Caractérisation biochimique et biophysique des hémoglobines 2/2 HbN et HbO de mycobacterium tuberculosis

Le génome de l'agent étiologique de la tuberculose humaine, Mycobacterium tuberculosis, a été séquencé complètement et l' analyse a révélé l'existence de deux gènes, glbN et glbO, codant respectivement pour les hémoglobines 2/2 Mt-HbN et Mt-HbO. Chez M bovis BCG, l'homologue de Mt-HbO est exprimé de façon constitutive tandis que l'homologue de MtHbN est exprimé seulement en phase stationnaire, suggérant des fonctions différentes. MtHbN fixe l'O2 avec une grande affinité (8 nM) et possède une cavité optimisée pour la dioxygénation du NO en N03. En effet, les travaux présentés au chapitre 2 ont permis de démontrer, par inactivation génique, complémentation et spectrophotométrie ± stopped-flow ¿ que Mt-HbN possède une activité NO-dioxygénase très efficace et capable de protéger la respiration cellulaire contre les effets toxiques du NO. Mt-HbO possède, en plus d'une tyrosine en BIO, une tyrosine en CD 1 et un tryptophane en G8. Les chapitres suivants (3 - 5) décrivent mes travaux portant sur la caractérisation bio~himique et biophysique de Mt-HbO, et plus spécifiquement les réactions avec les ligands gazeux O2, CO et -NO et le H20 2. Par spectrophotométrie ± stopped-flow ¿, nous avons déterminé les constantes d'association et de dissociation de ligands gazeux chez la protéine sauvage ainsi que chez les variants Y(B1O)F, Y(CD1)F et W(G8)F. Les cinétiques d'association et de dissociation des ligands sont complexes et modulées par ces trois résidus distaux. Toutefois, le remplacement des résidus Y(CD1) et W(G8) montre davantage d'impact que celui de la Y(B10) avec, par exemple, des augmentations respectives de 25 à 115 fois pour la constante d'association de l'O2. De plus, des analyses de spectroscopie de résonance Raman (chapitre 4) ont démontré, en accord avec la structure cristalline, la participation des résidus Y(CD1) et W(G8) dans la formation d'un réseau complexe de ponts H stabilisant l'O2 lié au fer de l'hème. Le chapitre 5 décrit les travaux publiés portant sur la réaction entre la forme férrique de Mt-HbO et le H2O2. Cette étude a permis de constater que Mt-HbO possède une activité peroxydase significative, impliquant vraisemblablemnt des espèces spectrales différentes comparées à d'autres peroxydases très connues. Ces dernières observations ainsi que les fortes interactions avec 1'02 suggèrent que Mt-HbO et les autres trHbs du groupe II soient capables d'activer l'O2 et de catalyser des réactions d'oxydo-réduction de types peroxydase et oxygénase

Pulse-shape discrimination against low-energy Ar-39 beta decays in liquid argon with 4.5 tonne-years of DEAP-3600 data

The DEAP-3600 detector searches for the scintillation signal from dark matter particles scattering on a 3.3 tonne liquid argon target. The largest background comes from 39Ar beta decays and is suppressed using pulse-shape discrimination (PSD). We use two types of PSD estimator: the prompt-fraction, which considers the fraction of the scintillation signal in a narrow and a wide time window around the event peak, and the log-likelihood-ratio, which compares the observed photon arrival times to a signal and a background model. We furthermore use two algorithms to determine the number of photons detected at a given time: (1) simply dividing the charge of each PMT pulse by the mean single-photoelectron charge, and (2) a likelihood analysis that considers the probability to detect a certain number of photons at a given time, based on a model for the scintillation pulse shape and for afterpulsing in the light detectors. The prompt-fraction performs approximately as well as the log-likelihood-ratio PSD algorithm if the photon detection times are not biased by detector effects. We explain this result using a model for the information carried by scintillation photons as a function of the time when they are detected

Cholesterol Degradation Pathways as Therapeutic Targets in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen responsible for about 2 million deaths worldwide annually. During infection, Mtb faces the nutrient-deficient environment of the macrophage phagosome and switches to lipids, including cholesterol, as its main source or carbon and energy. Extensive work has been done on the cholesterol AB rings degradation, but much less is known about the degradation of the CD rings and aliphatic side-chain, which are thought to be shortened by β-oxidation reactions. In this study, we investigate the role of four fadD (fadD3, fadD17, fadD19 and fadD36) and eight fadE (fadE26-fadE33) genes, encoding for probable acyl-CoA ligases and acyl-CoA dehydrogenases, respectively. To elucidate the catalytic function and determine the essentiality/redundancy of these genes in the cholesterol degradation pathways, we will generate deletion mutant strains by homologous recombination using specialized transducing mycobacteriophages. Allelic exchange substrate (AES) at specific loci will be constructed by amplifying the flanking regions by PCR from Mtb genomic DNA, followed by their insertion into two alternative recombineering vectors (pMSG360 and pJSC407). The recombinant vectors will be converted into thermosensitive phage particles in non-pathogenic Mycobacterium smegmatis cells at the permissive temperature of 30 °C. High-titer solutions of specialized transducing mycobacteriophages will then be used to infect Mtb cells at the non-permissive temperature of 39°C, and deletion mutant clones will be obtained after 3-4 weeks of incubation at 37 °C. Progress in the construction of AES will be presented

