Triclosan (TCS) and triclocarban (TCC), widely used antimicrobial agents found in numerous consumer products, are incompletely removed by wastewater treatment plant (WWTP) processing. Methyl-triclosan (M-TCS) is a more lipophilic metabolite of its parent compound, TCS. The focus of this study was to quantify bioaccumulation factors (BAFs) for TCS, M-TCS, and TCC in Pecan creek, the receiving stream for the City of Denton, Texas WWTP by using field samples mostly composed of the alga Cladophora sp. and the caged snail Helisoma trivolvis as test species. Additionally, TCS effects on E. coli and Arabidopsis have been shown to reduce fatty acid biosynthesis and total lipid content by inhibiting the trans-2 enoyl- ACP reductase. The lipid synthesis pathway effects of TCS on field samples of Cladophora spp. were also investigated in this study by using [2-14C]acetate radiolabeling procedures. Preliminary results indicate high TCS concentrations are toxic to lipid biosynthesis and reduce [2-14C]acetate incorporation into total lipids. These results have led to the concern that chronic exposure of algae in receiving streams to environmentally relevant TCS concentrations might affect their nutrient value. If consumer growth is limited, trophic cascade strength may be affected and serve to limit population growth and reproduction of herbivores in these riparian systems.

Os protocolos de Avaliação de Risco Ambiental (ERA) de Fármacos e Produtos de Cuidados Pessoais (FPCP) recomendam o uso de ensaios ecotoxicológicos tradicionais (por exemplo algas, bactérias, invertebrados, peixes) e a avaliação de efeitos em um único nível de organização biológica para a determinação dos efeitos potenciais dos FPCP à biota. Considerando que efeitos em nível de sub-indivíduo pode afetar igualmente a aptidão ecológica de organismos marinhos, e que os mesmos estão cronicamente expostos aos FPCP, o objetivo do presente estudo foi avaliar o risco ambiental de triclosan (TCS), ibuprofeno (IBU) e 17&alpha;-etinilestradiol (EE2) em sedimentos marinhos utilizando respostas de efeitos sub-individuais e populacionais. Por meio do HPLC-ESI-MS/MS, as concentrações ambientais de TCS e IBU foram quantificadas em sedimentos marinhos coletados no entorno do emissário submarino de esgoto de Santos (Baía de Santos, São Paulo - Brasil) com 15,14 e 49,0 ng.g-1, respectivamente, enquanto o EE2 não foi detectado (<33 ng.g-1). Uma bateria de ensaios de toxicidade crônica (desenvolvimento embriolarval) com ouriços-do-mar (Lytechinus variegatus) e bivalves (Perna perna) foi realizada (efeito a nível de indivíduo) após exposição a sedimentos contaminados com os FPCP. Além disso, foram analisados alguns biomarcadores de Fase I (etoxiresorufina O-deetilase EROD e dibenzilfluoresceína DBF), de Fase II (glutationa S-transferase GST) do metabolismo, do sistema antioxidante (glutationa peroxidase GPx), de neurotoxicidade (colinesterase ChE), de estresse oxidativo (peroxidação lipídica LPO e danos em DNA) e de citotoxicidade que foram selecionados para avaliação das respostas a nível de sub-indivíduo em mexilhões Mytella charruana. Todos os compostos analisados apresentaram efeitos sobre o desenvolvimento embriolarval de L. variegatus e P. perna em concentrações ambientalmente relevantes. Em nível de sub-indivíuo foi possível observar que o TCS causou efeitos cito-genotóxicos (diminuição da estabilidade da membrana lisossomal, peroxidação lipídica e danos em DNA) e neurotóxicos. O IBU causou efeitos citotóxicos e neurotóxicos, enquanto o EE2 apresentou efeitos citotóxicos e danos em DNA. Nesse sentido, mesmo em baixas concentrações os FPCP são potencialmente capazes de alterar os mecanismos de manutenção da homeostase. Os dados químicos e ecotoxicológicos foram integrados e os quocientes de risco estimados para TCS, IBU e EE2 apresentaram valores superiores a 1,0, indicando alto risco ambiental destes compostos em sedimentos marinhos. Estes são os primeiros dados de avaliação de risco ambiental de FPCP em sedimentos de uma zona costeira brasileira. Os resultados sugerem que a ERA de fármacos e produtos de cuidados pessoais deve contemplar, além dos ensaios de toxicidade tradicionais