Long-distance translocation (LDT), the relocation of an animal outside of its home range, is a popular strategy for mitigating conflict between humans and venomous snakes. While LDT has been demonstrated to prevent a snake’s return to the location of capture, it may result in increased mortality, magnitude and frequency of movements, and activity range sizes. Thus, it has generally been discouraged. However, the effects of LDT on stress physiology and thermoregulation have gone largely unstudied in reptiles. To elucidate these effects, we conducted an experimental LDT on Western Rattlesnakes (Crotalus oreganus) on Vandenberg Air Force base in California. Fourteen snakes were monitored, beginning in mid July 2012 and ending in early September. Each was implanted with a radio transmitter and iButton temperature data logger within the coelomic cavity. In late August, seven snakes were translocated to similar habitat, approximately 30 kilometers away, where they were monitored for 9-13 days. Prior to translocation, all snakes were tracked every other day, while after translocation all snakes were tracked every day. A ‘Before-After Control-Impact’ (BACI) experimental design was used, with a dedicated control group, but also with the translocated group serving as control prior to the act of translocation. We collected data on snake body temperatures (T­b) and temperatures (T­e) of physical operative temperature models (OTMs) that simulated non-thermoregulating snakes and allowed for a comparison of habitat thermal quality between our two study sites. Together, T­b and T­e allowed for a formal assessment of thermoregulatory effectiveness. Additionally, blood concentrations of corticosterone (CORT), the primary stress hormone in reptiles, and testosterone (T), a metric of male reproductive ability that is often negatively associated with CORT, were assayed just prior to translocation and again at the end of the study. During each of the two sampling periods, in addition to baseline hormone concentrations, stressed hormone concentrations were assayed following the application of an acute stressor (the baseline blood draw plus one hour’s captivity in a plastic bucket). We also studied the effect of LDT on the CORT and T response (stressed concentration minus baseline concentration). Furthermore, we evaluated how LDT impacted a suite of behaviors related to defense and movement, as well as snake body mass and body condition index (BCI). Finally, we assessed the effects of LDT on movements and spatial use (activity range size). We sought to assess the effects of LDT on movements, spatial use, and behavior in order to facilitate comparison with other translocation studies, as well as to evaluate those impacts in a physiological context. In addition to assessing the impact of LDT on CORT and T separately, we evaluated a relationship between the two steroid hormones, and, using a model selection approach, we evaluated relationships between CORT and T and movements and spatial use. The thermal quality of the habitat at our two sites did not differ and we found no effect of LDT on snake body temperature or thermoregulatory effectiveness. We found that spatial use increased following LDT, since the average 100% minimum convex polygon (MCP) activity range size of our translocated snakes was greater than that of our control snakes during the post-translocation period. However, movements (mean distance moved per day and total distance moved) were unaffected by LDT. Translocation was not found to affect snake body mass or BCI, indicating that snake energy demands did not increase as a result of LDT. Of the behaviors that we recorded, many (rattling, tongue-flicking, fleeing, moving vs. stationary when found) were observed too infrequently to carry out satisfactory parametric analyses, though a qualitative assessment leads to the conclusion that LDT did not affect them. The effect of LDT on how often our snakes were visible was marginally significant, but post-hoc testing found no differences. Nonetheless, the translocated snakes tended to be visible more often than the controls, during the post-translocation period. We found no effect of LDT on the percentage of a snake’s body sunlit when visible. Our translocation was not found to any CORT metric, while for T, the only metric that was affected was the baseline concentration. Baseline T was found to be higher in translocated snakes than in control snakes following translocation. We found some evidence for a positive relationship between CORT and T for baseline and stressed concentrations. Our model selection procedure found little evidence for a relationship between snake movements and spatial use and either CORT or T. Our results are encouraging in that we did not find that LDT disrupts thermoregulation or results in a condition of chronic stress, as indicated by the CORT results. The positive effect of LDT on baseline T is somewhat mysterious, but studies on mammals suggest that increased T may aid in spatial learning and memory. Since the site that a snake is translocated to is entirely novel, an elevated capacity for spatial learning and memory could be beneficial to a translocated snake. Our finding that spatial use increased following LDT agrees with other studies of LDT in snakes, but some studies have found that movements increased following LDT, while we did not. In addition to snake movements being unaffected, we translocated our snakes within a relatively thermally benign climate in coastal central California. Translocations carried out in more extreme climates, with either cold winters or hot summers may indeed result in an effect on thermoregulation and a state of chronic stress. Consequently, we advocate further study of the physiological effects of LDT on other snake species in a variety of climates. Additionally, while it was our goal to study the short-term effects of LDT, more long-term studies, which follow the snakes through the entire active season and the winter hibernation period, may be informative.

