acase@tulane.edu / 1 / Rakesh Gurrala

Cyanobacterial blooms, such as those recently reported in Great Slave Lake (GSL, NWT), have sparked concern over the occurrence of toxic blooms in the North. This study investigated past and present incidences of cyanobacteria in lakes above latitude 60° N. The abundance of the toxin (microcystin) gene mcyE, as well as genes common to all cyanobacteria (16S rRNA) and bacteria (glnA) were quantified from lake sediment cores using ddPCR. Individual colony isolates from a surface bloom in Yellowknife Bay (GSL) in August 2015 were amplified and identified as non-toxigenic Dolichospermum lemmermannii. Very low levels of microcystin genes were detected through the sediment archives (over ~100-150 yr) of GSL and other lakes, as well as in the plankton of GSL. While recent increases in mcyE were not observed, an increase in the cyanobacterial 16S rRNA and glnA genes was seen through time. In the high Arctic Meretta Lake, gene abundance profiles reflected the effects of past eutrophication and recovery.

L'infection par le virus de l'hépatite B est un problème de santé publique avec 250 millions de porteurs chroniques et cela malgré l'existence d'un vaccin préventif. Les traitements actuellement utilisés sont les analogues de nucléos(t)ide et/ou l'interféron a. Bien qu'ils permettent une diminution de la charge virale, ils ne permettent pas d'éradiquer la maladie du fait de la persistance de l'ADNccc, le minichromosome de l'hépatite B. Cet ADN sert de matrice à la transcription virale, et la présence d'une seule copie permet la réactivation de l'infection. En prenant en compte la longue demi-vie des hépatocytes et de la stabilité de l'ADNccc dans leur noyau, un modèle mathématique suggère que de nombreuses années de traitement seraient nécessaires pour éliminer complètement cet ADN du foie des patients infectés chroniquement. Les techniques utilisées en routine pour la quantification de l'ADNccc ne sont pas assez sensibles pour pouvoir détecter des faibles concentrations de cet ADN, notamment dans des biopsies de patients infectés chroniquement et traités à long terme. Il est nécessaire de développer de nouvelles stratégies permettant de cibler directement l'ADNccc afin d'éliminer le virus. Ainsi les travaux de cette thèse reposent sur le développement d'une nouvelle technologie : la Droplet Digital PCR (ddPCR) pour permettre la quantification de l'ADNccc dans les biopsies de patient. Cette technique permet un gain de 2 log au niveau de la sensibilité par rapport à la qPCR, technique utilisée actuellement en routine. Elle nous a ainsi permis de constater la présence de cet ADN chez des patients traités à long terme par des analogues de nucléos(t)ides et même en présence d'interféron. La présence d'ARNpg et les expériences de ChIP ont également confirmé que l'ADNccc était encore transcriptionnellement actif. Ces résultats confirment d'autant plus la nécessité d'élaborer de nouvelles thérapeutiques pour permettre l'inactivation voire l'élimination de l'ADNccc. L'une des stratégies envisagées est le système CRISPR/Cas 9. Ainsi le dernier axe de cette thèse a été de développer ce système dans des modèles d'infection du virus de l'hépatite B. Pour vérifier l'efficacité de ce système sur le VHB, nous avons testé 8 ARN guide différents incorporer via des ribonucléoprotéines dans des cellules HepG2-NTCP. Les résultats préliminaires ont ainsi démontré que ce système pouvait réduire le pool d'ADNccc dans ces cellules et ouvre des perspectives intéressantes pour le développement de nouveaux traitements / Hepatitis B virus {HBV) is a major health problem with 250 million chronic carriers, despite the existence of a preventive vaccine. Currently the treatments used are nucleos{t)ide analogues and / or interferon a. Although they efficiently reach a decrease of the viral load, they do not allow the eradication the disease due to the persistence of the cccDNA, the minichromosome of the hepatitis B. This DNA serves as a template for the viral transcription and only a single copy suffice for the infection rebound. However, the techniques used routinely for the quantification of the cccDNA are not sensitive enough to be able to detect low concentrations of this DNA, in particular in biopsies of patients chronically infected and long term treated. ln addition, it is necessary to develop new strategies to target the cccDNA in order to clear the infection. Thus, my thesis work is based on the development of a new technology: the Droplet Digital PCR {ddPCR) to allow the quantification of cccDNA in patient biopsies. This technique allows a gain of 2 log in sensitivity compared to the qPCR technique currently used in routine. lt allowed us to see the presence of this DNA in long-term treated patients even in the presence of interferon. The presence of pgRNA and ChlP experiments also confirmed that the cccDNA was still transcriptionally active.These results confirm the requirement to develop new therapeutics to allow the inactivation or even the elimination of the cccDNA. One of the strategies envisaged is the CRlSPR / Case 9 system. Thus, the following part of this thesis was to develop this system in hepatitis B virus infection models. To reduce off-target effect we tested 8 different guide RNAs incorporated via ribonucleoproteins into HepG2- NTCP cells. Preliminary results have shown that this system can reduce the pool of cccDNA in these cells and open up the possibilities to test this model on PHH and opens interesting perspectives for the development of new treatments

