Les NAD(P)H déshydrogénases de type II (Ndh-II) sont des enzymes de faible poids moléculaire capables doxyder le NAD(P)H et de transférer les électrons à un groupement quinone (plastoquinone ou ubiquinone). On les appelle « de type II » par opposition aux déshydrogénases de type I qui correspondent au complexe I mitochondrial. Chez Arabidopsis thaliana, des protéines Ndh-II ont été identifiées sur les faces interne et externe de la membrane interne mitochondriale, sur la membrane des peroxysomes, et au niveau de la membrane thylacoïdale du chloroplaste. Au niveau de la chaîne de transport délectrons mitochondriale, les protéines Ndh-II constituent une voie alternative aux complexes I et II pour lapport des électrons au pool dubiquinones. Cette voie alternative permettrait une adaptation de la chaîne de transport délectrons en fonction du métabolisme de lalgue. Au niveau de la chaîne de transport délectrons chloroplastique, les protéines Ndh-II participeraient à plusieurs mécanismes dadaptation de la chaîne à la quantité et à la qualité de la lumière disponible : transitions détats, transport cyclique délectrons autour du photosystème II. Leur fonction serait de catalyser la réduction non-photochimique du pool de plastoquinones. En 2005, sept open reading frame correspondant à des NAD(P)H déshydrogénases de type II hypothétiques (NDA1 à NDA7) ont été identifiées dans le génome nucléaire de Chlamydomonas. Ces séquences étaient cependant largement incomplètes du fait de régions non séquencées dans le génome de Chlamydomonas. Les données récoltées au cours de ce travail ont permis lobtention dune version complète de la séquence codante des gènes NDA de Chlamydomonas. Ces analyses ont démontré que le gène putatif NDA4 correspondait, en fait, à des régions internes non attribuées au gène NDA2. Chez Arabidopsis thaliana et Solanum tuberosum, une corrélation entre le positionnement phylogénétique des gènes NDH-II et la localisation subcellulaire de la protéine correspondante a été mise en évidence. Lanalyse phylogénétique des séquences des protéines Nda de Chlamydomonas montre que les gènes NDA1, 2 et 3 seraient proches phylogénétiquement et seraient à positionner dans le clade des protéines Ndh-II mitochondriales des plantes supérieures. A linverse, la protéine Nda5 serait dorigine cyanobactérienne et se positionne dans le même clade que les protéines identifiées dans le chloroplaste des plantes supérieures. Les protéines Nda6 et 7 sont très proches du point de vue de la séquence, suggérant une duplication récente des gènes NDA6 et 7. Ces deux protéines se positionnent dans un nouveau clade, apparemment intermédiaire entre le domaine eucaryote et le domaine procaryote. Une étude dexpression des gènes NDA de Chlamydomonas a permis de mettre en évidence lexpression apparemment majoritaire du gène NDA2. Pour étudier la fonction spécifique de NDA2, nous avons inactivé lexpression de ce gène par RNA interférence afin détudier le phénotype des mutants obtenus. Contrairement aux prédictions in silico, il est apparu que la protéine Nda2 se localise au niveau du chloroplaste. Létude de la fluorescence chlorophyllienne de deux mutants montre que la capacité de ces mutants à réduire de manière non-photochimique le pool de plastoquinones est largement diminuée. Dautre part, les mutants sont largement affectés dans leur capacité à modifier la distribution de lénergie dexcitation entre les deux photosystèmes (transition détat) lorsque la respiration mitochondriale est inhibée. Il est connu que les transitions détat sont initiées par des changements de létat rédox du pool de plastoquinones, qui est lui-même dépendant de létat rédox de la cellule. Dans ce cadre, nous proposons que la protéine Nda2 pourrait servir de « senseur » du métabolisme cellulaire de lalgue et permettrait dadapter les flux délectrons chloroplastiques en réponse aux changements du contexte énergétique cellulaire.

