides implies that this iron is in a mineralized storage form, likely ferritin. Dietary iron supplementation induced the appearance of ferric phosphates in fumble, malvolio, and wildtype. The subcellular location of this iron is unknown. It may be non-transferrin bound iron in the hemolymph, or a cytosolic intermediate in the labile iron pool. Also of note was the presence of transferrin-bound iron in wildtype heads on normal diet that was not seen after iron supplementation or in the heads of the fumble mutant. The presence in fumble of the kind of ferrihydrite characteristic of the mitochondrial protein frataxin may indicate that iron is accumulating in mitochondria.<p>The upregulation of MVL in the fumble mutant is of significant interest because it is the first protein involved in iron metabolism found to be altered with mitochondrial PANK deficiency. A disruption in MVL could be relevant to the brain iron accumulation in fumble and could be a treatment target for human PKAN.

Sindbis virus (SINV) is an arthropod-borne Alphavirus in the family Togaviridae. Sindbis virus has a broad host range that includes avian, mammalian, and human hosts; therefore, its receptor(s) is/are highly conserved. When the mosquito imbibes a viremic blood meal, the virus infects the midgut cells, disseminates into the hemolymph, and eventually infects the salivary glands. The midgut is an organ of transmission and the virus must overcome the midgut epithelia infection- and escape-barriers. Sindbis virus infection is determined by the chance collision of the glycoproteins with a compatible receptor. Research has supported the involvement of high-affinity laminin receptor and heparan sulfate in SINV binding to host cells. However, it has been suggested that not all strains of SINV are dependent on heparan sulfate for attachment/entry and that SINV could be utilizing multiple receptors. A study using Drosophila demonstrated that, of the nine genes that encode for proteins that enhance SINV infection, only natural resistance-associated macrophage protein (NRAMP) was conserved. A symporter of divalent metals and hydrogen ions, NRAMP is ubiquitously expressed. Overexpression of NRAMP led to an increase in SINV infection of human cells while deletion of NRAMP in mouse and Drosophila decreased SINV infection. Sindbis virus could be utilizing this protein to overcome the infection barriers of mosquito midgut epithelia. In this study, NRAMP was localized to Aedes aegypti and Anopheles quadrimaculatus tissues via immunofluorescence assay and TR339-TaV-eGFP was detected in the midgut epithelia and visceral muscles. We suspect that NRAMP was detected on midguts and/or Malpighian tubules of Aedes aegypti and Anopheles quadrimaculatus. The similarities between the pattern of NRAMP labeling and TR339-TaV-eGFP infection of the midgut suggest that SINV infection is influenced by NRAMP in the midgut epithelia. Because NRAMP is ubiquitously expressed, this research provides insight into the attachment and entry phase of the arbovirus lifecycle.