The hippocampus of birds and mammals has been shown to play a crucial role in spatial memory and navigation. The hippocampus exhibits plasticity in adulthood in response to diverse environmental factors associated with spatial demands placed on an animal. The cortical telencephalon of squamate reptiles has been implicated as a functional homologue to the hippocampus. This study sought to experimentally manipulate the navigational demands placed on free-ranging northern Pacific rattlesnakes (Crotalus o. oreganus) to provide direct evidence of the relationship between spatial demands and neuroplasticity in the cortical telencephalon of the squamate brain. Adult male rattlesnakes were radio-tracked for two months, during which one of three treatments was imposed weekly: 225 meter translocation in a random direction, 225 meter walk and release at that day’s capture site (handling control), and undisturbed control. Snakes were then sacrificed and brains were removed and processed for histological analysis of cortical features. The volume of the medial cortex was significantly larger in the translocated group compared to undisturbed controls. No differences in dorsal or lateral cortical volume were detected among the groups. Numbers of 5-Bromo-2’-deoxyuridine (BrdU) -labeled cells in the medial and dorsal cortices three weeks after BrdU injection were not affected by treatment. The activity range was larger in the translocated group compared to handled and undisturbed controls. A causal relationship between increased navigation in a free-ranging reptile and changes in brain morphology was established. The use of translocation as a conservation strategy for reptiles is a controversial topic revisited many times. Previous studies have demonstrated the aberrant movement patterns and mortality caused by translocation and have established that short-distance translocation within an animal’s home range is best for the animal. In conjunction with the neuroplasticity study, we examined the physiological impacts that repeated short-distance translocation and handling have on reptiles. This is essential knowledge if the efficacy of the technique is to be properly evaluated. Baseline and stressed concentrations of corticosterone and testosterone were assayed in blood taken immediately upon capture and following one hour of confinement in a bucket. Neither baseline nor stressed concentrations of either hormone were impacted by translocation or handling. Body condition and change in mass were not affected. Translocated animals had larger MCP activity ranges than handled and undisturbed animals at the 95%, but not 100% levels, while an interaction between time and treatment impacted other movement parameters.Treatment had no effect on a number of behaviors observed during visits to each animal. We suggest that rattlesnakes are quite resistant to potential impacts on their physiology and behavior enacted by frequent short-distance translocation or handling. Additionally, studies that require frequent handling of reptilian subjects are not likely to severely alter stress physiology.

