Per- and polyfluoroalkyl substances (PFASs) are persistent organic pollutants used in industrial applications and are globally distributed in the environment. A group of PFASs that are difficult to measure with today’s method are perfluoroalkyl acid precursors (PFAA precursors) that, when degraded, serves as indirect sources of PFAAs. This study has optimized a previously developed method for quantification of PFAA precursors in soil; through total oxidizable precursor assay (TOP assay) under alkaline conditions, to be applicable on sewage sludge. To achieve and maintain an alkaline environment during the entire oxidative treatment, several parameters were tested: concentrations of NaOH, persulfate and sample; additional clean-up with graphitized non-porous carbon and reaction time. Solid phase extraction-weak anion exchange (SPE-WAX) was used for clean-up and separation of analytes, and LC-MS/MS was used for quantification. The optimal conditions with the highest levels of PFAAs detected was obtained with 1.33 M NaOH, 60 mM persulfate, 3.57 g/L sludge with a reaction time of 6 hours. The use of graphitized non-porous carbon reduced matrix effects on oxidative conversion resulting in a higher pH as well as a higher degree of oxidation, but with some analyte loss.

The detection by serology of nematode-transmitted polyhedral viruses (nepoviruses) in grapevines is often unreliable. Nepoviruses were detected by enzyme-linked immunosorbent assay (ELISA) in tissue-cultured and field-grown grapevines. Nepovirus detection in in vitro plants (plantlets) was affected by virus distribution and growth room temperature. The reliability of virus detection in field-grown grapevines was improved when modified grinding buffers were used. Arabis mosaic virus (AMV) was detected by ELISA, for the first time, in in vitro grapevines initiated from field-and screenhouse-grown plants throughout the summer. The virus was not reliably and repeatedly detected in in vitro plantlets grown at 25°C. AMV and grapevine fanleaf virus (GFLV) distribution was not uniform throughout the plantlets. This distribution was affected by the culture room temperature. The best plant parts to sample for virus detection came from the zones of rapidly proliferating shoots. The viruses were sometimes not detected in samples taken from other tissues. Growth room temperature had an important effect on virus detection in plantlets. The highest virus titres were found in plants growing at 15°C. Temperature increases in 5°C steps to 30°C reduced virus titre. AMV became undetectable in nearly all plantlets growing at 30°C for as little as 30 days. Growth at 30°C reduced ELISA absorbance values by 76% after 8 days and after 21 days the values were at 4% of pre-treatment levels. The virus titre dropped below detectable levels in most plantlets. AMV could not be detected in plantlets or rooted explants after being placed in a 30°C treatment for 2 months. Tomato ringspot virus was detected by ELISA, for the first time, in in vitro grapevine plants. The virus was repeatedly detected in in vitro plants growing at 20°C. Under the typical summer conditions experienced at Sidney, B.C., modifying the standard ELISA grinding buffer (0.01 M phosphate buffered saline, pH 7.4, 0.05% Tween-20, 0.2% ovalbumin, 2% polyvinylpyrrolidone) was essential for reliable detection of AMV or GFLV. AMV was reliably detected by ELISA in foliar samples from field or screenhouse plants throughout the summer when the grinding buffer was modified by increasing the pH to at least 8.2 and adding 1% nicotine or 0.15 M phosphate buffered saline. The most reliable results with GFLV were obtained with the nicotine enhanced buffers. In comparison, because of the increased workload associated with growing plants in vitro and the unreliable detection of viruses in these plants, it remained preferable to detect nepoviruses in field plants by ELISA. / Land and Food Systems, Faculty of / Graduate