o uso de biomarcadores como indicadores dos primeiros sinais de efeitos e, assim, estabelecer uma avaliação de risco mais efetiva que assegure a proteção e funcionamento dos ecossistemas aquáticos. / The guidelines for the Environmental Risk Assessment (ERA) of pharmaceuticals and personal care products (PPCPs) usually recommend the use of standard ecotoxicity assays (e.g. algae, bacteria, invertebrate, fish) and the assessment of endpoints at individual level for the evaluation of potential effects of PPCPs on biota. Considering that effects at sub-individual level can also affect the ecological fitness of marine organisms, and that marine organisms are chronically exposed to PPCPs, the aim of the current study was to evaluate the environmental risk of triclosan (TCS), ibuprofen (IBU) and 17&alpha;-ethynylestradiol (EE2) in marine sediments using sub-individual and population endpoints. Using LC-ESI-MS/MS, the environmental levels of TCS and IBU were quantified in marine sediments from the vicinities of the Santos submarine sewage outfall (Bay of Santos, São Paulo, Brazil) at 15.14 and 49.0 ng g-1, respectively, while EE2 was not detected (<33ng g-1). A battery (n=3) of chronic bioassays (embryo-larval development) with a sea urchin (Lytechinus variegatus) and a bivalve (Perna perna) were performed at populational level after exposure to spiked sediment. Phases I (ethoxyresorufin O-deethylase EROD and dibenzylfluorescein dealkylase DBF) and II (glutathione S-transferase GST) of the metabolism, antioxidant system (glutathione peroxidase GPX), neurotoxicity (cholinesterase ChE), oxidative effects (lipid peroxidation LPO and DNA damage strand breaks) and cytotoxicity were selected to evaluate the sublethal responses in the bivalve Mytella charruana. These compounds showed developmental effects on L. variegatus and P. perna at environmentally relevant concentrations. At sub-individual level TCS induced cyto-genotoxic (reduction on stability of lysosome membrane, lipid peroxidation and DNA damage) and neurotoxic effects. IBU caused cyto and neurotoxic effect, while EE2 caused cytotoxic and DNA damage. Chemical and ecotoxicological data were integrated and the quotient risk estimated for TCS, IBU and EE2 showed values higher than 1.0, indicating high environmental risks of these compounds in sediments. These are the first data of risk assessment of pharmaceuticals and personal care products in sediments of a Brazilian coastal zone. The results suggests that the ERA of pharmaceuticals and personal care products must include, in addition to the standard toxicity tests, the use of biomarkers as indicators of the early warning signs and thus provide a more effective risk assessment to security the protection and functioning of aquatic ecosystems.

Os protocolos de Avaliação de Risco Ambiental (ERA) de Fármacos e Produtos de Cuidados Pessoais (FPCP) recomendam o uso de ensaios ecotoxicológicos tradicionais (por exemplo algas, bactérias, invertebrados, peixes) e a avaliação de efeitos em um único nível de organização biológica para a determinação dos efeitos potenciais dos FPCP à biota. Considerando que efeitos em nível de sub-indivíduo pode afetar igualmente a aptidão ecológica de organismos marinhos, e que os mesmos estão cronicamente expostos aos FPCP, o objetivo do presente estudo foi avaliar o risco ambiental de triclosan (TCS), ibuprofeno (IBU) e 17&alpha;-etinilestradiol (EE2) em sedimentos marinhos utilizando respostas de efeitos sub-individuais e populacionais. Por meio do HPLC-ESI-MS/MS, as concentrações ambientais de TCS e IBU foram quantificadas em sedimentos marinhos coletados no entorno do emissário submarino de esgoto de Santos (Baía de Santos, São Paulo - Brasil) com 15,14 e 49,0 ng.g-1, respectivamente, enquanto o EE2 não foi detectado (<33 ng.g-1). Uma bateria de ensaios de toxicidade crônica (desenvolvimento embriolarval) com ouriços-do-mar (Lytechinus variegatus) e bivalves (Perna perna) foi realizada (efeito a nível de indivíduo) após exposição a sedimentos contaminados com os FPCP. Além disso, foram analisados alguns biomarcadores de Fase I (etoxiresorufina O-deetilase EROD e dibenzilfluoresceína DBF), de