The pursuit of agricultural sustainability is necessary to ensure global food security into the future. To achieve sustainability, production systems around the world use different approaches. Utilizing several biological and physical indicators, this study investigates two agricultural production systems and assesses how management has affected the long-term health and sustainability of the soils. The first study assessed the effect of variable intensities of tillage on three Saskatchewan soils under low-disturbance (LD) management for the ten years prior to tillage. The soils represented were in the Grey, Black and Brown soils zones at sites located near Tisdale, Rosthern and Central Butte, Saskatchewan, respectively. A completely randomized block design utilized four treatments of varying tillage intensity. Samples were taken in spring before planting and after harvest at all sites. The soils were analyzed for microbial indicators of health by assessing dehydrogenase, urease, protease, and alkaline phosphatase activities. Microbial biomass carbon (MBC), microbial biomass nitrogen (MBN) and microbial quotient nitrogen (MQN) also were analyzed. Traditional soil nutrient and physical parameters were measured. The tillage intensities affected each parameter differently likely due to the differences in litter quality at each site. The high intensity tillage treatment decreased dehydrogenase activity at Tisdale at May, while in Rosthern dehydrogenase activity was increased in the moderate intensity tillage treatment and decreased by the high intensity tillage treatment. At Central Butte no effect was detected until October when dehydrogenase activity was increased by the low and moderate tillage intensity treatments. Protease and urease activities were affected at Rosthern only where the moderate intensity tillage treatment decreased activity relative to the control treatment. Soil physical parameters were not affected by tillage intensity; however nutrient levels were impacted by the increasing tillage intensity. Specifically, NO3- was reduced at Tisdale and was increased at Rosthern. Phosphate levels were reduced by the high tillage intensity in Rosthern whereas, with increasing tillage, the opposite occurred at Tisdale and Central Butte. The responses were strongly influenced by site characteristics, especially soil zone, organic matter content and surface litter abundance and quality. These effects were short-term, having no long-term impact on the agricultural sustainability or health of the soil, although knowledge of litter condition and quality is agronomically beneficial in order to predict soil responses to intense tillage events. iii The second part of the study was to assess the success of grass terraces on preserving the soil health of hillslope farm plots with Oxisolic soils in southern Ethiopia. Soil erosion has a devastating impact on hillslope agriculture in Ethiopia causing severe land degradation. An adjacent terraced and unterraced hillslope was chosen and sampled, along with a second unterraced slope for comparison. These soils were analyzed for dehydrogenase, alkaline phosphatase, and urease activities, as well as total C and total N. The plots above the terraces [terraced upper and unterraced upper] had higher urease activities than the plots below [terraced lower and unterrraced lower]. The impact of a vegetative strip that had formed a terrace 20 years ago was still evident in consistently higher alkaline phosphatase, urease, and dehydrogenase activities than the other plots. Simple methods of erosion prevention on erosion prone hill-slopes indicated that vegetative strips leading to terracing have a positive effect on soil health and functionality, promoting the long-term agricultural productivity and sustainability of these landscapes.

Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (<i>Nicotiana tabacum L.</i>), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, indicating the possibility of two tandem targeting signals. In this work the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) to generate a series of constructs and introduced into tobacco leaves by <i>Agrobacterium</i>-mediated transient transfection. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined under the confocal microscope. Constructs predicted to be targeted to the chlroplast all localized to the chloroplast. However, this was not the case for constructs that were predicted to be mitochondrial targeted. While some constructs localized to mitochondria, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein which was present in those constructs. In addition, during the process of generating these constructs our sequence analysis indicated a stop codon present at amino acid position 161 of the mature NADP+-ICDH protein from both Xanthi and Petit Havana cultivars of tobacco. This was confirmed by multiple sequencing reactions and created discrepancies with the reported sequence present in the database. The results of this study raise interesting questions with regard to the targeting and processing of NADP+-ICDH.