Le cancer du pancréas est un cancer très agressif et de pronostic très sombre. Il est diagnostiqué tardivement et se montre résistant aux traitements. Dans ce contexte, il est nécessaire de mettre en évidence de nouveaux marqueurs spécifiques de cette pathologie dévastatrice. Le but de notre étude était de rechercher un marqueur « génétique » de ce cancer au sein du gène de la lipase sels biliaires-dépendante (BSDL) localisé en position 9q34.3 et plus particulièrement au niveau de l'exon 11 de ce gène codant pour le domaine C-terminal de la protéine et constitué de séquences répétées (séquences VNTR). L’analyse des électrophérogrammes obtenues après séquençage de Sanger à partir des amplicons de PCR réalisées sur l’ADNg extraits de tissus de patients atteints de cancer du pancréas, a permis d’identifier deux altérations génétiques : (i) la présence d’un SNP (Single Nucleotide Polymorphism) synonyme référencé rs488087, impliquant la transition c.1719C>T ; qui semble être associée au développement d’un cancer du pancréas sporadique et pourrait être un potentiel facteur prédictif de ce cancer permettant de cibler des populations à risque, (ii) la présence d’une insertion d’un nucléotide C induisant l'apparition d'une protéine BSDL tronquée présentant une séquence C-terminale modifiée contre laquelle ont été développé des anticorps polyclonaux. Cette nouvelle séquence pourrait constituer un potentiel marqueur diagnostique et/ou thérapeutique du cancer du pancréas. Ces deux altérations génétiques constituent ainsi de potentiels marqueurs du cancer du pancréas. / Pancreatic cancer is a devastating disease progressing asymptomatically until death within months after diagnosis. In this context, it is necessary to identify new specific markers to develop diagnostic tools and to target an at risk population. The aim of our study was to find a "genetic" marker in the bile salt-dependant lipase gene (BSDL). The human BSDL gene is located on the long arm of chromosome 9 in 9q34.3 with a variable number of tandem repeats (VNTR) in the coding region of exon 11. The electropherograms obtained after Sanger sequencing analysis of gDNA amplified from pancreatic cancer tissue samples allowed us to highlight: (i) the presence of a SNP (Single Nucleotide Polymorphism) involved in c.1719C>T transition which is referenced rs488087. rs488087 seems to be associated with the sporadic pancreatic cancer development and may be a predictive factor of pancreatic cancer for targeting an at risk population, (ii) the presence of a C nucleotide insertion leading to a premature stop codon with truncated protein and to the modification of the C-terminal sequence end. This new C-terminal sequence, alteration could be used as a potential diagnostic and/or therapeutic marker. Finally, these two genetic alterations identified in BSDL gene could constitute potential markers of pancreatic cancer.