Bispyribac-sodium is an acetolactate synthase (ALS) inhibiting herbicide introduced for control of grasses, broadleaves, and sedges in rice and turf. However, Mississippi and Arkansas rice producers have reported inconsistent barnyardgrass (Echinochloa crus-galli Beauv.) control with bispyribac-sodium. Experiments were conducted to determine if inconsistent barnyardgrass control with bispyribac-sodium could be alleviated through adjuvant technology. Absorption of 14C-bispyribac-sodium was greatest with a proprietary blend of methylated seed oil/organosilicone (MSO/OSL) (0.37 L ha-1) plus urea ammonium nitrate (UAN) (2% v/v) and the proprietary blend of MSO/OSL/UAN (2% v/v) at 80 and 74% of applied 14C-bispyribac-sodium, respectively. The addition of 32% UAN to MSO/OSL and non-ionic organosilicone (OSL/NIS) adjuvant systems resulted in a four- to fiveold increase in absorption compared to treatments without UAN. Maximum absorption was achieved 12 h after application and therefore, bispyribac-sodium should be rainfast at this time. MSO/OSL adjuvants supplied from multiple manufacturers were also examined to determine which provided the highest levels of 14C-bispyribac-sodium absorption as well as herbicidal efficacy and rainfastness. The addition of Rivet® plus UAN or Sil-MES 100® plus UAN to 14C-bispyribac-sodium resulted in the highest levels of absorption among adjuvants supplied. Bispyribac-sodium applied with no adjuvant or with 32% UAN only resulted in significant reductions in control compared to MSO/OSL adjuvant formulations. Addition of MSO or MSO/OSL adjuvants and UAN to bispyribac-sodium resulted in greater than 85% freshweight reduction of barnyardgrass. Bispyribac-sodium applied with MSO or Dyne-A-Pak® was rainfast by two and four hours after application, respectively. Addition of Sil-MES 100® plus UAN or Inergy® plus UAN to bispyribac-sodium reduced the rainfastness interval to 30 minutes and one hour after herbicide application, respectively. Further experiments were conducted to determine if individual components of 32% UAN as well as ammonium sulfate (AMS) would provide control similar to that of 32% UAN. The addition of ammonium chloride, calcium nitrate, and ammonium sulfate at selected rates provided barnyardgrass control similar to that of 32% UAN.

No description available.

Les translocations chromosomiques du gène MLL sont connues pour mener au développement de leucémies aiguës. La translocation avec un de ses partenaires de fusion les plus communs, ENL, peut engendrer des leucémies aiguës de plusieurs types différents pour cette même translocation. Une fois la leucémogenèse initiée par la fusion MLL-ENL, son rôle quant au maintien du phénotype leucémique n’est pas encore bien connu à ce jour. Pour mieux comprendre l’importance de MLL-ENL après la leucémogenèse, des cellules souches/progénitrices de sang de cordon ombilical humain purifiées ont ainsi été transduites par un virus exprimant le gène de fusion MLL-ENL bordé par des sites LoxP ainsi que le marqueur eGFP. Ces cellules infectées ont ensuite été injectées dans notre modèle de souris immunodéficientes irradiées et placées sous observation pendant 24 semaines pour voir le développement de leucémies aiguës. Elles ont alors été sacrifiées et les cellules la moelle osseuse et de la rate ont ensuite été analysées par cytométrie en flux pour déterminer si la xénogreffe a engendré une leucémie dans notre modèle ainsi que le phénotype de celle-ci. Les souris injectées par les cellules infectées par le MLL-ENL ont généré uniquement des leucémies lymphoïdes aiguës de type B. Les cellules de ces leucémies primaires isolées ont été par la suite infectées par un lentivirus exprimant la cre-recombinase et le marqueur BFP afin d’exciser le gène MLL-ENL des cellules leucémiques grâce aux sites LoxP. Les cellules ont ensuite été triées pour le marqueur BFP et injectées dans des souris secondaires pour de voir si les cellules leucémiques souches pouvaient toujours régénérer la leucémie. Les conséquences de l’absence de MLL-ENL dans le maintien du phénotype leucémique n’ont cependant pas pu être vérifiées à cause d’une erreur dans la séquence de la cre-recombinase, mais nous avons observé la régénération des leucémies secondaires. / Chromosome translocations of the MLL gene which fusions with different other genes in hematopoietic stem and progenitors cells are well characterized and known to induce the development of acute leukemias. Translocation with one of its most common fusion partner, ENL, induces different acute leukemias but once the leukemia is fully developped, the role of MLL-ENL regarding the maintenance of the leukemic phenotype/disease is still uncertain. To get a better understanding of MLL-ENL’s long term role in leukemias, stem cells and progenitor cells from human umbilical cord blood purified by negative selection have been transduced by a virus expressing the fusion gene MLL-ENL flanked by LoxP sites and the eGFP marker. These infected cells were then transplanted by intra-femoral injection in sub-lethally irradiated immunodeficient mouse and observed for 24 weeks to see the development of acute leukemias. They were then euthanized and bone marrow/spleen cells were analyzed by flow cytometry to determine whether the xenograft generated leukemia and determine their phenotype. Mice injected with MLL-ENL transduced cells only generated type B acute lymphoblastic leukemias in our model. The primary leukemia cells isolated were subsequently infected with a lentivirus expressing the cre recombinase and a BFP marker, aiming to excise the MLL-ENL gene of leukemic cells through the LoxP sites. The cells were then sorted for the BFP marker and injected into secondary mouse to see if the leukemic stem cells could still regenerate leukemia. Due to an error on the sequence of the cre recombinase, the outcomes of MLL-ENL removal could not be verified, but we still managed to regenerate secondary leukemias in our model.

