The Chattahoochee River is an essential surface water source as it provides over 70 percent of Metro Atlanta’s drinking water, amounting to over 300 million gallons. In addition to serving as Metro Atlanta’s primary source of drinking water, the Chattahoochee River serves as a major point of discharge for industrial and municipal waste as well as urban runoff. The primary goal of this study was to assess the presence of Pepper Mild Mottle Virus in the Chattahoochee River. During a five-month period in 2014, water samples were collected at fifteen sample sites and two outfall sites in the Chattahoochee River. PMMoV was tested for in 6 out of 17 sample. A one-way ANOVA analysis (p<0.05), of concentrations across sampling locations resulted in a p-value of 0.044. As a result, it can be determined that the location of the sampling sites does result in a statistically significant difference in the PMMoV values observed. Furthermore, a one-way ANOVA analysis (p<0.05), of concentrations across sampling dates resulted in a p-value of 0.063. Therefore, it is determined that the dates on which sampling took place did not result in a statistically significant difference in the PMMoV values observed across time . Furthermore, the MS2 virus was also detected in these samples. Through a paired t-test (p<0.05), between the sample concentrations with and without MS2 presence, it was determined that there was no statistical difference in concentration of PMMoV when MS2 is present since p=0.0740 The results indicate that PMMoV was present in the Chattahoochee River due to the detection of PMMoV in the samples collected. However, additional investigations, using a larger sample size, are needed to assess PMMoV as a viable indicator of fecal contamination of ambient surface waters and recreational waters.

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La régulation de l’expression génétique est essentielle afin qu’un organisme puisse s’adapter aux changements environnementaux. Chez les bactéries, la régulation peut s’effectuer à plusieurs étapes de l’expression des gènes (transcription, stabilité de l’ARN, traduction, maturation et dégradation des protéines) et par des mécanismes impliquant différents types de molécules (ADN, ARN, protéines, métabolites ou ions inorganiques). Traditionnellement, les protéines se situaient au centre de ces mécanismes de régulation. On sait maintenant que certains ARN, dont les riborégulateurs, ont également un grand rôle à jouer dans ce processus. Un riborégulateur est un élément génétique retrouvé dans une région non-codante de certains ARN messagers (ARNm), qui peut lier directement un ligand spécifique afin de réguler l’expression de son transcrit. Chez Escherichia coli (E. coli), trois riborégulateurs lient la thiamine pyrophosphate (TPP). Un d’entre eux, le riborégulateur thiB, n’a toujours pas été étudié. On croit qu’il contrôlerait, au niveau de l’initiation de la traduction, l’expression de l’opéron thiBPQ encodant un transporteur ABC de la thiamine. De plus, un petit ARN nommé SroA a précédemment été identifié grâce à des techniques de séquençage d’ARN. SroA correspond à l’aptamère du riborégulateur thiB, mais aucun rôle ne lui a été attribué à ce jour. Le présent mémoire porte sur la caractérisation du riborégulateur thiB d’E. coli. Dans un premier temps, nous avons démontré que le riborégulateur est fonctionnel. Le mécanisme permettant la régulation génétique en cis est cependant plus complexe que seulement la régulation prédite au niveau traductionnel. Dans un second temps, nous avons utilisé deux approches différentes à l’échelle transcriptomique afin de vérifier si SroA régule l’expression de certains ARNm en trans. Plusieurs cibles potentielles découlent de cette étude. Une caractérisation préliminaire de certaines d’entre elles est présentée ici et devra être poursuivie par des travaux subséquents. Les résultats présentés ici suggèrent que le riborégulateur thiB régulerait l’expression de l’opéron thiBPQ (en cis) et d’autres gènes en trans.

