Mycobacterium tuberculosis atteint le tiers de la population mondiale et cause plus de 1.5 millions de décès chaque année. L’augmentation des infections chez les patients immuno-compromis et l’émergence d’infections à de nouvelles souches multirésistantes aux antibiotiques somme la communauté scientifique à la découverte de nouvelles cibles thérapeutiques ainsi qu’au développement de nouveaux antibiotiques, vaccins et thérapies. Parmi les ~ 4000 gènes du génome de Mycobacterium tuberculosis, un d’entre eux, glbN, code pour l’hémoglobine tronquée N. Les hémoglobines sont de petites métalloprotéines qui fixent réversiblement l’oxygène et qui effectuent plusieurs activités catalytiques importantes. Ainsi, nos recherches s’inscrivent dans le cadre d’une approche biochimique qui vise à définir une fonction pour l’hémoglobine tronquée N de Mycobacterium tuberculosis à l’aide de techniques biochimiques modernes et de la spectroscopie à flux-arrêté. Nos recherches nous amènent également à résoudre la structure tridimensionnelle du complexe oxygéné de trHbN, à caractériser les interactions protéines-ligands au site actif de l’hémoglobine et à définir leurs rôles dans l’établissement du potentiel fonctionnel de l’enzyme en utilisant les spectroscopies d’absorption, de résonance Raman et de diffraction des rayons X. Nous avons découvert que l’activité rapide et efficace de détoxication aérobie du •NO de l’hémoglobine tronquée N mesurée chez Mycobacterium bovis BCG pourrait remplir un rôle similaire chez Mycobacterium tuberculosis et permettre la persistance de l’infection tuberculeuse dans l’hôte par la prévention des effets cytotoxiques associés au •NO et à ses dérivés réactifs azotés. De plus, l’architecture et la polarité du site actif de l’hémoglobine tronquée N définissent le potentiel fonctionnel de la protéine et contrôlent la diffusion, la fixation, la stabilisation, l’activation des ligands coordonnées au fer de l’hème et assurent le maintien d’une activité catalytique rapide et efficace. Finalement, nos découvertes suggèrent que l’hémoglobine tronquée N pourrait constituer une nouvelle cible thérapeutique pour le développement éventuel de drogues inhibitrices qui inactiveraient la première ligne de défense du parasite et perturberaient son adaptation métabolique face aux stress oxydatifs. / Mycobacterium tuberculosis infects over one-third of the human population, causing 1.5 millions deaths each year. The increase incidence of infections among immunocompromised patients and the emergence of strains with resistance to multiple antibiotics urge the scientific community to discover new therapeutic targets as well as develop new antibiotics, vaccines and therapies. Among the ~ 4000 genes that compose the genome of Mycobacterium tuberculosis, one of them, glbN, encodes the truncated hemoglobin N. Hemoglobins are small metalloproteins that reversibly bind oxygen and perform a wide array of important catalytic activities. Thus, our research aims at defining a function for Mycobacterium tuberculosis truncated hemoglobin N using modern biochemical techniques and stopped-flow spectroscopy. Our research also leads us to solve the three-dimensional structure of the oxygenated complex of trHbN, characterize the proteins-ligands interactions within the active site of the hemoglobin and define their roles in establishing the functional potential of the enzyme using absorption, resonance Raman and x-rays diffraction spectroscopies. We discovered that the fast and efficient •NO detoxification activity of truncated hemoglobin N measured in Mycobacterium bovis BCG could fulfill a similar role in Mycobacterium tuberculosis and allow the persistence of the tuberculous infection in the host by preventing the cytotoxic effects associated with •NO and its reactive nitrogen derivatives. Moreover, the architecture and polarity of truncated hemoglobin N active site define the functional potential of the protein and control the diffusion, binding, stabilization, and activation of the heme-iron coordinated ligands and ensure the maintenance of a fast and efficient catalytic activity. Finally, our discoveries suggest that truncated hemoglobin N could constitute a new therapeutic target for the development of inhibitors that would inactivate the first line of defence of the parasite and disturb its metabolic adaptation to nitrosative stresses.