Field experiments were conducted in Virginia to evaluate the efficacy of AEF-130060 03 plus AEF-107892 applied postemergence (POST) for the control of Italian ryegrass [Lolium multiflorum (Lam)] in barley [Hordeum vulgare (L.)] in comparison to other herbicides currently registered for use in wheat [Triticum aestivum (L.)] and barley. Laboratory experiments were also conducted to evaluate absorption, translocation and metabolism of AEF-130060 03 plus AEF-107892 in wheat, barley, and Italian ryegrass with or without the addition of dicamba. AEF-130060 03 plus AEF-107892 was applied alone at three POST timings. All of these applications were effective in controlling Italian ryegrass. The third application timing of AEF-130060 03 plus AEF-107892 commonly resulted in lower yields than the first or second application timing, due to increased duration of Italian ryegrass competition, increased barley injury and insufficient time for barley recovery from this injury. In the field experiment, significantly greater barley injury was observed when AEF-130060 03 plus AEF-107892 was combined with 2,4-D and dicamba. Early postemergence (EP) treatments of AEF-130060 03 plus AEF-107892 provided control of Italian ryegrass equivalent to that of delayed preemergence (DPRE) applications of flufenacet plus metribuzin when rainfall was received. However, when rainfall was not received AEF-130060 03 plus AEF-107892 provided superior control. Because the efficacy of AEF-130060 03 plus AEF-107892 is not dependant on rainfall, it should provide more consistent control of Italian ryegrass than DP treatments. Laboratory experiments indicated that Italian ryegrass absorbed greater than 2.5, 2.0, and 1.5 times the amount of applied radioactivity at 24, 48, and 96 hours after treatment (HAT), respectively, than wheat or barley. Metabolism experiments indicated that quantity of parent compound in the three species was greatest in Italian ryegrass, followed by barley and then wheat. However, the sum total of metabolites was not different between species. A higher rate of metabolism of AEF-130060 03 plus AEF-107892 was also observed in wheat and barley than in Italian ryegrass. Thus, lower absorption of herbicide by wheat and barley, coupled with a more rapid rate of metabolism, most likely accounts for differential selectivity between these plant species. No differences in absorption, translocation or metabolism were observed within the three plant species due to the addition of dicamba. An additional experiment was conducted in 2000 and 2001 to evaluate the efficacy of preemergence (PRE) and EP applications of DPX-R6447 for the control of Italian ryegrass in wheat and barley in comparison to other herbicides currently registered for use in these crops. Barley and wheat injury and yield were similar with treatments of DPX-R6447 at rates below 176 g ai/ha and treatments of flufenacet plus metribuzin applied alone in both years. Consistent Italian ryegrass control with DPX-R6447 occurred only with rates of 176 g ai/ha or greater in both years. However, these rates resulted in variable injury in both wheat and barley between years. Rates higher than 176 g ai/ha of DPX-R6447 resulted in unacceptable barley and wheat injury. The lack of consistency with regard to barley and wheat injury could limit the utility of this compound in these crops. Field trials were conducted in Virginia to evaluate herbicide programs for control of Italian ryegrass in no-till corn [Zea mays (L.)] establishment. Herbicide programs using transgenic corn hybrids were compared to standard programs that utilize non-selective herbicides in combination with high rates of triazine herbicides. Italian ryegrass control and corn yields similar to those provided by standard programs could be attained through the use of paraquat plus atrazine, and glyphosate applied in combination with atrazine or rimsulfuron plus thifensulfuron-methyl. In glyphosate-tolerant corn, EP applications of glyphosate controlled Italian ryegrass, but yield did not differ from yields of corn treated with standard PRE applications of glyphosate plus atrazine. The use of glufosinate, imazethapyr plus imazapyr, or sethoxydim with appropriate herbicide-tolerant hybrids did not demonstrate potential for improved control of Italian ryegrass. Field experiments were also conducted to