Contamination of drinking water sources, such as groundwater, by pathogens (protozoa, bacteria and viruses) is of major concern globally. Due to their small size, mobility and high infectivity, enteric viruses have been a focus of groundwater research. However, the behaviour of enteric viruses in aquifer media is still poorly understood, which is partially attributable to the lack of reliable surrogates for these viruses. In the study reported in this thesis, a new type of surrogate was characterised and validated for its use in studying virus fate and transport in groundwater. The surrogates developed were composed of 70 nm carboxylated silica nanoparticles, labelled with dsDNA tags for sensitive detection, and coated with selected proteins to mimic the physico-chemical characteristics (size, charge, density) of two enteric viruses, human rotavirus and adenovirus, frequently found in faecal-contaminated groundwater. The selected enteric viruses and a commonly used virus surrogate, the MS2 bacteriophage, were purified and characterised in terms of size, surface charge, hydrophobicity and aggregation. For validation, the characteristics, the adsorption, degradation and transport of the surface-modified nanoparticles and the viruses were investigated in laboratory studies and compared. The characterisation of the viruses and particles revealed that the modified silica nanoparticles resemble the size and negative surface charge of the rotavirus and adenovirus. In general, the nanoparticles were found to be less hydrophobic than the enteric viruses, thus presumably less interactive with hydrophobic media. In contrast, the MS2 bacteriophage was smaller in size than the enteric viruses studied and considerably more hydrophobic implying stronger interactions with hydrophobic media. The surface-modified nanoparticles were found to be more stable and remained more monodispersed over time than the purified enteric viruses. In laboratory studies using simulated groundwater, the DNA-labelled nanoparticles were more stable over time than the rotavirus, the adenovirus or a plasmid DNA on its own. Interestingly, the study revealed that rotavirus was more persistent than the adenovirus over time in terms of degradation and aggregation, however, day light considerably enhanced rotavirus degradation. The adsorption studies revealed strong interactions between the enteric viruses and natural aquifer media (gravel and sand), whereas most of the surface-modified nanoparticles adsorbed weakly to these media. Only the casein-coated nanoparticles adsorbed strongly to the sand. The MS2 adsorbed to the gravel strongly, but weakly to the sand implying different interactions. The studies on virus and nanoparticle adsorption to hydrophobic-coated and non-modified Ottawa sand supported the results of characterisation. Column studies investigating the transport of the viruses and the nanoparticles in gravel and sand showed that even though gravel had high adsorption capacity in the adsorption tests, all viruses and nanoparticles travelled though the gravel columns with little retention, probably due to insufficient interaction time. This highlights the vulnerability of gravel aquifers to virus contamination. Experiments using sand columns showed great differences in the transport of the particles. Results suggested that the recovery of the DNA-labelled nanoparticles was similar to the recovery of the adenovirus, however, their transport pattern was different. The glycoprotein-, the protein A- and the AMBP-coated nanoparticles mimicked the transport pattern and low recovery of the rotavirus. In contrast, the streptavidin- and casein-coated nanoparticles were not recovered, emphasising the great importance of surface structure in particle transport. The results of this study demonstrated the usefulness of protein-coated silica nanoparticles as virus surrogates in groundwater studies. Surface-modified nanoparticles are able to mimic the surface characteristics of viruses. The glycoprotein-, protein A- and AMBP-coated particles were found to be suitable surrogates for rotavirus, whereas the DNA-labelled nanoparticles resembled adenovirus behaviour in hydrophilic media. Using particles with different material, size and protein-coating other pathogens can be modelled as well. Furthermore, these particles are expected to besafe to humans and the environment, thus can be used in a great variety of experiments in environmental research.