Data from: Caribou avoiding wolves face increased predation by bears – caught between Scylla and Charybdis

1. Prey may trade off resource acquisition with mortality risk by using various habitat-selection strategies. Empirical assessments have shown that the functional and numerical responses of predators to human disturbances are variable, yet spatial changes in predation risk by two predators have seldom been studied for prey occurring in human-modified landscapes. Using the boreal caribou (Rangifer tarandus caribou) – grey wolf (Canis lupus) – black bear (Ursus americanus) system in eastern Canada, we investigated whether responses of prey towards one predator could concomitantly increase risk of predation from another predator exhibiting a different foraging tactic. 2. We investigated trade-offs made by solitary caribou females and mothers accompanied by their calf during the period of highest calf vulnerability, and compared the behaviour of mothers that would eventually lose their calf to predation to that of mothers whose calf survived until the following year. We modelled habitat selection using different metrics of forage based on field measurements and digital maps, and developed empirical models of predation risk and prey behaviour using GPS data collected on both predators and prey. 3. Mothers accompanied by their calf seemed to compromise foraging opportunities for safety, as opposed to solitary females who showed no particular avoidance of areas used by predators. Although caribou mothers adopted selection strategies that could have protected their offspring from wolves, females that eventually lost their calf to predation selected for vegetative associations that were favourable to bears. 4. Synthesis and applications. We determined that mothers that most strongly avoided suitable wolf habitat were also those that most strongly selected suitable bear habitat, suggesting that by using anti-predator strategies aimed at reducing predation risk from wolves, caribou exposed their offspring to increased predation risk from bears. This result is of paramount conservation value as bears were responsible for 94% of caribou calf kills in this system. In the short term, conservation efforts for boreal caribou may benefit from the management of bear populations by means of liberal hunting regulations or predator control. In the long term, however, these actions should be used in conjunction with the protection of potential calving areas away from cutblocks and roads

Cyanide binding to truncated hemoglobins: a crystallographic and kinetic study

Cyanide is one of the few diatomic ligands able to interact with the ferric and ferrous heme-Fe atom. Here, the X-ray crystal structure of the cyanide derivative of ferric Mycobacterium tuberculosis truncated hemoglobin-N (M. tuberculosis trHbN) has been determined at 2.0 A (R-general = 17.8% and R-free = 23.5%), and analyzed in parallel with those of M. tuberculosis truncated hemoglobin-O (M. tuberculosis trHbO), Chlamydomonas eugametos truncated hemoglobin (C. eugametos trHb), and sperm whale myoglobin, generally taken as a molecular model. Cyanide binding to M. tuberculosis trHbN is stabilized directly by residue TyrB10(33), which may assist the deprotonation of the incoming ligand and the protonation of the outcoming cyanide. In M. tuberculosis trHbO and in C. eugametos trHb the ligand is stabilized by the distal pocket residues TyrCD1(36) and TrpG8(88), and by the TyrB10(20) - GlnE7(41) - GlnE11(45) triad, respectively. Moreover, kinetics for cyanide binding to ferric M. tuberculosis trHbN and trHbO and C. eugametos trHb, for ligand dissociation from the ferrous trHbs, and for the reduction of the heme-Fe(III)-cyanide complex have been determined, at pH 7.0 and 20.0 degrees C. Despite the different heme distal site structures and ligand interactions, values of the rate constant for cyanide binding to ferric (non)vertebrate heme proteins are similar, being influenced mainly by the presence in the heme pocket of proton acceptor group(s), whose function is to assist the deprotonation of the incoming ligand (i.e., HCN). On the other hand, values of the rate constant for the reduction of the heme-Fe(III)-cyanide (non)vertebrate globins span over several orders of magnitude, reflecting the different ability of the heme proteins considered to give productive complex(es) with dithionite or its reducing species SO(2)(-). Furthermore, values of the rate constant for ligand dissociation from heme-Fe(II)-cyanide (non)vertebrate heme proteins are very different, reflecting the different nature and geometry of the heme distal residue(s) hydrogen-bonded to the heme-bound cyanide