Loblolly pine (Pinus taeda L.) occupies 13 million hectares in the United States and represents a critical component of the global carbon (C) cycle. Forest management alters C dynamics, affecting the C sequestration capacity of a site. Identifying drivers that influence C cycling, quantifying C fluxes, and determining how management alters processes involved in C cycling will allow for an understanding of C sequestration capacity in managed forests. Objectives of the first study included (1) investigating environmental, soil C, root, and stand influences on soil CO2 efflux on the South Carolina coastal plain and (2) quantifying soil CO2 efflux over a rotation in loblolly pine stands located on the South Carolina coastal plain and the Virginia piedmont. In relation to the first objective, temporal variation in soil CO2 efflux was most highly related to soil temperature. Spatial and temporal variability in soil CO2 efflux was weakly related to soil C and root biomass, and not related to coarse woody debris, stand age, stand volume, or site index [Chapter 2]. Soil CO2 efflux was not related to stand age on the South Carolina sites while efflux was positively related to age on the Virginia sites. Cumulative soil C efflux on the South Carolina sites over 20 years is an estimated 278.6 Mg C/ha compared with an estimated 210.9 Mg C/ha on the Virginia sites [Chapter 3]. Objectives of the second study were (1) to investigate short-term effects of fertilization on processes permitting enhanced growth in loblolly pine and (2) to determine the short-term effects of fertilization on autotrophic, heterotrophic, and soil respiration. Major results from the study include the finding that fertilization caused a transient rise in photosynthetic capacity, which paralleled changes in foliar nitrogen. Leaf area accumulation and enhanced growth following fertilization was partly due to enhanced C fixation capacity [Chapter 4]. Fertilization altered the contribution of autotrophic and heterotrophic respiration to total soil CO2 efflux. Enhanced specific root respiration was short-lived while suppressed microbial respiration following fertilization was maintained over the course of the nearly 200-day study. Respiring root biomass growth increased total soil respiration over time [Chapter 5]. / Ph. D.

Soils are a major component of the global carbon budget and may serve an important role in mitigating increasing atmospheric CO2 through their capacity to store carbon. In this regard, it is important to evaluate the implications of forest management on changes in carbon cycling and sequestration and to determine the magnitude by which the efflux of CO2 from the soil surface can vary in time and space. For this study, soil CO2 efflux was measured in 5 replicate blocks of naturally regenerated and planted loblolly pine (Pinus taeda), shortleaf pine (Pinus echinata), and eastern white pine (Pinus strobus) in a 50-acre clear-cut on the Virginia Piedmont. Rates of CO2 efflux were measured every 2 weeks immediately adjacent and away (1m) from newly planted seedlings and cut stumps using a dynamic, closed-chamber infrared gas analyzer system. For each measurement date, volumetric water content was taken in the top 17cm, using time domain reflectometry (TDR) and soil-surface temperature was recorded in the top 7cm, using a temperature probe. For the October measurement a 12cm depth soil core (7cm diameter) was collected for each location. Carbon, nitrogen, coarse fragments, roots, surface litter and coarse woody debris were measured separately for each core. Position (near and away from seedling) had a strong effect on soil CO2 efflux rates. For the first measurement date, rates were higher near the newly planted seedlings (3.09μmol/m2/s) than those taken away from the seedlings (2.29 μmol/m2/s).. The same trend was maintained for the CO2 efflux rates measured near a cut stumps (3.51μmol/m2/s) and those taken away from the stump base (2.56μmol/m2/s). Species proved to have no significant effect on respiration rates for any date and no interaction between species and position was observed. Regression analysis was used to model the influence of soil and plant factors on efflux rates. Temperature (29.2%), position (near and away from the seedlings and stumps base)* temperature, (7.7%), soil carbon (4.1%), organic matter (1.6%), and soil moisture (0.7%) proved