IntroductionAcute myeloid leukemia (AML) is caused by a wide range of genetic aberrations, includingmutations within the genes that encode the enzymes isocitrate dehydrogenase 1 and 2(IDH1/2). Drugs that target mutant IDH1/2 are now available, which makes assessment of themutational status of IDH1/2 important in clinical diagnostics of AML. A promising method todetect these mutations is the droplet digital polymerase chain reaction (ddPCR), which showsadvantages of a high sensitivity and a simple workflow.AimTo evaluate ddPCR as method of choice to detect hotspot mutations in IDH1 (codon R132)and IDH2 (codon R140 and R172) in patients with AML.MethodsFifteen AML patients known to be positive for IDH1/2 diagnosed by a previously performednext generation sequencing (NGS) were selected for evaluation of ddPCR. Diagnosticsamples were tested for 14 patients, whereas follow-up samples were tested for one patient.ddPCR was performed using QX200™ Droplet Digital PCR system and data were presentedas fractional abundance of mutant allele.ResultsThe amount of mutant IDH1/2 in samples reported by ddPCR correlated well with the resultsfrom NGS when using probes that target their specific mutation. The detection limit formutant allele in the background of wild type IDH1/2 was 0,5% for IDH2 p.R140Q and 0.1%for IDH1 p.R132C/H.ConclusionddPCR that target specific mutations shows a great potential in measuring minimal residualdisease during follow-up. However, its use in screening for mutant IDH1/2 at the time ofdiagnosis is limited and alternative approaches should be considered.

Climate change and eutrophication, accelerated by anthropogenic activities, have impacted aquatic ecosystems worldwide. These impacts have stimulated the expansion of cyanobacterial blooms which pose severe threats to ecosystem functioning, environmental health, and the economy. However, the long-term effects of environmental change on bloom-forming cyanobacteria are not well understood as traditional paleolimnological approaches are of limited use in the reconstruction of cyanobacterial dynamics through time. Here, sediment DNA (sedDNA) was used to investigate long-term cyanobacterial trends using sediments from two experimental (fertilized L227 and acidified L223) and two reference (L224 and L442) lakes in the Experimental Lakes Area, Canada. First, to determine whether taxonomic bias might arise from the cyanobacterial sediment record, I performed a 1-year incubation experiment comparing the degradation rates of selected cyanobacterial genes under contrasting environmental conditions. Based on first-order linear decay models, Synechococcus sp. (Synechococcales) decayed the slowest under cold, anoxic conditions, followed by Trichormus (Nostocales), then Microcystis (Chroococcales), suggesting differential preservation of DNA. I then compared the quantitative performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the analysis of sedDNA and found that the ddPCR results were more consistent with the known history of the lakes. Furthermore, ddPCR showed that cyanobacterial abundance increased over the past century in all study lakes, but the greatest increase was observed in experimentally fertilized L227. HTS revealed shifts in the cyanobacterial community towards Nostocales dominance and a decrease in alpha diversity in response to phosphorus-only additions. An increase in abundance of the mcyE gene (indicative of microcystin producing taxa) was uniquely observed in L227 when nitrogen additions ceased. Heating degree days were important in explaining variation in the cyanobacterial community composition in all lakes, but nutrients had a greater influence on the L227 community. When sediment data were compared to historical surface water phytoplankton records, moderate to strong correlations between the two archives were found, validating the use of sedDNA. This research demonstrated that sedDNA can elucidate cyanobacterial trends at the community, population, and species level over multidecadal timescales in response to environmental change.