Fase II (glutationa S-transferase GST) do metabolismo, do sistema antioxidante (glutationa peroxidase GPx), de neurotoxicidade (colinesterase ChE), de estresse oxidativo (peroxidação lipídica LPO e danos em DNA) e de citotoxicidade que foram selecionados para avaliação das respostas a nível de sub-indivíduo em mexilhões Mytella charruana. Todos os compostos analisados apresentaram efeitos sobre o desenvolvimento embriolarval de L. variegatus e P. perna em concentrações ambientalmente relevantes. Em nível de sub-indivíuo foi possível observar que o TCS causou efeitos cito-genotóxicos (diminuição da estabilidade da membrana lisossomal, peroxidação lipídica e danos em DNA) e neurotóxicos. O IBU causou efeitos citotóxicos e neurotóxicos, enquanto o EE2 apresentou efeitos citotóxicos e danos em DNA. Nesse sentido, mesmo em baixas concentrações os FPCP são potencialmente capazes de alterar os mecanismos de manutenção da homeostase. Os dados químicos e ecotoxicológicos foram integrados e os quocientes de risco estimados para TCS, IBU e EE2 apresentaram valores superiores a 1,0, indicando alto risco ambiental destes compostos em sedimentos marinhos. Estes são os primeiros dados de avaliação de risco ambiental de FPCP em sedimentos de uma zona costeira brasileira. Os resultados sugerem que a ERA de fármacos e produtos de cuidados pessoais deve contemplar, além dos ensaios de toxicidade tradicionais o uso de biomarcadores como indicadores dos primeiros sinais de efeitos e, assim, estabelecer uma avaliação de risco mais efetiva que assegure a proteção e funcionamento dos ecossistemas aquáticos. / The guidelines for the Environmental Risk Assessment (ERA) of pharmaceuticals and personal care products (PPCPs) usually recommend the use of standard ecotoxicity assays (e.g. algae, bacteria, invertebrate, fish) and the assessment of endpoints at individual level for the evaluation of potential effects of PPCPs on biota. Considering that effects at sub-individual level can also affect the ecological fitness of marine organisms, and that marine organisms are chronically exposed to PPCPs, the aim of the current study was to evaluate the environmental risk of triclosan (TCS), ibuprofen (IBU) and 17&alpha;-ethynylestradiol (EE2) in marine sediments using sub-individual and population endpoints. Using LC-ESI-MS/MS, the environmental levels of TCS and IBU were quantified in marine sediments from the vicinities of the Santos submarine sewage outfall (Bay of Santos, São Paulo, Brazil) at 15.14 and 49.0 ng g-1, respectively, while EE2 was not detected (<33ng g-1). A battery (n=3) of chronic bioassays (embryo-larval development) with a sea urchin (Lytechinus variegatus) and a bivalve (Perna perna) were performed at populational level after exposure to spiked sediment. Phases I (ethoxyresorufin O-deethylase EROD and dibenzylfluorescein dealkylase DBF) and II (glutathione S-transferase GST) of the metabolism, antioxidant system (glutathione peroxidase GPX), neurotoxicity (cholinesterase ChE), oxidative effects (lipid peroxidation LPO and DNA damage strand breaks) and cytotoxicity were selected to evaluate the sublethal responses in the bivalve Mytella charruana. These compounds showed developmental effects on L. variegatus and P. perna at environmentally relevant concentrations. At sub-individual level TCS induced cyto-genotoxic (reduction on stability of lysosome membrane, lipid peroxidation and DNA damage) and neurotoxic effects. IBU caused cyto and neurotoxic effect, while EE2 caused cytotoxic and DNA damage. Chemical and ecotoxicological data were integrated and the quotient risk estimated for TCS, IBU and EE2 showed values higher than 1.0, indicating high environmental risks of these compounds in sediments. These are the first data of risk assessment of pharmaceuticals and personal care products in sediments of a Brazilian coastal zone. The results suggests that the ERA of pharmaceuticals and personal care products must include, in addition to the standard toxicity tests, the use of biomarkers as indicators of the early warning signs and thus provide a more effective risk assessment to security the protection and functioning of aquatic ecosystems.