to be the major drivers for soil respiration (R2 = 0.4329). When only data near seedlings or stumps were modeled, species had a significant effect on soil CO2 efflux rates. The largest seedlings, loblolly pine (100 cm3 seedling value), had on average the highest rates followed by shortleaf pine (30 cm3 seedling value) and eastern white pine, which were the smallest (9 cm3 seedling value). Stumps had the highest efflux rates. The mean soil respiration rate measured over a seven month sampling period was 2.58 μmol/m2/s,, while the calculated carbon loss from the soil over the same period added up to 575 g C/ m2. The annual carbon loss was estimated to be 675 g C/ m2. / Master of Science

Oxidative stress-inducing chemical contamination in the environment is a significant concern for public health. The depletion of antioxidants by these chemicals results in oxidative stress which may cause detrimental effects in many cell types. For example, multiple stress responses may be activated in bacteria and several disorders including neurodegenerative disorders may occur in mammalian organisms. Oxidative chemicals also have negative effects on engineered water systems as an oxidative stress response in bacteria has been implicated to cause process failure in wastewater treatment facilities. Therefore, it is essential to monitor oxidative chemical contamination in water environments to provide early warning of potential negative effects. Whole-cell biosensors that indicate bacterial stress responses to oxidative toxic agents can be powerful tools in environmental monitoring. An oxidative stress response found in many Gram-negative heterotrophic bacteria called the glutathione-gated potassium efflux (GGKE) mechanism is a good biological indicator to be used in a biosensor designed to detect the presence of oxidative chemicals in water. The authors of this study propose the development of a GGKE biosensor using an environmental strain of Pseudomonas aeruginosa. The abundance of the global antioxidant glutathione, the gating compound in GGKE, in various cell types suggests that there may be connections between the responses of the different cell types to oxidative stress. In this study, specific oxidative stress responses in two distantly related cell types were studied: the GGKE mechanism in Gram-negative heterotrophic bacteria, and mitochondrial dysfunction in rat brain cells. Furthermore, the use of an octanol-based emulsification method for the immobilization of P. aeruginosa in calcium alginate microbeads was evaluated for long-term mechanical stability, viability, and GGKE response of the immobilized cells. The immobilization of cells is an important factor in the design of a whole-cell biosensor, and must yield viable and active cells over time. This study showed that the dose-dependent responses of GGKE in Pseudomonas aeruginosa cells and of mitochondrial dysfunction in a mixed culture of rat brain cells to a model oxidative electrophilic chemical, N-ethylmaleimide, correspond well to each other. We also showed that both responses are accompanied by the depletion of intracellular glutathione, which precedes the GGKE response in P. aeruginosa as well as mitochondrial damage in rat brain cells. Thus, this study suggests that bacterial responses to oxidative stress involving glutathione, such as GGKE, could potentially be used as an early warning to predict the presence of bioavailable oxidative chemicals that can induce oxidative stress in eukaryotic systems. Although further research is needed, this suggests that bacterial stress response biosensors may be used to predict oxidative stress responses in mammalian brain cells. The octanol-based emulsification method produced P. aeruginosa encapsulated alginate microbeads with an average diameter of 200 μm. The microbeads were mechanically stable in solutions containing up to 20 mg/L K+ for 15 days. LIVE/DEAD® and specific oxygen uptake rate (SOUR) analyses showed that the microbead-immobilized cells recovered their membrane integrity within 5 days but not their net respiration potential. The microbead immobilized cells had no net GGKE potential in response to 50 mg/L N-ethylmaleimide after 14 days whereas water-based alginate bead (2mm) immobilized cells did, albeit at a reduced level to planktonic cells. Confirmation experiments revealed that octanol impeded cellular activities of the immobilized cells. Overall, this study showed that the octanol-based emulsification method is not suitable for the immobilization of P. aeruginosa for use in the GGKE biosensor and other microscale immobilization methods should be evaluated. / Master of Science