Den vanligaste cancerformen hos män är prostatacancer, med 10 000 nya dödsfall årligen. Vid prostatacancerdiagnostik utförs prostata biopsi. För att minska risken för komplikationer i samband med biopsi ges i Sverige en singeldos av antibiotikapreparatet Ciprofloxacin. Andelen bakterier som är resistenta mot ciprofloxacin har ökat. För detektion av genmutationer som orsakar antibiotikaresistens kan droplet digital PCR (ddPCR) användas. Det är en metod som ger en absolut kvantifiering av antalet DNA-sekvenser. Den är baserad på vatten olja-emulsionsdroppsystem.  Syftet med denna studie var att optimera och validera en digital droplet PCR för att detektera och kvantifiera mutationen S83L i gyrA genen från faecesprover, samt att jämföra digital droplet resultat från studieprover med odlingsresultat från resistensbestämning och ration mellan S83L allelen och vildtyps allelen i prover tagna före och efter biopsi. För att optimera och validera metoden användes prover tagna före och efter biopsi från nio patienter som genomgått en transrektal prostatabiopsi och fått en dos antibiotika profylax i samband med ingreppet.  Den optimala annealingtemperaturen bedömdes vara 60°C och den optimala  primer- och probekoncentrationen bestämdes till 1,2 µM respektive 0,4µM. Dessa koncentrationer gav lägst antal falskt positiva droppar. Den minsta detektionsnivån för S83L gyrA (EC40) var 160 kopior/ml och för vildtyps gyrA (EC108) var det 78 kopior/ml. Resultatet från valideringen visade att både vildtyps gyrA och S83L gyrA kunde detekteras och kvantifieras i rektalprover från samtliga patienter. / The most common form of cancer in men is prostate cancer, with 10,000 new deaths annually. In prostate cancer diagnosis, prostate biopsy is performed. To reduce the risk of complications in connection with biopsy, a single dose of the antibiotic drug Ciprofloxacin is given in Sweden. The proportion of bacteria that are resistant to ciprofloxacin has increased. For the detection of gene mutations that cause antibiotic resistance, droplet digital PCR (ddPCR) can be used. It is a method that provides an absolute quantification of a DNA sequence in a sample. It is based on water oil emulsion drop system.  The purpose of this study was to optimize and validate a digital droplet PCR to detect and quantify the S83L mutation in the gyrA gene from faecal samples and to compare digital droplet results from study samples with culture results from resistance determination and the ration between the S83L allele and the wild-type allele in samples taken before and after biopsy. To validate the method, samples taken before and after biopsy were used from nine patients who had undergone a transrectal prostate biopsy and received a dose of ciprofloxacin or trimethoprim in connection with the procedure.  The optimal annealing temperature was determined to be 60 °C and the optimal primer and probe concentrations were determined to be 1.2 µM and 0.4µM, respectively. These concentrations gave the lowest number of false positive droplets. The minimum detection level for S83L gyrA (EC40) was 160 copies/ml and for wild-type gyrA (EC108) it was 78 copies/ml. The results showed that both wild-type gyrA and S83L gyrA could be detected and quantified in rectal samples from all nine patients.

No description available.

Plasmids are small circular DNA molecules within bacterial cells that are separated from the bacterial chromosome and replicate independently. Also, they play a crucial role in the dissemination of antibiotic resistance genes among bacteria through horizontal gene transfer. They can be present in many copies within host cell, which is known as plasmid copy number. Plasmids can regulate their own copy number by different mechanisms. Additionally, the selective pressure can also play a pivotal role in determining plasmid copy number. The presence of antibiotics in the surrounding environment can drive variations of plasmid copy number. In this study, we examined plasmid copy number variations of multidrug resistance plasmids in presence of antibiotics by using EvaGreen® - based multiplexed digital droplet PCR. We could observe that cultures of Klebsiella pneumoniae and Escherichia coli harboring multidrug resistance plasmids grown in presence of sub-MIC concentrations of the antibiotics did not show high variations in plasmid copy numbers. On the other hand, mutants of K. pneumoniae selected for increased antibiotic resistance showed high increases in copy number of a multidrug-resistance plasmid.