Cette thèse porte sur l’évaluation de l’impact de certains composés environnementaux sur la fécondité féminine, tel que mesuré par le délai de conception (« time to pregnancy » en anglais, ou TTP). Cette recherche a été réalisée dans le cadre de l’Étude mère-enfant sur les composés chimiques de l’environnement (MIREC), une cohorte de grossesse de 2001 femmes recrutées durant le premier trimestre dans dix villes canadiennes de 2008 à 2011. Les données des questionnaires et les échantillons biologiques ont servi à évaluer l’effet de deux groupes de composés : les persistants [composés perfluorés – perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA) et perfluorohexane sulfonate (PFHxS)] et les non persistants (bisphénol A, triclosan et phtalates). Cette thèse comprend également une analyse du potentiel du ratio index-annulaire (2D:4D) comme mesure de sensibilité endocrinienne. À ce jour, des mesures anthropométriques ont été collectées sur environ 800 mères-enfants dans le cadre de l’Étude mère-enfant sur les composés chimiques de l’environnement : biomonitoring et neurodéveloppement à la petite enfance (MIREC CD Plus), un suivi de la cohorte MIREC portant sur la croissance et le développement des enfants jusqu’à 5 ans. Sur l’ensemble, les résultats de cette thèse permettent d’étoffer les preuves concernant les effets adverses potentiels de plusieurs contaminants environnementaux sur la fécondité féminine, telle que mesurée par le TTP. Dans le premier article, nous avons montré une association entre les PFOA et les PFHxS et une baisse de fécondité, ce que d’autres recherches avaient déjà révélé. Dans le deuxième article, nous avons évalué l’effet du triclosan sur le TTP, ce qui n’avait jamais été examiné, pour montrer un délai plus élevé chez les femmes du quartile supérieur d’exposition. De plus, nos résultats sont en accord avec ceux de la seule étude ayant évalué l’effet du Bisphénol A sur la fécondité féminine, qui n’avait pas détecté d’effet. Finalement, nos données semblent indiquer une association entre l’exposition des femmes aux phtalates et un TTP plus court, mais ces résultats ne sont pas statistiquement significatifs. En ce qui a trait au potentiel du ratio index-annuaire (2D:4D) pour mesurer la sensibilité endocrinienne chez les femmes, nos données ne permettent pas d’établir une association entre ce ratio et le TTP. Pour ce qui est des enfants, nous n’avons pas trouvé d’effet adverse entre le tabagisme de la mère durant la grossesse et leur ratio 2D:4D. Par conséquent, nos données ne semblent pas justifier l’utilisation du ratio 2D:4D pour mesurer la sensibilité endocrinienne en lien avec le potentiel reproducteur (basé sur le TTP) ou l’exposition des enfants au tabac durant le premier trimestre de grossesse. / In this thesis, we aimed to evaluate the impact of selected environmental compounds on female fecundity as measured by time to pregnancy (TTP). This research was conducted in the framework of the Maternal-Infant Research on Environmental Chemicals (MIREC) study, a pregnancy cohort of 2001 women recruited during the first trimester of pregnancy in ten cities across Canada between 2008 and 2011. Questionnaire data and biological samples were analyzed to assess the effect of two groups of compounds: persistent [perfluorinated compounds - perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and perfluorohexane sulfonate (PFHxS)-] and nonpersistent chemicals (Bisphenol A, Triclosan, and phthalates). In addition, this thesis aimed to examine the potential of the second to fourth finger digit ratio (2D:4D) as a sensitive-endocrine endpoint. To this end, anthropometric measurements were obtained in about 800 children and their mothers during the Early Childhood Biomonitoring and Neurodevelopment Study (MIREC-CD Plus), a MIREC follow-up conducted to measure growth and development up to age five. Overall, the results of this thesis have contributed to the evidence regarding the potential adverse effect of several environmental contaminants (ECs) on female fecundity as measured by TTP. In the first article, we found that PFOA and PFHxS were associated with diminished fecundity, supporting previous evidence that suggested a similar effect. In the second article, we assessed for the first time the effect of Triclosan on TTP, presenting evidence of delayed fecundity at the highest quartile of exposure. In addition, our findings agreed with those of the only study that has assessed the effect of Bisphenol A on female fecundity, and which showed no effect. Finally, we found some indication that female exposure to phthalates might be associated with a shorter TTP, although this finding did not reach statistical significance. With regard to the potential of the digit length ratio (2D:4D) as an endocrine-sensitive endpoint in women, our data do not support a strong association between 2D:4D and TTP. In children, we did not find an adverse impact of maternal smoking during pregnancy on children’s 2D:4D. Thus, our data do not support evidence to suggest that 2D:4D could be used as a potential reproductive endocrine-sensitive endpoint in women as measured by TTP, and in their offspring as measured by exposure to maternal smoking during the first trimester of pregnancy.