Les polyomavirus sont des virus très prévalents dans la population générale. Chez les sujets immunodéprimés, quatre polyomavirus sont associés à des pathologies. Parmi ces virus, le MCPyV est quant à lui responsable du carcinome à cellules de Merkel. Son implication dans un cancer humain a conduit à un regain d’intérêt pour la famille des Polyomaviridae. Au cours de ma thèse, nous nous somme intéressés à l’épidémiologie de six nouveaux polyomavirus humains (MCPyV, HPyV6, HPyV7, TSPyV, HPyV9 et MWPyV). Ces études ont montré que ces virus sont très répandus dans la population générale et que les infections surviennent dès l’enfance. Par ailleurs, nous nous sommes intéressés aux réactivités croisées entre polyomavirus humains et simiens phylogénétiquement proches. Nous avons montré qu’il existe des cross réactions entre le LPyV et l’HPyV9, entre le MCPyV et deux virus de chimpanzé (PtvPyV1 et PtvPyV2). Ces polyomavirus simiens ne circulent donc pas chez l’Homme. D’autre part, afin de mieux comprendre le cycle du MCPyV, nous avons initié l’identification des partenaires cellulaires de l’ensemble de ses protéines. Ce travail a tout d’abord été réalisé sur les protéines mineures de capside VP2/VP3. Des cribles en double hybride en levure ont permis d’identifier les partenaires cellulaires des VP2/3. L’interaction effective entre les protéines virales et cellulaires a ensuite été validée en cellules de mammifères par une approche de complémentation de la luciférase Gaussia princeps. Les partenaires cellulaires des protéines VP2/VP3 identifiés sont impliqués dans les voies de prolifération cellulaire, d’apoptose, NF?B et dans le transport intracellulaire du virus. / Polyomaviruses are ubiquitous in the general population and in immunocompromised patients, and four are associated with disease. 0f these viruses, MCPyV is responsible for Merkel ceil carcinoma, and its involvement in human cancer has led to a renewed interest in the Polyomaviridae family. My thesis work has focused on the epidemiology of six new human polyomaviruses (MCPyV, HPyV6, HPyV7, TSPyV, HPyV9 and MWPyV). These studies have shown that these viruses are widespread in the genera population and that infection occurs in early childhood. We have also focused on cross-reactivity between phylogenetically closely related human and simian polyomaviruses. We have shown that there is cross-reactivity between sirnian virus LPyV and HPyV9 and between MCPyV and two chimpanzee viruses (PtvPyVl and PtvPyV2). However, these simian polyomaviruses do not circulate in humans. Moreover, in order to improve understanding of the cycle of MCPyV, we set out to identify the cellular partners of its proteins. This work was initially performed on minor capsid proteins VP2 and VP3. Screening in yeast two-hybrid identified cellular partners of VP2 and VP3. Interactions between viral and cellular proteins were then validated in mammalian celis by complementation assay using Gaussia princeps luciferase. Cellular patners of VP2 and VP3 are involved in ceil proliferation, apoptosis, NFkB and intracellular transport of the virus.

Cancer immunotherapy is an encompassing term referring to therapeutic strategies that aim to boost the immune system to fight cancer. These strategies include administering immune cells that have been altered to have greater anti-tumor activity or using biologics and small molecules that target immune components to also promote tumor clearance. Natural Killer (NK) cells are cells of the innate immune system that recognize and kill abnormal cells such as cancer cells and play an important role in the anti-tumor response. Because of their crucial role in tumor immunity, NK cells are prime targets for immunotherapies. Repurposing small molecule drugs is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we screened 1,200 approved drugs from the Prestwick Chemical Library to identify drugs that increase NK cell cytotoxicity. We used a high-throughput luciferase-release cytotoxicity assay to measure the killing of the myeloid leukemia cell line, K562 cells expressing nano luciferase (NL) by NK92 cells, a human NK cell line. From the drug candidates identified from the screening assay, the antibiotic colistin sulfate increased cytotoxicity of the NK92 cell line and unstimulated human NK cells towards K562-NL cells. This increase in NK cytotoxicity was short-lived as pre-treating NK92 cells with colistin for 1 hour or 24 hours did not increase cytotoxicity. Also, we show pre-treating K562-NL target cells with colistin does not sensitize them to NK-mediated killing. Further studies are needed to uncover the mechanism of action of colistin, thus contributing to knowledge of fundamental NK cell biology regarding NK cell cytotoxicity which will aid in identifying additional small molecule drugs that enhance NK cell activity.