Les travailleurs impliqués dans la gestion des matières résiduelles domestiques sont continuellement exposés à des bioaérosols pouvant entrainer des problèmes de santé de nature infectieux, allergène et cancérigène. Le but de cette étude était d’évaluer l’exposition aux bioaérosols des éboueurs et des chauffeurs de camion durant la collecte des déchets et des matières résiduelles à Montréal par une analyse multimétrique et multimédia. Les conditions de travail ont été évalués dans six camions (2 recyclages, 2 organiques et 2 domestiques). Pour chaque camion, des mesures ambiantes (cabine du chauffeur) et des mesures dans la zone respiratoire d’un collecteur et d’un chauffeur ont été réalisées. Des prélèvements sur la surface des sièges des chauffeurs ont aussi été effectués et les filtres à air de l’habitacle des camions ont été évalués. Les échantillons ont été analysés au laboratoire microbiologique de l’IRSST selon la méthode par culture microbienne et la méthode moléculaire Droplet Digital PCR. Les résultats indiquent que les collecteurs sont généralement les plus exposés aux bioaérosols. Les collecteurs des déchets domestiques étaient notamment les plus exposés avec des concentrations moyennes pour les bactéries (27 000 UFC/m3), les endotoxines (100 UE/m3) et les moisissures (5 900 UFC/m3) excédant les recommandations sanitaires. Néanmoins, les collecteurs du compost étaient les plus exposés aux moisissures (6 800 UFC/m3). E.coli et A.fumigatus ont été détectés dans tous nos échantillons avec des expositions plus importantes pour les travailleurs du domestique. De plus, les sièges des chauffeurs des camions domestiques avaient les niveaux de contamination les plus élevés. La concentration moyenne de A.fumigatus (2 500 UG/m3) dans l’air de ces camions était supérieure à celle des camions de recyclage et de compost. Les résultats des filtres n’ont pas permis de tirer de conclusions quant à l’exposition des chauffeurs aux bioaérosols. Les résultats de cette recherche ont permis de mettre en évidence que l’exposition des travailleurs lors de la collecte des déchets pourrait être influencée par le type de déchets, le poste de travail ainsi que les tâches exécutées. Cette étude confirme le potentiel élevé d’exposition aux bioaérosols des travailleurs attitrés au transport des matières résiduelles et des déchets. Elle valide aussi le besoin de réduire les expositions professionnelles par diverses stratégies, y compris les procédures de nettoyage de la cabine et l’utilisation d’équipement de protection respiratoire lors de tâches génératrices de bioaérosols (nettoyage des camions au jet, déchargement des camions). / Workers involved in the management of household residual materials are continuously exposed to bioaerosols which can cause them health problems of an infectious, allergenic and carcinogenic nature. The purpose of this study was to assess the exposure to bioaerosols of collectors and trucks drivers during the collection of domestic waste and residual materials in Montreal by a multimeric and multimedia analysis. A sampling campaign was conducted on six drivers and six collectors. A total of 6 trucks that collect recyclable (2), organic (2) and compost (2) waste were evaluated. Samples were also taken from the surface of the drivers' seats, and the cabin air filters were recovered. Samples were analyzed at the microbiology laboratory of the IRSST using the microbial culture method and the molecular method of Droplet Digital PCR (ddPCR). The results indicate that collectors are generally the most exposed to bioaerosols. Domestic waste collectors were in particular the most exposed with average concentrations for bacteria (27,000 CFU/m3), endotoxins (100 EU/m3) and fungi (5,900 CFU/m3) exceeding health recommendations. However, the compost collectors were the most exposed to fungi (6,800 CFU/m3). E. coli and A. fumigatus were detected in all of our samples, but exposures were greater for workers collecting domestic waste. In addition, the seats of drivers involved in domestic waste collection had the highest levels of contamination. The average concentration of A. fumigatus (2,500 UG/m3) in the air of the cabin of domestic waste trucks was higher than that of recycling and compost trucks. The results of the filters did not allow us to draw any conclusions on their role in terms of drivers exposure to bioaerosols. The results of this research highlighted that workers exposure to bioaerosols could be influenced by factors such as the type of waste, the workplace and workers’ tasks performed. This study confirms the high potential for exposure to bioaerosols of workers assigned to the transport of household residual materials and waste. It also validates the need to reduce workers exposures by various strategies including, cabin cleaning procedures and the use of respiratory protection equipment for specific tasks generating bioaerosols (water jet cleaning of trucks, unloading of trucks).