Forests of the southeastern U.S. play an important role in meeting the increasing demand for forest products, and represent an important carbon (C) sink that can be managed as a potential tool for mitigating atmospheric CO2 concentrations and global climate change. However, realizing this potential depends on full accounting of the ecosystem carbon (C) budget. The separate evaluation of root-derived, autotrophic (RA) and microbially-derived heterotrophic (RH) soil respiration in response to management and climate change is important, as environmental and ecological factors often differentially affect these components, and RH can be weighed against net primary productivity (NPP) to estimate the C sink or source status of forest ecosystems. The objective of this research was to improve the quantitative and mechanistic understanding of soil respiratory fluxes in managed loblolly pine (Pinus taeda L.) plantations of the southeastern U.S. To achieve this overall objective, three studies were implemented to: 1) estimate the proportion and seasonality of RH:RS in four stand age classes, and identify relationships between RH:RS and stand characteristics 2) evaluate the effects of forest nutrient management and throughfall reduction on factors that influence RH and decomposition dynamics, including litter quality, microbial biomass, and enzyme activity and 3) evaluate the sensitivity of sources of RH (mineral soil-derived heterotrophic respiration; RHM, and leaf litter-derived heterotrophic respiration; RHL) to varying soil and litter water content over the course of a dry down event, and assess whether fertilization influences RH. Stand age and measurement season each had a significant effect on RH:RS (P < 0.001), but there were no interactive effects (P = 0.202). Mean RH:RS during the 12-month study declined with stand age, and were 0.82, 0.73, 0.59, and 0.50 for 3-year-old, 9-year-old, 18- year-old, and 25-year-old stands, respectively. Across all age classes, the winter season had the highest mean RH:RS of 0.85 while summer had the lowest of 0.55. Additionally, there were highly significant (P < 0.001) and strong (r > 0.5) correlations between RH:RS and peak LAI, stem volume, and understory biomass. Fertilization improved litter quality by significantly decreasing lignin:N and lignin:P ratios, caused a shift in extracellular enzyme activity from mineral soil N- and P-acquiring enzyme activity to litter C-acquiring enzyme activity, and increased microbial biomass pools. Throughfall reduction decreased litter quality by increasing lignin:N and lignin:P, but also increased C-acquiring enzyme activity. RHL was more sensitive to water content than RHM, and increased linearly with increasing litter water content (R2 = 0.89). The contribution of RHL to RH was greatest immediately following the wetting event, and decreased rapidly to near-zero between three – 10 days. RHM also had a strong relationship with soil water content (R2 = 0.62), but took between 200 – 233 days to attain near-zero RHM rates. Fertilization had no effect on RHM (P = 0.657), but significantly suppressed RHL rates after the wetting event (P < 0.009). This research provides estimates of RH:RS in managed loblolly pine systems that can be used to improve regional ecosystem C modeling efforts, and demonstrates the need to consider the impact of stand age and seasonal patterns to identify the point at which plantations switch from functioning as C sources to C sinks. Additionally, it demonstrates that the controls over RH are dynamic and influenced in the short-term by fertilization and changed precipitation regimes, with the greatest impact on properties affecting litter RH compared to mineral soil. Future research should work to improve the mechanistic understanding of the seasonal and spatial variability of RH and related controlling biotic and abiotic parameters to remedy the variability in existing RS and ecosystem C models. Understanding how management and climate change may impact factors that control RH will ultimately improve our understanding of what drives changes in forest C fluxes. / Ph. D.