Inflammatory and neuropathic pains arise in part from sensitization at nociceptive synapses in the spinal cord. Activity-dependent signaling cascades converge onto the tyrosine kinase Src, which participates in augmenting the function of N-methyl-D-aspartate receptors (NMDARs) and thus potentiates the nociceptive system. Src is capable of these effects because it is anchored to the NMDAR complex via an adaptor protein called NADH dehydrogenase subunit 2 (ND2). There is evidence that this interaction occurs between amino acids 40-49 of Src and amino acids 310-321 of ND2. I have determined that a peptide consisting of amino acids 310-321 of ND2, and affixed to the HIV Tat domain for cell permeability, is capable of alleviating tactile allodynia induced by Complete Freund's Adjuvant (CFA) in rats. Src40-49Tat was not effective in two models of inflammatory pain. This work further implicates the Src-ND2 interaction in pain hypersensitivity and suggests that Tat ND2 310-321 may alleviate it.

The shikimate pathway links carbohydrate metabolism to biosynthesis of the aromatic amino acids in plants, fungi, bacteria and apicomplexan parasites. The pathway has seven enzymatic steps which convert erythrose-4-phosphate and phosphoenolpyruvate to chorismate, the precursor of tyrosine, tryptophan and phenylalanine. Due to the absence of the pathway in mammalian species, the enzymes are attractive targets for herbicides and antimicrobials. Shikimate dehydrogenase (SDH) catalyses the fourth step, the NADP-dependent reversible reduction of 3-dehydroshikimate to shikimate. Five SDH homologs – AroE, Ael1, YdiB, RifI and SdhL – have been identified through kinetic analysis and phylogenetic studies in the bacterium Pseudomonas putida. SDH homolog gene knockouts (KO) were used to characterize their functions. The AroE KO and Ael1 KO were successfully constructed via gene SOEing of the SDH homolog with a gentamycin antibiotic cassette and homologous recombination via electroporation into WT P. putida KT2440. Preliminary characterization tested KO growth, auxotroph recovery and fluorescent activity.

Inflammatory and neuropathic pains arise in part from sensitization at nociceptive synapses in the spinal cord. Activity-dependent signaling cascades converge onto the tyrosine kinase Src, which participates in augmenting the function of N-methyl-D-aspartate receptors (NMDARs) and thus potentiates the nociceptive system. Src is capable of these effects because it is anchored to the NMDAR complex via an adaptor protein called NADH dehydrogenase subunit 2 (ND2). There is evidence that this interaction occurs between amino acids 40-49 of Src and amino acids 310-321 of ND2. I have determined that a peptide consisting of amino acids 310-321 of ND2, and affixed to the HIV Tat domain for cell permeability, is capable of alleviating tactile allodynia induced by Complete Freund's Adjuvant (CFA) in rats. Src40-49Tat was not effective in two models of inflammatory pain. This work further implicates the Src-ND2 interaction in pain hypersensitivity and suggests that Tat ND2 310-321 may alleviate it.

An intriguing mystery about tryptophan 2, 3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2, 3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔEQ = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism.

3,3'-Neotrehalosadiamine (NTD) is a diaminosugar that possesses a rare alpha,beta-1,1'-linked glycosidic bond and has been reported to possess antimicrobial activity against Staphylococcus aureus. The ntdABC operon contains three structural genes that are necessary for the production of NTD in certain mutants of Bacillus subtilis. The gene predicted to be the first in the NTD biosynthetic pathway, ntdC, was subcloned into pET-28b as the hexa-histidine tagged fusion. The gene product was expressed, purified to homogeneity, and found to be an NAD+-dependent glucose 6-phosphate 3-dehydrogenase, likely operating according to a ternary complex mechanism and possessing a catalytic dyad composed by D176 and H180. The advent of this knowledge suggests that additional genes are required for the biosynthesis of NTD aside from the three encoded by the ntdABC operon.

Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (<i>Nicotiana tabacum L.</i>), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, indicating the possibility of two tandem targeting signals. In this work the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) to generate a series of constructs and introduced into tobacco leaves by <i>Agrobacterium</i>-mediated transient transfection. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined under the confocal microscope. Constructs predicted to be targeted to the chlroplast all localized to the chloroplast. However, this was not the case for constructs that were predicted to be mitochondrial targeted. While some constructs localized to mitochondria, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein which was present in those constructs. In addition, during the process of generating these constructs our sequence analysis indicated a stop codon present at amino acid position 161 of the mature NADP+-ICDH protein from both Xanthi and Petit Havana cultivars of tobacco. This was confirmed by multiple sequencing reactions and created discrepancies with the reported sequence present in the database. The results of this study raise interesting questions with regard to the targeting and processing of NADP+-ICDH.

The pursuit of agricultural sustainability is necessary to ensure global food security into the future. To achieve sustainability, production systems around the world use different approaches. Utilizing several biological and physical indicators, this study investigates two agricultural production systems and assesses how management has affected the long-term health and sustainability of the soils. The first study assessed the effect of variable intensities of tillage on three Saskatchewan soils under low-disturbance (LD) management for the ten years prior to tillage. The soils represented were in the Grey, Black and Brown soils zones at sites located near Tisdale, Rosthern and Central Butte, Saskatchewan, respectively. A completely randomized block design utilized four treatments of varying tillage intensity. Samples were taken in spring before planting and after harvest at all sites. The soils were analyzed for microbial indicators of health by assessing dehydrogenase, urease, protease, and alkaline phosphatase activities. Microbial biomass carbon (MBC), microbial biomass nitrogen (MBN) and microbial quotient nitrogen (MQN) also were analyzed. Traditional soil nutrient and physical parameters were measured. The tillage intensities affected each parameter differently likely due to the differences in litter quality at each site. The high intensity tillage treatment decreased dehydrogenase activity at Tisdale at May, while in Rosthern dehydrogenase activity was increased in the moderate intensity tillage treatment and decreased by the high intensity tillage treatment. At Central Butte no effect was detected until October when dehydrogenase activity was increased by the low and moderate tillage intensity treatments. Protease and urease activities were affected at Rosthern only where the moderate intensity tillage treatment decreased activity relative to the control treatment. Soil physical parameters were not affected by tillage intensity; however nutrient levels were impacted by the increasing tillage intensity. Specifically, NO3- was reduced at Tisdale and was increased at Rosthern. Phosphate levels were reduced by the high tillage intensity in Rosthern whereas, with increasing tillage, the opposite occurred at Tisdale and Central Butte. The responses were strongly influenced by site characteristics, especially soil zone, organic matter content and surface litter abundance and quality. These effects were short-term, having no long-term impact on the agricultural sustainability or health of the soil, although knowledge of litter condition and quality is agronomically beneficial in order to predict soil responses to intense tillage events. iii The second part of the study was to assess the success of grass terraces on preserving the soil health of hillslope farm plots with Oxisolic soils in southern Ethiopia. Soil erosion has a devastating impact on hillslope agriculture in Ethiopia causing severe land degradation. An adjacent terraced and unterraced hillslope was chosen and sampled, along with a second unterraced slope for comparison. These soils were analyzed for dehydrogenase, alkaline phosphatase, and urease activities, as well as total C and total N. The plots above the terraces [terraced upper and unterraced upper] had higher urease activities than the plots below [terraced lower and unterrraced lower]. The impact of a vegetative strip that had formed a terrace 20 years ago was still evident in consistently higher alkaline phosphatase, urease, and dehydrogenase activities than the other plots. Simple methods of erosion prevention on erosion prone hill-slopes indicated that vegetative strips leading to terracing have a positive effect on soil health and functionality, promoting the long-term agricultural productivity and sustainability of these landscapes.