This research aimed to address the knowledge gaps in the literature regarding the removal of waterborne pathogens (viruses and protozoa) by modified multistage slow sand filtration. In the current study, two pilot-scale multistage slow sand filtration systems were operated continuously for over two years. The pilot systems treated agricultural- and urban-impacted raw river water of variable quality with turbidity peaks over 300 NTU and seasonal cold temperatures <2??C. The first system (Pilot 1) consisted of two independent trains that included pre-ozonation, shallow-bed upflow gravel roughing filtration, and shallow-bed slow sand filtration. Pilot 1 was a pilot-scale version of an innovative, commercially available full-scale system. The second system (Pilot 2) included a full-depth upflow gravel roughing filter, a full-depth slow sand filter, and a second shallow-depth slow sand filter in series. The SSFs of both pilots were operated at high hydraulic loading rates (typically 0.4 m/h) at the upper limit of the literature recommended range (0.05 to 0.4 m/h). Both pilot systems provided excellent turbidity removal despite the high filtration rates. Effluent turbidity of all multistage SSF pilot systems were within the regulated effluent limits in Ontario for full-scale SSFs (below 1 NTU at least 95% of the time and never exceeded 3 NTU), despite raw water turbidity peaks over 100 NTU. The roughing filters contributed to approximately 60-80% of the full-train turbidity removal, compared to and 20-40% for the slow sand filters. On average, the second slow sand filter in pilot 2 provided almost no additional turbidity removal. The slow sand filter run lengths were short because of frequent high raw water turbidity, with about 50-80% of the runs in the range of 1-3 weeks. To prevent excessive SSF clogging and maintenance, filtration rates should be decreased during periods of high turbidity. Seven Cryptosporidium and Giardia challenge tests were conducted on the slow sand filters of both pilot systems at varying filtration rates (0.4 or 0.8 m/h), temperatures (2 to 25??C), and biological maturities (4 to 20 months). Removal of oocysts and cysts were good regardless of sand depth, hydraulic loading rate, and water temperature in the ranges tested. Average removals in the SSFs ranged from 2.6 to >4.4 logs for Cryptosporidium oocysts and ranged from >3.8 to >4.5 logs for Giardia cysts. This was consistent with findings in the literature, where oocyst and cyst removals of >4 logs have been reported. Cryptosporidium oocyst removals improved with increased biological maturity of the slow sand filters. At a water temperature of 2??C, average removal of oocysts and cysts were 3.9 and >4.5 logs, respectively, in a biologically mature SSF. Doubling the filtration rate from 0.4 to 0.8 m/h led to a marginal decrease in oocyst removals. Sand depths in the range tested (37-100 cm) had no major impact on oocyst and cyst removals, likely because they are removed primarily in the upper section of slow sand filter beds by straining. In general, good oocyst and cyst removals can be achieved using shallower slow sand filter bed depths and higher filtration rates than recommended in the literature. There are very few studies in the literature that quantify virus removal by slow sand filtration, especially at high filtration rates and shallow bed depths. There are no studies that report virus removal by slow sand filtration below 10??C. As such, 16 MS2 bacteriophage challenge tests were conducted at varying water temperatures (<2 to >20??C) and filtration rates (0.1 vs. 0.4 m/h) between February and June 2006 on biologically mature slow sand filters with varying bed depths (40 vs. 90 cm). Biologically mature roughing filters were also seeded with MS2. Average MS2 removals ranged from 0.2 to 2.2 logs in the SSFs and 0.1 to 0.2 logs in the RFs under all conditions tested. Virus removal by slow sand filtration was strongly dependant on hydraulic loading rate, sand depth, and water temperature. Virus removal was greater at a sand depth of 90 cm vs. 40 cm, at an HLR of 0.1 m/h vs. 0.4 m/h, and at warm (20-24??C) vs. cold (<2-10??C) water temperatures when sufficient warm water acclimation time was provided. Increased sand depth likely increased MS2 removal because of greater detention time for predation and greater contact opportunities for attachment to sand grains and biofilms. A lower HLR would also increase MS2 removal by increasing detention time, in addition to decreasing shear and promoting attachment to filter media and biofilms. Greater MS2 removal at warmer water temperatures was attributed to improved biological activity in the filters. Schmutzdecke scraping was found to have only a minor and short-term effect on MS2 removals. Virus removal can be optimized by providing deep SSF beds and operating at low filtration rates. Virus removal may be impaired in cold water, which could affect the viability of using SSF/MSF at northern climates if communities do not use disinfection or oxidation. As a stand-alone process, slow sand filtration (with or without roughing filtration) may not provide complete virus removal and should be combined with other treatment processes such as disinfection and oxidation to protect human health.