To improve the process stability of wastewater treatment plants, the construction of a whole-cell bacterial biosensor is explored to harness the natural stress response of the bacterial cells. The stress response selected in this work is the glutathione-gated potassium efflux (GGKE) system, which responds to electrophilic stress by effluxing potassium from the interior to the exterior of the cell. Thus, the bulk potassium in solution can be monitored as an indicator of bacterial stress. By utilizing this stress response in a biosensor, the efflux of potassium can be correlated to the stress response of the immobilized culture, providing an early warning system for electrophilic shock. This type of shock is a causative factor in many process upset events in wastewater treatment plants, so the application of the sensor would be an early warning device for such plants. The research conducted here focused on the biological element of the biosensor under development. Three immobilization matrices were explored to determine the cell viability and potassium efflux potential from immobilized cells: a calcium alginate, a photopolymer, and a thermally reversible gel. The calcium alginate was unstable, and dissolved after five days, such that the long-term impact of immobilization on the cells could not be determined in the matrix. The photopolymer resulted in very low actvity and viability of immobilized cellsOf the three matrices tested, indicating that the composition of the polymer was toxic to the cells. Of the matrices tested, the thermally-reversible gel showed the best response for further study, in that the matrix did not inhibit cell activity or potassium efflux. / Master of Science

Bacterial antibiotic resistance is a globally recognized problem that has prompted extensive research into novel antimicrobial compounds. This dissertation describes research focusing on two types of potential antimicrobial molecules, organometallic compounds (OMC) and peptide nucleic acids (PNA). Organometallic compounds show promise as antimicrobial drugs because of their structural difference from conventional antibiotics and antimicrobials, and because of the ability to "tune" their chemical and biological properties by varying ligand attachments. Peptide nucleic acids, when linked to a cell-penetrating peptide (CPP), can suppress bacterial gene expression by an antisense mechanism and are attractive candidates for antimicrobial drugs because they bind strongly to target nucleic acids and are resistant to nucleases. Chapters 1 and 2 of the dissertation provide an introduction and broad literature review to frame the experimental questions addressed. Chapter 3 describes work to test the cytotoxicity and cellular penetration capabilities of novel OMCs by evaluating their effects on J774A.1 murine macrophage-like cells that were either uninfected or were infected with Mycobacterium bovis BCG. Results indicate that OMCs with an iridium (Ir) metal center and an amino acid ligand show minimal cytotoxicity against eukaryotic cells but likely do not penetrate the intracellular compartment in significant amounts. Chapter 4 presents research into in vitro effects of CPP-PNAs targeting the tetA and tetR antibiotic resistance genes (CPP-anti-tetA PNA and CPP-anti-tetR PNA, respectively) in tetracycline-resistant Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Through the use of modified minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays it was shown that both the CPP-anti-tetA PNA and CPP-anti-tetR PNA increase tetracycline susceptibility in DT104. Chapter 5 explores the molecular mechanism of the CPP-anti-tetA PNA and CPP-anti-tetR PNA through the use of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results indicate good specificity of the CPP-anti-tetA PNA for its nucleic acid target as evidenced by suppression of tetA mRNA expression in DT104 cultures treated with a combination of tetracycline and the PNA. Chapter 6 describes the development of a mouse model of DT104 infection using BALB/c mice, followed by implementation of that model to test in vivo antimicrobial effects of the CPP-anti-tetA PNA and the CPP-Sal-tsf PNA, which targets expression of the essential tsf gene. An optimal dose of DT104 was identified that causes clinical illness within 2-4 days. At the doses tested, concurrent treatment of infected mice with tetracycline and the CPP-anti-tetA PNA or with the CPP-Sal-tsf PNA alone did not have a protective effect. Final conclusions are 1) that further research with the OMCs should focus on compounds with an Ir center and an amino acid ligand, and should explore ways to enhance intracellular penetration, 2) that the in vitro results of the PNA studies suggest that PNAs targeting expression of antibiotic resistance genes could allow for repurposing of antibiotics to which bacteria are resistant, and 3) additional study of the behavior of PNAs in vivo is advised. / Doctor of Philosophy / Antibiotic-resistant bacteria are increasingly recognized as a threat to global health, and new antibacterial drugs are urgently needed. Before a chemical compound can advance far in the journey to becoming a new drug it must be tested for toxicity against mammalian cells. A portion of this dissertation research involved testing the toxicity of several organometallic compounds (OMCs) previously shown to have antibacterial potential. Mouse-derived mammalian cells