investigate the incidence and severity of maize chlorotic dwarf virus (MCDV) and maize dwarf mosaic virus (MDMV) in response to POST johnsongrass control in two corn hybrids. Recent observations have indicated a lack of virus-tolerance in glyphosate-tolerant corn hybrids in Virginia. The rapidity of virus disease development in corn resulting from application of glyphosate or nicosulfuron was also investigated. The virus-susceptible glyphosate-tolerant hybrid developed significantly higher levels of virus incidence three weeks after treatment than the virus-tolerant, non-transgenic hybrid, and virus incidence and severity increased throughout the duration of the growing season. Little or no disease incidence occurred in the virus-tolerant hybrid. The virus-susceptible hybrid exhibited significant increases in disease incidence in response to any herbicide treatment applied to johnsongrass-containing plots relative to the same treatment applied to weed free plots. Johnsongrass control with nicosulfuron or glyphosate caused similar disease incidence and severity in the virus-susceptible hybrid, regardless of application method. Results of these experiments indicated that growers' choice of hybrid should focus primarily on disease resistance rather than herbicide resistance. / Ph. D.

Isoxaben is a preemergence herbicide used for broadleaf weed control in turf and ornamentals. Although isoxaben can be used on a number of ornamentals, certain species are injured by isoxaben applications. The objectives of this research were: a) to evaluate the tolerance of ajuga, wintercreeper and dwarf burning bush to isoxaben applications, b) to compare the absorption, translocation and metabolism of isoxaben following root and shoot application in these ornamentals, and c) to examine the effect of isoxaben on glucose incorporation in the roots of these species. Greenhouse and lathhouse studies demonstrated that ajuga was the most sensitive species compared to wintercreeper and dwarf burning bush following root and shoot exposure to isoxaben at 0.84, 1.69 and 3.39 kg ai/ha. Following root and shoot application, isoxaben at 3.39 kg/ha caused approximately 50% shoot injury in ajuga at 2 months after treatment compared to approximately 30% in dwarf burning bush in sand culture. Wintercreeper was not visually injured by any isoxaben rate. Isoxaben at 3.39 kg/ha reduced wintercreeper root weight by 15% following root application and shoot weight by 10% following shoot application. Field studies showed that isoxaben applications made one month after bud-break caused 30 to 45% injury to dwarf burning bush. However, the plants outgrew the injury in the following year. Dwarf burning bush was not injured from applications of isoxaben made at the dormant stage or two months after the bud-break stage. Studies with root-applied radiolabeled isoxaben showed that ajuga and dwarf burning bush had absorbed 34 and 41% of the applied radioactivity, respectively, while wintercreeper had absorbed only 21% at 14 days after treatment (DAT). The percent of absorbed radioactivity which translocated was greater in ajuga (58%) and wintercreeper (50%) than in dwarf burning bush (28%). In the root extracts, metabolism of isoxaben was greater in ajuga than wintercreeper or dwarf burning bush at 3, 7 and 14 DAT. Most of the radioactivity recovered from the shoots of the three species appeared to be polar metabolites of isoxaben, possibly conjugates. In studies with shoot-applied radiolabeled isoxaben, radioactivity recovered from the treated leaf of ajuga increased from 46% of applied at 3 days to 64% at 14 days after treatment. In wintercreeper, the most tolerant species, approximately 40% of the applied radioactivity was recovered in the treated leaf at each harvest date. Radioactivity recovered from the treated leaflet increased from 45 at 3 DAT to 70% at 14 DAT in both growth stages of dwarf burning bush. Ajuga and wintercreeper metabolized isoxaben faster than dwarf burning bush. There was no difference in the metabolism of isoxaben between the two growth stages of dwarf burning bush. Incorporation of glucose in the roots of wintercreeper and dwarf burning bush was not inhibited by isoxaben (1 mM). Approximately 10% inhibition of glucose incorporation by isoxaben was observed in the roots of the sensitive species ajuga. / Ph. D.