The Chattahoochee River is the most utilized surface water in Georgia, and it and Utoy Creek are receiving waters for Atlanta stormwater and wastewater effluent. Population growth and record-breaking rainfall in 2013 has led to potential stress from stormwater runoff and nonpoint source loading. The goals of this research are to examine spatial and temporal variations in E. coli and the bacteriophage MS2 and relationships with DO, turbidity, rainfall, and riverflow; to determine if E. coli in water is correlated with E. coli in sediment; and to determine if wastewater effluent discharges influence downstream sample sites. Water samples were collected at fifteen sample sites and two outfall sites in the Chattachoochee, and ten sites in Utoy Creek. No significant spatial variation in E. coli was found for the Chattahoochee, although there was significant temporal variation in mean E. coli concentrations. The lowest mean DO values and the highest mean turbidity levels both occurred on the date of the highest mean E. coli concentrations. Effluent from the two outfalls did not contaminate downstream sample sites. In Utoy Creek, E. coli concentrations showed spatial and temporal variation in water samples, but not for sediment samples. Turbidity was found to be positively correlated with both E. coli in sediment and MS2. These findings suggest that nonpoint source loading is a potential cause of contamination. Since DO, turbidity, and rainfall were correlated with E. coli and MS2, these parameters could be used as indicators of pollution for future monitoring of the Chattahoochee River and Utoy Creek.

Protein analysis is important to understanding biological systems, but sample diversity necessitates a multitude of analysis techniques and methods. Challenges that are addressed include analysis of low abundance samples, fractionation to reduce sample complexity, and automation to reduce time and cost. Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important technique for protein characterization. In Paper I, the sensitivity of MALDI-MS was enhanced through the fabrication of a hydrophobic coating for the MALDI target plate, yielding analyte concentration. The plate outperformed a commercial concentration plate. Capillary electrophoresis (CE) separation offers low sample consumption and high efficiency, and in Paper II, offline CE-MALDI-MS fractionation was employed. A robot system for automation was constructed and used in analysis of spermatophore proteins from the butterfly Pieris napi. The robot was also used in automated on-target trypsin digestion under a lid of liquid fluorocarbons, a simpler and cheaper alternative to controlled humidity chambers. An indication of indigenous proteolysis of the sample was seen. Electrospray ionization (ESI) is the other technique for protein analysis in MS. In Paper III, the biomarker protein osteopontin (OPN) was analyzed by ESI-MS in order to find suitable conditions for its detection. A preliminary optimization of solvents and ionization conditions was done, and tandem MS (MSn) performed to increase the reliability of identification. / Proteinanalys är viktigt för att förstå biologiska system, men mångfalden av prov kräver en mängd olika analystekniker och metoder. Utmaningar som tas upp inkluderar analys av små provmängder, fraktionering för att minska provkomplexiteten, och automatisering för att minska tidsåtgång och kostnad. Matris-assisterad laserjoniserings-masspektrometri (MALDI-MS) är en viktig teknik för proteinkarakterisering. I Artikel I förbättrades känsligheten i MALDI-MS genom tillverkning av en hydrofob beläggning på MALDI-provplattan, vilket gav en koncentration av provet. Provplattan gav bättre resultat än en kommersiell koncentrationsprovplatta. Kapillärelektroforesseparation (CE) har låg provåtgång och hög separationseffektivitet och i Artikel II användes offline CE-MALDI-MS-fraktionering. Ett robotsystem för automatisering konstruerades och användes för analys av spermatoforproteiner från fjärilen Pieris napi. Roboten användes även i automatiserad trypsinklyvning under en yta av en flytande fluorkolförening, ett billigare alternativ tilli nkubationskammare med kontrollerad luftfuktighet. En indikation på naturlig enzymatisk proteinklyvning i provet hittades. Elektrospray jonisering (ESI) är den andra tekniken för proteinanalys i MS. I Artikel III analyserades biomarkören osteopontin (OPN) med ESI-MS för att hitta lämpliga förhållanden för dess detektion. En preliminär optimering av lösningsmedel och jonisationsförhållanden gjordes, och tandem-MS (MSn) utfördes för att öka identifikationens tillförlitlighet. / <p>Full text will not be uploaded due to unpublished results. QC 20181121</p>