were treated with several of the OMCs, and initial results indicated that one of the OMCs is non-toxic and is likely a safe option for additional analysis. This OMC was further tested to see if it could inhibit mycobacterial growth inside of the mammalian cells. It did not effectively clear bacteria from inside of the mammalian cells, likely because of poor penetration of the cell membrane. Further research with this compound should focus on ways to effectively transport the OMC inside infected mammalian cells so that it can reach the bacteria it is meant to target. A second portion of this research involved using a peptide nucleic acid (PNA) to try and reverse tetracycline antibiotic resistance in the bacterial strain Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Peptide nucleic acids are short linear molecules that can bind strongly to complementary DNA and RNA sequences and thus be used to interfere with expression of specific genes. A PNA was designed to inhibit expression of the bacterial tetA gene that codes for a protein called the TetA tetracycline efflux pump, which imparts resistances to tetracycline. Treating the bacteria with the PNA resulted in a lower dose of tetracycline needed to inhibit bacterial growth, indicating a successful increase in tetracycline susceptibility. By using a molecular analysis technique called reversetranscriptase quantitative polymerase chain reaction (RT-qPCR), it was possible to measure the amount of tetA messenger RNA (mRNA) in cultures of DT104 treated only with tetracycline or with a combination of tetracycline and the PNA. As expected, bacteria treated with both the antibiotic and the PNA had less tetA mRNA than the cultures treated only with tetracycline, supporting the hypothesis that the PNA prevents the bacteria from effectively expressing the tetA gene. The PNA was next used in conjunction with tetracycline as an experimental treatment for mice infected with DT104. The PNA did not provide the expected protective effect under these circumstances. The overall conclusion for this part of the research is that PNAs offer an exciting potential avenue for counteracting antibiotic resistance, but additional experimentation is needed. Future research should focus on investigating more effective ways to get the PNAs inside the bacteria and on understanding more about how the PNAs behave in live animals. Several other PNAs targeting different genes involved in antibiotic resistance or essential bacterial functions were also tested against DT104 with variable success.

Conversion of native forests to turfgrass-dominated residential landscapes under a wide range of management practices results in dramatic changes to vegetation and soils, which may affect soil carbon storage. To better understand the effects of landscape conversion and management on soil carbon, we conducted a study on residential properties in the Valley and Ridge physiographic province of southwest Virginia to compare soil carbon storage and dynamics between turfgrass landscapes and the surrounding mixed oak forests from which they were developed. Sixty-four residential properties ranging from 5 to 52 years since site development were investigated. Soil samples were collected from lawns and adjacent forest stands to a depth of 30 cm and analyzed for carbon and nitrogen content. Additional measurements taken were soil bulk density, temperature, moisture, and total soil CO₂ efflux rate. Homeowners participating in the study completed a survey on their lawn management practices so that the effects of specific practices (e.g. fertilization) and intensity levels on carbon dynamics could be analyzed. Also included in the survey were 11 questions regarding the homeowners' commitment to the environment. Homeowners were assigned an environmental commitment score based on their responses which was compared with lawn management practices in order to identify any connection between environmental attitude and lawn management practices. Total soil carbon content to 30 cm depth of lawn (6.5 kg C/m²) and forest (7.1 kg C/m²) marginally differed (P=0.08); however, lawn soil contained significantly greater C than forest soil at the 20-30 cm depth (0.010 vs. 0.007 g C/cm³, P=.0137). There was a weak negative relationship between carbon in the lawn and time since development at the 20-30 cm depth (P=0.08), but no significant relationship between time and C content at shallower depths. We found a positive relationship between time since development and percent C of lawn at the 0-5 cm depth (P=0.04), whereas there was a negative relationship with percent C and time at the 20-30 cm depth (P=0.03). Based on the homeowner survey, we found a positive correlation between lawn fertilization frequency and both lawn nitrogen content (P=.07) and lawn carbon content (P=.0005) in the top 0-5 cm of soil. Nitrogen content was greater in lawn than forest soil at the 0-5 cm depth (0.0025 vs. 0.0018 g/cm³³, P<.0001) and the 5-10 cm depth (0.0013 vs. 0.0009 g/cm³, P <.0001). There was a positive relationship (P=0.059) between overall environmental commitment score and level of management intensity. Higher environmental commitment (EC) score corresponded with a higher level of management intensity (fertilizer and pesticide use). Our results indicate that converting unmanaged Appalachian hardwood forest into managed, turf-grass dominated residential homesites results in similar soil organic concentration and depth distribution as the previous forest within a short period of time following development. Although total soil carbon does not differ between lawn and forest, lawn may develop greater density at 20-30cm depth over time. Fertilization enhances carbon and nitrogen content in the upper 0-5cm in lawns. Homeowners who feel that they are more strongly committed to the environment are more likely to apply higher levels of fertilizer to their lawn. / Master of Science