In freshwater, there is more opportunity for silver (Ag) to interact with organic ligands than in seawater. Triclosan is an antibiotic agent which resembles thyroid hormone T3 and is finding its way into aquatic systems. Preliminary toxicology studies for the frogSCOPE program suggest that triclosan and nanosilver (nanoAg), also used as an antibiotic agent, may be chemically interacting, as they seem to synergistically increase the endocrine-disrupting abilities already observed independently in each chemical. Ag speciation methods can be used to determine if triclosan or thyroid hormone T3 are interacting with Ag ion (Ag+), which gets released over time by nanoAg. To fully utilize Ag speciation methods, however, total Ag in the sample must also be independently analyzed. Here we investigated a new total Ag analysis using cadmium sulfide quantum dots (CdS QDs) as fluorescence probes in solution. This method promises results in a fraction of the time of the established competitive ligand equilibration-solvent extraction (CLE-SE) technique utilizing PDC- and DDC- to bind Ag and bring it out of solution. Following this investigation were a series of experiments using CLE-SE for total Ag and Ag speciation in well water used to house bullfrog tadpoles in frogSCOPE Ag exposure studies. CLE-SE for Ag speciation was also applied to well water samples containing the two levels of nanoAg or Ag+ used in frogSCOPE Ag exposures, and used in ligand competition experiments to examine the potential of triclosan or T3 to act as strong Ag-binding ligands, as compared to glutathione and EDTA, two known Ag-binding ligands. The results of the latter experiments could be used to determine if either of these could be forming complexes with Ag which increase or decrease their delivery to amphibian cells. The fluorometric method using CdS QDs showed no ideal analytical response to nanomolar Ag+, even when commercial QDs were modified and used, so it could not be applied to our samples. Using CLE-SE for total Ag, the well water used as a base for toxicity studies in frogSCOPE contained Ag below the method detection limit of 5 pM. Using the speciation variation of the CLE-SE method, no evidence of naturally-occurring ligands which could produce extractable (hydrophobic) or non-extractable (hydrophilic) Ag complexes was found in this well water. EDTA and glutathione responded as model Ag-binding ligands to form non-extractable hydrophilic Ag complexes in fresh water. T3 behaved like these model ligands, while triclosan enhanced the extractability of Ag in the presence of certain concentrations of the added ligand, DDC-. In another set of experiments, coordination of Ag by triclosan or T3 was not detectable within that analytical window. These results suggest that ionic Ag released over time by nanoAg may be binding T3 and preventing it from reaching its receptor, but confirming the interaction of triclosan and Ag+ will require additional experiments using different analytical windows.

L’émergence de la résistance à une multitude d’agents antimicrobiens chez des bactéries pathogènes est considérée comme une menace majeure pour la santé publique (2). Ces souches sont reconnues comme des organismes multirésistants aux médicaments ou MDR (multidrug-resistant) (4). Les recherches progressent chez les bactéries, à Gram positif, à Gram négatif et acido-alcoolo-résistantes au vu de l’ampleur de la menace pour la santé publique, ces bactéries multirésistantes sont devenues les cibles potentielles à cette fin de recherche. De ce fait, les objectifs de la présente étude ont consisté en la caractérisation structurale et fonctionnelle de différents transporteurs transmembranaires de la famille des RND (Resistance-Nodulation-Division) encore énigmatiques, à savoir: le MmpL3 chez Mycobacterium tuberculosis (Mtb) via l’étude de son orthologue CmpL1 chez Corynebacterium glutamicum (Cgl) et le TriAxBC chez Pseudomonas aeruginosa (P. aeruginosa). Ainsi, comme première démarche présentée dans le chapitre 2, la structure du transporteur MmpL3 Mtb (un transporteur d'acides mycoliques – sous forme de tréhalose de monomycolates (ou TMM) - essentiel pour la viabilité de Mtb) (5) et celle de son orthologue CmpL1 Cgl ont été prédites via le serveur I-TASSER (6-8). Ces structures ont été validées par la suite en comparant à la carte électronique générée pour CmpL1 (18 Å) par des analyses de microscopie électronique en transmission à coloration négative (TEM). La caractérisation du transporteur CmpL1 purifié par chromatographie à exclusion stérique a confirmé le complexe trimérique de taille avoisinant les 315 KDa (incluant la couronne du détergent) en accord avec des analyses par gel SDS-PAGE. Des études génétiques et biochimiques en collaboration ont d’autre part identifié des résidus engagés dans le transport du TMM chez MmpL3 ainsi que d’autres