Les systèmes de sécrétion de NO3– de la membrane plasmique des cellules végétales jouent un rôle important dans l'activité stomatique et la réponse des plantes à des stress biotiques et abiotiques. Malgré quelques avancées récentes, ces systèmes restent mal connus sur le plan moléculaire. Mon travail de thèse a consisté à caractériser le rôle physiologique de NAXT2, un membre du sous-groupe NAXT (NitrAte eXcretion Transporter) de la famille des transporteurs NRT1/PTR chez Arabidopsis thaliana. Mes résultats montrent que NAXT2 est un transporteur de NO3– et qu'il est principalement exprimé dans les cellules du péricycle de la racine, au voisinage des vaisseaux xylémiens. Sous contrainte saline et comparativement aux plantes sauvages (WT), un mutant knock-out pour NAXT2 (naxt2-1) présente un défaut de distribution du NO3– vers les feuilles et de sécrétion du NO3– dans la sève xylémienne, qui se traduisent par une diminution des teneurs en NO3– foliaires. NAXT2 est donc impliqué dans la charge du xylème en NO3– sous contrainte saline. Aucune des différences phénotypiques mises en évidence entre le mutant et WT sous contrainte saline n'a été observée sous stress osmotique ou en condition standard, suggérant que NAXT2 est essentiellement impliqué dans la réponse à la composante ionique du stress salin. Enfin, après un traitement salin prolongé, la biomasse foliaire de naxt2-1 est inférieure à celle de WT, ce qui indique que NAXT2 joue un rôle important dans l'adaptation des plantes aux contraintes salines modérées. Dans l'ensemble, ce travail suggère que NAXT2 est impliqué dans une fonction physiologique majeure, la translocation du NO3–, point de contrôle de la distribution du nitrate, et dans l'adaptation de la plante aux contraintes salines. / NO3– secretion systems at the plasma membrane of plant cells play an important role in stomata activity and plant response to biotic and abiotic stresses. Despite of few recent advances, these systems are still poorly known at the molecular level. During my thesis, I worked on the characterization of the physiological role of NAXT2, a member of the NAXT (NitrAte eXcretion Transporter) sub-group from the large NRT1/PTR transporters family in Arabidopsis thaliana. The results presented here show that NAXT2 is a NO3– transporter and that it is mainly expressed in root pericycle cells, close to the xylem vessels. Under salinity constraint and relatively to wild type plants (WT), a NAXT2 knock-out mutant (naxt2-1) displayed a defect in NO3– distribution towards the shoots and in NO3– secretion into the xylem sap, which lead to a decrease in shoot NO3– content. Thus, NAXT2 is involved in NO3– xylem loading under salinity constraint. None of the phenotypic differences described in this work between WT and mutant was observed under osmotic stress or standard culture conditions, suggesting that NAXT2 is specifically involved in response to the ionic component of salt stress. Finally, after a prolonged salt treatment, naxt2-1 shoot biomass was lower than that of WT, indicating that NAXT2 plays an important role in plant adaptation to mild salinity constraint. Altogether, this work suggests that NAXT2 is involved in a major physiological function, the NO3– translocation, control point of nitrate distribution and in plant adaptation to salinity constraint.