In an effort to provide wastewater treatment operators with the capability to be proactive in assessing and solving deflocculation events, this study has tested the components of a biosensor to predict deflocculation and investigated the mechanistic cause of deflocculation relating to chlorination of activated sludge cultures. In order to effectively manage upset events, it is necessary to know the source of an upset and the causative mechanism that the source initiates. The Glutathione-gated potassium efflux (GGKE)induced activated sludge deflocculation biosensor incorporates novel microtechnology with a whole cell biological element to predict deflocculation from electrophilic sources. This sensor utilizes microfluidic channels to conduct influent wastewater across a biofilm of Eschericia coli K 12 and monitors the bacterial response to the influent. The bacterial response, which is efflux of K+ ion from the cytoplasm, is monitored with a fluorescence-based sensor called an optode. The components of the system satisfy the project requirements, which include minimal expense (both operation and manufacture), on-line capability and minimal maintenance. The research conducted to date demonstrates the ability of the components of the biosensor to fulfill the design requirements. The optode K+ detector successfully measured an increase in soluble K+ following the exposure of E. coli K-12 to the electrophile N ethyl malemide. The manufacture of the microfluidic device has been completed and the device has demonstrated the ability to conduct influent under negative pressure across an established biofilm with the optode in place. The establishment of a biofilm under expected hydrodynamic conditions has also been completed. Future research efforts will include integrating the components of the biosensor into a working prototype that will be capable monitoring the reaction of bacteria to the presence of electrophilic compounds in wastewater. Sensors of this nature will provide operators with the early warning necessary to be proactive against toxic upsets rather than reactive. The knowledge needed to create a biosensor resides in the identification of bacterial response mechanisms that cause upset events in wastewater treatment facilities. The biosensor that has been developed relies on the discovery of the link between electrophile-induced GGKE and activated sludge deflocculation. Research has been concluded, which expands the role of GGKE and activated sludge deflocculation to include chlorine-induced GGKE. Through a series of laboratory-scale reactors, a relationship has been established between chlorine addition to control filamentous bulking, increased soluble K+ levels and an increase in effluent suspended solids . The results demonstrate that the addition of chlorine to control filamentous bulking may elicit the GGKE mechanism, initiating activated sludge deflocculation, similar to observations of chlorination at full-scale activated sludge wastewater treatment facilities. Establishing a mechanistic cause of deflocculation related to chlorination will permit operators to apply chlorine in a manner that may avoid deflocculation, rather than reacting to deflocculation after it has occurred. / Master of Science

Yes / Adequate drug distribution through tumours is essential for treatment to be effective. Palbociclib is a cyclin-dependent kinase (CDK) 4/6 inhibitor approved for use in patients with hormone receptor (HR) positive, HER2 negative metastatic breast cancer (BC). It has unusual physicochemical properties, which may significantly influence its distribution in tumour tissue. We studied the penetration and distribution of palbociclib in vitro, including the use of multicellular three-dimensional models and mathematical modelling. MCF-7 and DLD-1 cell lines were grown as single cell suspensions (SCS) and spheroids; palbociclib uptake and efflux were studied using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Intracellular concentrations of palbociclib for MCF-7 SCS (Cmax 3.22 µM) and spheroids (Cmax 2.91 µM) were 32 and 29 fold higher and in DLD-1, 13 and 7 fold higher, respectively than the media concentration (0.1 µM). Total palbociclib uptake was lower in DLD-1 cells than MCF-7 cells both in SCS and in spheroids. Both uptake and efflux of palbociclib were slower in spheroids than SCS. These data were used to develop a mathematical model of palbociclib transport that quantifies key parameters determining drug penetration and distribution. The model reproduced qualitatively most features of the experimental data and distinguished between SCS and spheroids, providing additional support for hypotheses derived from the experimental data. Mathematical modelling has the potential for translating in vitro data into clinically relevant estimates of tumour drug concentrations. / Grant for Translational Research and a grant from Leeds NHS Charitable Trustees.