impliqués dans la résistance à des inhibiteurs ciblant ce transporteur. L’ensemble de ces données a mis en évidence la localisation des résidus essentiels au transport et à la résistance au niveau du canal central du modèle trimérique de MmpL3. La région de MmpL3 activant le transport par force protomotrice a été localisée au niveau d’une cavité centrale qui est une caractéristique intrinsèque de la famille des RND. Les cartes électroniques de faible résolution déjà obtenues pour la protéine CmpL1 font de ce projet une des directions futures du laboratoire. Dans le chapitre 3, nous illustrons le deuxième aspect du présent projet qui repose sur l’extension de l’étude du potentiel thérapeutique du ciblage du transporteur transmembranaire MmpL3 chez les différentes souches de Mycobacterium. Nos collaborateurs ont effectué une analyse biochimique de l’effet thérapeutique des inhibiteurs les plus prometteurs du transporteur MmpL3 Mtb sur certaines souches mycobactériennes non-tuberculeuses (NTB) multi-résistantes. Basés sur nos modélisations structurales comparatives obtenues par I-TASSER (6-8), nous avons pu complémenter les informations biochimiques en soulignant les similitudes et les différences de structure entre les souches TB et NTB ainsi que leurs impacts fonctionnels. Ce chapitre met en évidence l’intérêt du ciblage thérapeutique de MmpL3 chez les espèces NTB. En effet, l’efficacité de certains inhibiteurs de MmpL3 Mtb sélectionnés sur le traitement des infections pulmonaires NTB promet de pouvoir généraliser cette nouvelle voie de traitement pour d’autres souches multi-résistantes NTB voire à contribuer à remédier à la problématique de la résistance aux antibiotiques et décomplexifier le traitement actuel. D’autres études en collaboration entreprenant les mêmes approches d’études structurales ont été réalisées pour les transporteurs tripartites TriAxBC (P. aeruginosa), des pompes à efflux appartenant à la famille des RND. Le but du chapitre 4 était de générer une structure du complexe et de déchiffrer son mode d’assemblage et d’expulsion des antibiotiques vers le milieu externe. Un modèle à structure quaternaire de TriAxBC a été prédit par I-TASSER (6-8) et validé contre sa carte électronique à 4.3 Å générée en Cryo-EM. Le complexe TriAxBC a été également caractérisé par filtration sur gel confirmant une taille approximative de 620 KDa et sa composition en trimère par visualisation sur gel SDS-PAGE. En conclusion, nous avons pu à travers cette étude combiner différentes approches biochimiques, génétiques et structurales soutenant la nécessité d’une approche multidisciplinaire pour l’approfondissement de la compréhension de la structure et du mode de fonctionnement des transporteurs RND. Ces derniers demeurent toujours énigmatiques; toutefois, nos avancées et d’autres à venir permettront la génération de nouveaux médicaments spécifiques traitant les bactéries multirésistantes. / The emergence of resistance to a multitude of antimicrobial agents in pathogenic bacteria is considered a major threat to public health (2). These strains are recognized as multidrug resistant organisms (MDR) (4). Research is progressing in Gram positive, Gram positive high GC and Gram negative bacteria, and given the scale of the public health threat, these MDR have become potential targets for this research. The objectives of the present study consist of the structural and functional characterization of various transmembrane transporters of the still enigmatic RND (Resistance-Nodulation-Division) family, namely: MmpL3 in Mycobacterium tuberculosis (Mtb) via the study of its ortholog CmpL1 in Corynebacterium glutamicum (Cgl) and TriAxBC in Pseudomonas aeruginosa (P. aeruginosa). The first component of this project, presented in Chapter 2, studies the structure of the transporter MmpL3 Mtb (a TMM mycolic acid transporter essential for the viability of Mtb (5) and that of its CmpL1 Cgl orthologue, which have been predicted via the I- Tasser Pack (6-8). These structures were subsequently validated by comparing to the electronic map generated for CmpL1 (18 Å) by negative staining transmission electron microscopy (TEM). Characterization of the purified CmpL1 transporter by size exclusion chromatography confirmed the trimeric complex size around 315 KDa (including the detergent crown) corroborated by SDS-PAGE gel analyses. Collaborative genetic and biochemical studies have also identified residues involved in the transport of TMM in MmpL3 as well as those residues conferring antibiotic resistance. This data highlighted the location of the essential residues of transport and resistance in the central channel of the trimeric Mmpl3 model. The MmpL3 region activating proto-motor transport has been located at a central cavity, which is an intrinsic feature of the RND family. The low-resolution electronic maps obtained for the protein