Lors d’une infection, les polynucléaires neutrophiles phagocytent l’agent pathogène et le détruisent grâce à la production de formes réactives de l’oxygène (FRO) par la NADPH oxydase. Cette enzyme est constituée de sous-unités membranaires (Nox2, p22phox) et cytosoliques (p67phox, p47phox,p40phox, Rac) qui s’assemblent soit à la membrane plasmique, lors de l’activation des cellules par un stimulus soluble comme le fMLF, soit à la membrane du phagosome, lors de la phagocytose de particules. La régulation de la NADPH oxydase implique divers facteurs comme la signalisation calcique et les lipides, notamment les phospholipides anioniques. En effet, il a été montré que l’activation et la translocation de la petite protéine G Rac peuvent être dépendantes du calcium. D’autre part les sous unités Rac et p47phox peuvent interagir avec les phospholipides anioniques tels que la phosphatidylsérine,grâce à des interactions stéréosélectives et/ou électrostatiques.L’objectif de ce travail est donc d’évaluer le rôle du calcium et de la phosphatidylsérine dans le recrutement de p47phox et/ou Rac lors de l’assemblage de NADPH oxydase. Pour suivre la dynamique des deux protéines, nous avons exprimé ces sous-unités marquées avec des protéines fluorescentes dans des lignées phagocytaires mimant les neutrophiles (HL-60 et PLB-985). Nous avons ainsi pu suivre, par vidéomicroscopie, le déplacement des sous-unités marquées lors d’une stimulation par fMLF ou PMA etdurant la phagocytose de particules opsonisées. Après stimulation par fMLF, Rac1 transloque du cytosol à la membrane plasmique, alors que le mutant constitutivement actif de Rac1 est constamment localisé àla membrane plasmique, indépendamment de la stimulation par fMLF. De plus après stimulation parPMA, le mutant constitutivement actif de Rac2 transloque à la membrane plasmique, suggérant que sa translocation pourrait être possible en absence d’un influx de calcium extracellulaire. Lors de la phagocytose, en masquant la phosphatidylsérine avec le domaine C2 discoïdine de la lactadhérine qui lie spécifiquement ce phospholipide, nous avons pu montrer que la phosphatidylsérine régule la production initiale des FRO en favorisant le recrutement de p47phox et de Rac2 au phagosome. De plus, ces deux sous-unités se détachent du phagosome alors que la production intraphagosomale de FRO continue,suggérant que leur départ n’est pas un signal de terminaison pour l’activité oxydase. Plus précisément,p47phox et Rac2 sont recrutées de manière transitoire, pendant seulement 1 à 3 minutes après la fermeture du phagosome. Ceci est en accord avec le modèle qui propose que p47phox servirait principalement à transporter p67phox au phagosome, et que les deux sous-unités p47phox et Rac2 faciliteraient le positionnement de p67phox dans le complexe membranaire. / During phagocytosis, neutrophils internalize pathogens in a phagosome and kill them through the production of reactive oxygen species (ROS) by the NADPH oxidase enzyme. The cytosolic NADPHoxidase subunits (p67phox p47phox, p40phox, Rac2) and the membranous subunits (Nox2, p22phox) assemble either at the plasma membrane after stimulation with soluble agonist, or at the phagosomal membrane during particle phagocytosis. The regulation of this enzyme involves several actors like calciumsignalling and anionic phospholipids. Actually Rac activation and translocation was found to be calciumdependent and, on the other hand, p47phox and Rac can interact with anionic phospholipids such asphosphatidylserine through stereoselective and/or electrostatic interactions.Therefore we wanted to investigate the role of calcium and phosphatidylserine in p47phox and Rac membrane recruitment. To study this dynamic, we expressed the subunits tagged with a fluorescent protein in neutrophil-like cells (HL-60 and PLB-985), and followed them by videomicroscopy