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Call number: LD2668 .T4 1933 B72

Call number: LD2668 .T4 1955 K64 / Master of Science

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Twelve F(,1) families of alfalfa (Medicago sativa L.) plants having different potential for nitrogenase activity, and the two parental populations were tested for response to mycorrhizal inoculation in a low-phosphate soil mixture in the greenhouse. The purpose of this study was to: (a) determine the effects of vesicular-arbuscular mycorrhizae on growth, nutrition and nodulation of these 14 populations, (b) determine if differences existed between the populations with regard to several morphological characteristics, and (c) determine if certain characteristics can be transmitted across generations. The 14 populations were evaluated under four treatments: control no Myorrhizae, no Rhizobium; Rhizobium alone; Mycorrhizae alone; and the combination of Mycorrhizae and Rhizobium. The growth parameters measured differed significantly among the treatments and among the 14 populations studied, and no significant interaction between the populations and the treatments were found. The dual Mycorrhizae and Rhizobium treatment significantly increased plant height at 30 days and 60 days after planting, leaf area per plant, and plant top-dry-weight at two harvest dates. Mycorrhizal inoculation, however, decreased specific-leaf-weight significantly. Nitrogen fixation parameters such as nodule mass score, fibrous root score, and nitrogenase activity were increased significantly by the dual inoculation of mycorrhizae and rhizobium. The 14 populations differed significantly in nodule mass score, fibrous root score, and nitrogenase activity. Mycorrhizal inoculation increased nitrogen fixation more than plant growth. Correlation coefficients indicated that increased Nitrogenase activity is positively correlated with increased nodule mass, increased fibrous root mass, greater top-dry-weight, and leaf area. A step wise multiple regression showed that 49% of the variation in nitrogenase activity can be explained by the variation due to nodule mass, fibrous root mass, top-dry-weight, and leaf area. Several morphological characters showed a heritable response. Plants selected for high nitrogenase activity and high top dry weight transmitted these characteristics to their progenies.

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Global production of soybeans continues to increase, reflecting the value of soybeans in food products, soil amendments, and petroleum replacements. While the N2O aspect of the legume greenhouse gas (GHG) budget has been given substantial, and mostly negative, attention, the CO2 side needs to be explored. During nitrogen fixation, hydrogen is produced. As a result of hydrogen-coupled CO2 fixation, legume soils have the ability to increase the amount of atmospheric carbon fixed into the soil. However, the amount and fate of this fixed carbon has yet to be determined. Therefore, two experiments were conducted. The first experiment explored the methods of 13C labeling of four soil treatments over a period of 28 days. Results showed that most of this carbon uptake was through biotic processes, and that the pattern of carbon uptake was similar for all four soil treatments. The ideal labeling time for both ‘root’ and ‘nodule’ soil was determined to be between 7 and 14 days. The second experiment incubated four 13C labeled soil treatments in a continuous soybean field for 11 months. Throughout this time, bags of soil were removed and separated into three soil organic matter (SOM) fractions; the microbial biomass fraction (MBF), the light fraction (LF), and the acid stable fraction (ASF). These fractions were then measured for δ13C, providing a time series showing carbon movement through the soil which enabled an estimation of the MRT of fixed carbon in the soil. Results showed that the MBF was responsible for the initial flux of carbon uptake in all soil treatments, and that the ‘nodule + H2’ MBF continued to fix carbon at the end of the 11 months. While the LF was not ideal at representing the intermediate SOM pool, the ASF did increase in enrichment at the end of the experiment, showing the movement of newly fixed carbon into long-term carbon storage. Therefore, hydrogen-coupled CO2 fixation does contribute to long-term carbon storage in legume soils, however, longer study times with focus on different legume plants and soil types are required to determine the mean residence time (MRT) of fixed carbon in the ASF. / Thesis (Master, Geography) -- Queen's University, 2013-01-30 09:28:01.899

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