La multi-résistance aux drogues des bactéries est un problème majeur en clinique. L’un des mécanismes de résistance consiste à effluer les composés toxiques hors de la cellule grâce à des protéines de la membrane interne nommées pompes d’efflux. Ces protéines appartiennent à cinq familles (MFS, RND, MATE, SMR et ABC) et peuvent fonctionner en association avec deux autres types de protéines (protéine du périplasme et protéine de la membrane externe) pour former un canal. Dans le cadre d’une thématique de recherche basée sur l’étude des mécanismes de résistance auxdrogues de la bactérie pathogène Legionella pneumophila, une approche bioinformatique menée sur lesgénomes de trois souches séquencés (souches Lens, Paris et Philadelphia) a permis d’identifier des protéinespouvant participer à l’efflux. Notre but a été de vérifier l’implication de ces protéines dans la résistance auxdrogues et dans la virulence de Legionella en ciblant un ou plusieurs gènes codant pour des composants desystèmes d’efflux. Pour inactiver les gènes, nous avons choisi une stratégie de recombinaison homologue. Lesrecombinants ont été testés pour leur sensibilité à des composés toxiques afin de voir si les gènes ciblés jouentun rôle dans l’efflux d’E. coli. Un de ces mutants, le mutant MF201, altéré pour le gène codant pour une protéinehomologue à TolC chez E. coli s’est avéré être 2 à 16 fois plus sensible aux drogues testées comparé à lasouche sauvage. De plus, ce mutant présente un défaut important de virulence dans Acanthamoeba castellanii,Dictyostelium discoideum et les macrophages U937. Ce premier résultat implique que la protéine TolC-like deLegionella aurait un rôle clef dans la relation hôte pathogène et sous-tend un lien entre multi-résistance auxdrogues et virulence. Par ailleurs une étude de l’expression des gènes codant pour des pompes à efflux a étéinitiée afin de comprendre leur rôle au cours du cycle infectieux de Legionella. / Bacterial multi-drug resistance is of major concern in the case of clinic. One of the resistance mecanisms used by bacteria is the efflux of noxious compounds out of the cell thanks to inner membran proteins called efflux pumps. This proteins belong to five families (MFS, RND, MATE, SMR and ABC) and can function in close association with two partners (periplasmic protein and outer membrane protein) to form a canal. In our new research axis based on the study of the drug resistance of the bacterium Legionella pneumophila, we conducted a bioinformatical approach to identify efflux pumps proteins coded by the sequenced genome of three strains (strains Lens, Paris and Philadelphia). Our goal was to study the role of this proteins in Legionella drug resistance and in its virulence. The bioinformatic approach data allowed us to choose one or several genes coding for potential efflux pump components for genetic invalidation by an homologousrecombination strategy. The bacterial mutants were exposed to different noxious compounds in order to know ifthe target genes invalidated were implicated in the efflux of drugs. One of this mutants, strain MF201, which isdeleted for the gene encoding a protein homologous to E. coli TolC protein, revealed to be 2 to 16 times moresensitive to the drug tested compared to the wild-type strain. Furthermore, this mutant showed an importantvirulence defect in Acanthamoeba castellanii, Dictyostelium discoideum and U937 macrophages. This first resultsmeans that the TolC-like protein of Legionella could be a key factor in host-pathogen interaction and stronglysuggests a link between multi-drug resistance and virulence. We also initiated a transcriptomic approach to studyefflux pump genes expression in order to understand their role during the infectious cycle of Legionella.