duringfMLF or PMA stimulation or during phagocytosis of opsonised particles. After fMLF stimulation, Rac1translocated from the cytosol to the plasma membrane whereas constitutively active form of Rac1 was permanently located at the plasma membrane, independently of fMLF stimulation. In addition, afterPMA treatment, constitutively active form of Rac2 translocated to the plasma membrane, suggesting that its translocation could occur without extracellular calcium entry. By using the specific phosphatidylserine binding discoïdine C2 domain of lactadherin, we could mask this phospholipid and found that phosphatidylserine is involved in NADPH oxidase activity by participating in the phagosomal recruitment of p47phox and Rac2. In addition we show that these two subunits detached from the phagosome while ROS production continued for a longer period, suggesting that their dissociation from the complex is not a termination signal for oxidase activity. More precisely, p47phox and Rac2 were briefly recruited to the phagosomal membrane, for 1 to 3 minutes after the phagosome closure. These results support the model in which p47phox serves as a carrier for p67phox and both p47phox and Rac2 are adapters that correctly position p67phox in the complex.

Les translocations chromosomiques qui consistent en l’échange de morceaux de chromosomes sont une des caractéristiques génétiques de nombreux cancers. Les séquences des jonctions des chromosomes transloqués chez les patients correspondent à une réparation par NHEJ. Nous avons étudié le rôle du complexe de ligation XRCC4/LigaseIV du C-NHEJ dans la formation de ces réarrangements chromosomiques dans les cellules humaines. Nous avons utilisé différentes nucléases artificielles (ZFN, TALEN, et CRISPR/Cas9) afin d'introduire deux CDB sur deux chromosomes et nous avons ainsi réussi à générer différentes translocations. Des lignées sauvages et mutantes pour ce complexe de ligation ont été utilisées et la fréquence formation de translocations a été quantifiée par PCR. Nous avons pu observer que celle-ci est souvent diminuée dans les différentes lignées mutantes. Les jonctions des translocations obtenues par séquençage sont modifiées dans des cellules déficientes pour ce complexe. En effet, elles présentent de longues délétions et un biais d’utilisation de microhomologies, indiquant l’utilisation d’un mécanisme alt-NHEJ. Une altération de cette voie dans les cellules humaines n’affecte d’ailleurs pas la formation de ces réarrangements chromosomiques. Ainsi, contrairement aux cellules de souris, les translocations dans les cellules humaines sont générées par le C-NHEJ. / Chromosomal translocations involve the exchange of chromosome pieces and are often associated with oncogenesis. It has been shown that breakpoint junctions of translocated chromosomes found in patients are typical of a repair by NHEJ. Here we investigated the specific role of XRCC4/LigaseIV, the ligation complex of C-NHEJ, on chromosomal translocation formation in human cells. Using different nucleases (ZFN, TALEN, et CRISPR/Cas9) targeting two chromosomes, we studied the induction of translocation in wt and KO human cells, expressing or not the XRCC4/LigaseIV complex. We found that translocation frequency was mostly reduced in XRCC4/LigaseIV deficient cells when we quantified the induction of translocation by PCR. In addition, we analyzed the breakpoint junctions by sequencing. Strikingly, we found that junctions of translocations show large deletions, and a bias towards the use of longer microhomologies only in XRCC4/LigaseIV KO cells, signature of the alt-NHEJ activity. In contrast, translocation formation was not affected in alt-NHEJ deficient cells. Thus conflicting with results obtained in rodent cells where alt-NHEJ promotes translocation formation, translocations in human cells are generated by the C-NHEJ.