Si la découverte récente de la souche virale JFH1, capable de réaliser un cycle viral complet en culture cellulaire, a permis de mieux caractériser le déroulement de l’infection du virus de l’hépatite C VHC, de nombreux aspects de la biologie du virus demeurent méconnus. Parmi ceux-ci, les mécanismes gouvernant l’encapsidation de l’ARN génomique viral au sein des particules restent à élucider. Cette thèse décrit la mise point d’un système de mobilisation hétérologue permettant l’analyse de l’interaction de la protéine de capside Core avec l’ARN viral. Ce système nous a permis d’identifier les régions de la protéine impliquées dans la liaison au génome viral, tout en autorisant son transfert dans des cellules naïves. Cette approche de mobilisation dépendante de la Core est complétée d’une étude de recrutement hétérologue basée sur la reconnaissance de l’ARN du phage MS2 avec la protéine de manteau Coat. Cette stratégie novatrice permet le recrutement efficace des ARNs marqués tout en autorisant leur localisation cellulaire. / The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. To study packaging in vivo, we developed a novel heterologous system to evaluate the interactions of HCV Core capside with viral RNA. This system allowed us to pinpoint Core domains involved in RNA binding, and package and transfer HCV RNA into a lentiviral vector. Aside from this Core dependent recruitment, we engineered retoviral vectors to trans-package MS2-tagged RNAs using MS2 Coat protein interaction. This system allowed us to efficiently recruit MS2-tagged replicons into naive cells and offers the opportunity to visualize HCV RNAs in Huh7.5 cells.

Même si les traitements thermiques ou la désinfection par les oxydants ont démontré leur efficacité virucide, les mécanismes liés à la perte du caractère infectieux ne sont pas connus. Ceci pose un réel problème d’interprétation de la présence de génome viral en matière de risque infectieux dans les aliments. Ce travail de thèse a pour objectif d’étudier l’évolution des propriétés de surface (charge et hydrophobie) de virus modèles, bactériophage MS2 et norovirus murin, au cours de l’inactivation par la chaleur, l’hypochlorite de sodium et l’ozone. Pour nos deux virus, nous démontrons l’existence d’une température critique au-delà de laquelle la particule virale se déstructure en libérant son génome. Un simple traitement à la RNase permettrait alors de ne détecter que des virus infectieux par biologie moléculaire. Le traitement thermique implique aussi une augmentation de l’hydrophobie soulignant des modifications conformationnelles de la capside. L’hypochlorite de sodium ne modifie que peu les propriétés de surface mais des phénomènes d’oxydation ont lieu au niveau de la capside puisque la charge du bactériophage MS2 est légèrement modifiée. Ces modifications diminuent la résistance thermique du virus. Nous démontrons un effet synergique de l’hypochlorite de sodium et la chaleur sur le bactériophage MS2 (inactivation, RNase et hydrophobie). Quant à l’ozone gazeux, nous soulignons son intérêt pour le traitement virucide des aliments fragiles. Ainsi, ce travail précise les mécanismes d’inactivation des virus et ouvre de nouvelles perspectives tant pour discriminer les virus infectieux et non-infectieux que pour proposer l’exploration de nouveaux traitements technologiques / Although heat treatments or disinfections by oxidants have proven their virucidal efficiencies, mechanisms related to the loss of infectivity are not known. This statement could lead to a misinterpretation of the presence of viral genome on infection risk for humans in food matrices. This thesis aimed to study the evolution of surface properties (charge and hydrophobicity) for model viruses, bacteriophage MS2 and murine norovirus, during the heat, sodium hypochlorite and ozone inactivations. For both viruses, the existence of a critical temperature beyond which the viral particle was disrupted and released its genome was demonstrated. Simple treatment with RNase would then