CmpL1 may serve as the foundation of future studies. In Chapter 3 we explore the therapeutic potential of the targeting of the transmembrane transporter MmpL3 in different Mycobacterium strains. Our collaborators studied the therapeutic effect of the most promising inhibitors of the MmpL3 Mtb transporter on certain multi-resistant mycobacterial non-tuberculous (NTB) strains. Based on our comparative structural modeling obtained by I-TASSER (6-8), we supplemented the biochemical data by highlighting the structural similarities and differences between the TB and NTB strains as well as their functional impacts. This chapter highlights the interest of direct or indirect targeting of MmpL3 in NTB species. Indeed, the efficacy of certain selected MmpL3 Mtb inhibitors on the treatment of NTB pulmonary infection have potential as generalizable treatment options for other NTB multi-resistant strains, or even to help address the problem of resistance to antibiotics and simplify current combination approaches. Other collaborative studies undertaking the same structural approaches were carried out for TriAxBC tripartite carriers (P. aeruginosa), efflux pumps belonging to the RND family. The purpose of Chapter 4 was to generate a structure of the complex and decipher its mode of assembly and expulsion of antibiotics from the intracellular environment. A quaternary structure model of TriAxBC was predicted by I-TASSER (6-8) and validated against its 4.3 Å electronic map generated by Cryo-EM. The TriAxBC complex was also characterized by gel filtration confirming an approximate size of 620 KDa and its trimer composition by SDS-PAGE. In conclusion, this study is combining different biochemical, genetic and structural approaches to highlight the need for a multidisciplinary approach to characterizing the structure function of RND transporters. The latter remain enigmatic; however, our contribution and the progress of others will allow the generation of new specific drugs targeting multiresistant strains.

The Pecan Creek Waste Reclamation Plant in Denton, Texas, an activated sludge WWTP, was sampled monthly for ten months to determine seasonal and site variation in concentrations of triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol), an antibacterial additive. SNK separation after the highly significant ANOVA on ranked data were: summer = fall > winter = spring and influent > downstream = effluent = wetland inflow > wetland outflow (a=0.05). After the plant converted to ultraviolet disinfection, measurements were made before and after the UV basin to determine if significant amounts of triclosan were converted to dioxin. Percent loss at each of the treatment steps was determined. Concentrations of triclosan in the downstream site were below the published NOEC for the most sensitive species.

Indiana University-Purdue University Indianapolis (IUPUI) / Organic-rich substrates such as soil provide an excellent carbon source for bacteria. However, hydrocarbons such as those found in various ignitable liquids can also serve as a source of carbon to support bacterial growth. This is problematic for fire debris analysis as samples may be stored at room temperature for extended periods before they are analyzed due to case backlog. As a result, selective loss of key components due to bacterial metabolism can make identifying and classifying ignitable liquid residues by their chemical composition and boiling point range very difficult. The ultimate goal of this project is to preserve ignitable liquid residues against microbial degradation as efficiently and quickly as possible. Field and laboratory studies were conducted to monitor microbial degradation of gasoline and other ignitable liquids in soil samples. In addition to monitoring degradation in potting soil, as a worst case scenario, the effect of soil type and season were also studied. The effect of microbial action was also compared to the effect of weathering by evaporation (under nitrogen in the laboratory and by the passive headspace analysis of the glass fragments from the incendiary devices in the field studies). All studies showed that microbial degradation resulted in the significant loss of n-alkanes and lesser substituted alkylbenzenes predominantly and quickly, while more highly substituted alkanes and aromatics were not significantly affected. Additionally, the residential soil during the fall season showed the most significant loss of these compounds over the course of 30 days. To combat this problem, a chemical solution is to be immediately applied to the samples as they are collected. Various household and commercial products were tested for their efficacy at low concentrations to eliminate all living bacteria in the soil. Triclosan (2% (w/v) in NaOH) proved to be the most effective at preserving ignitable liquid residues for at least 30 days.