only detect infectious virus by molecular biology. The heat treatment also involved a transient increase in the hydrophobicity which highlighted conformational changes of the viral capsid. Sodium hypochlorite slightly modified the surface properties but oxidation phenomena occurred onto capsid since the bacteriophage MS2 charge has changed a little. These changes decreased the thermal resistance of the virus. Synergistic effects of both sodium hypochlorite and heat were observed on the inactivation of MS2 phages, the sensitivity of their genome to RNases and the increase in hydrophobicity of remaining infectious particles. Regarding gaseous ozone, we underlined its interest in the case of virucidal treatment of fragile food matrices. Therefore, this work specified the virus inactivation mechanisms and opened up new perspectives to discriminate infectious from non-infectious viruses but also to propose the exploration of new technological processes

L’exposition aux aérosols microbiologiques présents dans les environnements clos est susceptible de provoquer, chez les occupants, diverses pathologies telles que des infections, des toxi-infections et des allergies. Pour s’en prémunir, diverses stratégies passant notamment par l’emploi de dispositifs épurateurs d’air, ont été développées et commercialisées par les industriels de la ventilation et du traitement de l’air. Cependant, à ce jour, aucune méthodologie d’évaluation y compris normative ne permet d’évaluer la pertinence de ces stratégies. Ce travail de recherche se propose, d’une part, d’appréhender le devenir des aérosols microbiologiques au sein des espaces clos : de la source à l’individu exposé, en explorant le rôle de la ventilation dans ce transport et, d’autre part, d’explorer le gain apporté par les nouvelles technologies de traitement microbiologique de l’air sur l’exposition des occupants. Pour ce dernier point de l’étude, une démarche globale d’évaluation en 3 volets a été adoptée avec l’étude de l’efficacité du ou des principes d’épuration mis en oeuvre, la détermination du rendement intrinsèque en condition dynamique de ces systèmes et l’évaluation du gain apporté par ces derniers sur l’exposition des occupants. Les travaux menés avec les dispositifs épurateurs (filtration et photocatalyse) ont montré que les efficacités intrinsèques des systèmes ne permettent pas de préjuger de leur gain vis-à-vis du niveau de l’exposition des individus lorsqu’ils sont mis en oeuvre en environnement intérieur. Les résultats obtenus ont également mis en évidence que la prise en compte des flux aérauliques et du transport des particules induit par la ventilation et le dispositif épurateur est indispensable à la définition d’une stratégie cohérente de traitement d’air / Exposure to bioaerosols in indoor environments is associated with a wide range of adverse effects on health including infectious diseases, acute toxic effects and allergies. In order to guard against this phenomenon, the ventilation and air treatment industry has developed and marketed many air control strategies. However, at present, there is no methodology adapted to the evaluation of the relevance of these strategies. The aim of this research work was to characterize, in a first time, the progress of microbiological aerosol from the original source, to their eventual inhalation by person exposed, considering their dissemination through the indoor environments. Secondly, the work consisted of determining the efficiency of air cleaner devices applied to control indoor air quality. For this point, a global approach of evaluation in 3 steps was adopted, consisting of studying the efficiency of the epuration principle implemented, determining the intrinsic performance of the systems in dynamic conditions and their impact on the exposure level of the exposed persons. The tests carried out with air cleaner devices (filtration and photocatalysis) have shown that the intrinsic performance wasn’t able to estimate the beneficial impact of these systems on the exposure level of people when there were applied in indoor environments. So the intrinsic performance of devices is not the single impact factor, the airflow promoted by the device is also a factor to consider. Moreover, the characterization of indoor airflows and airborne particles transport is essential to define a coherent strategy of air treatment