The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidosis, which is most commonly, caused by aggregation of Immunoglobulin (Ig) light chains or transthyretin (TTR) in the cardiac muscle, represent an important and often underdiagnosed cause of heart failure. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. AG10 is a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of TTR in serum samples obtained from patients with amyloid cardiomyopathy. The oral bioavailability and selectivity of AG10, makes it a very promising candidate to treat TTR amyloid cardiomyopathy. Understanding the reason behind the potency of AG10 would be beneficial for designing stabilizers for other amyloid diseases. This would be possible by designing and synthesizing structural analogues of AG10. Here we report the synthesis, characterization and analysis of AG10 analogs and the comparison of the in vitro activities of the synthesized analogs. The tremendous therapeutic potential of peptides has not been fulfilled and potential peptide therapies that have failed far outnumber the successes so far. A major challenge impeding the more widespread use of peptides as therapeutics is their poor pharmacokinetic profile, due to short In vivo half-life resulting from inactivation by serum proteases and rapid elimination by kidneys. Extending the In vivo half-life of peptides is clearly desirable in order for their therapeutic potential to be realized, without the need for high doses and/or frequent administration. Covalent conjugation of peptides to macromolecules (e.g. polyethylene glycol or serum proteins such albumin) has been the mainstay approach for enhancing the In vivo half-life of peptides. However, the steric hindrance and immunogenicity of these large macromolecules often compromises the In vivo efficacy of the peptides. Recently, our laboratory established the first successful reversible method of extending the half-life of peptides using serum protein TTR. The approach involved the use of a TTR Ligand for Half-life Extension (TLHE-1) which binds to TTR with high specificity and affinity. We have shown that our technology extends the half-life of multiple peptides without seriously affecting their activity. Our main objective here is to modify the structure of TLHE1 using linkers with different length and composition to optimize its affinity and selectivity for TTR in human serum.

The genotoxicological effects of five intra-peritoneal administered oestrogens (17β- oestradiol, daidzein, diethylstilboestrol, genistein, and equol), were examined. Male hooded- Lister rats were used to examine to what extent DNA damage occurred. The alkaline Comet assay was the chosen method used to assess double-strand DNA breakage by examining the Olive tail moment and %age tail DNA. Tissues from the testis, bone marrow, liver and blood were analysed after an 8-day duration of exposure. Statistically significant increases in DNA damage were observed in the testis with daidzein and in the blood with diethylstilboestrol. In addition, a further study was carried out to examine the effects of bulk and nanotised forms of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin and ibuprofen, in the Comet and micronucleus assays, on whole blood taken from prostate cancer patients or volunteers. These were used because it is known that the sensitivity of DNA to genotoxins can be heightened in patients with cancer. Patients’ and volunteers’ blood was cultured with either the bulk or nano-forms for 44 hours at 37°C, 5% CO2. Data were obtained for the Comet assay as above and the number of binucleated cells scored for the micronucelus assay. The results show the nanotised forms of the NSAIDs decreased the levels of strand breakage and lowered the numbers of micronuclei generated compared with their bulk forms. There was no clear difference between the sensitivity of the healthy controls and the prostate cancer patients, with only one individual showing evidence of heightened sensitivity.

Chronic inflammation is intimately linked with cancer development and progression and therefore reducing or eliminating inflammation represents a logical treatment and prevention strategy. Studies have shown that anti-inflammatory agents have anti-tumour effects in cancers, with reduced metastases and mortality. Current use of anti-inflammatory agents in the treatment and prevention of cancer is limited by their toxicity and side effects. The emerging field of nanotechnology allows the fundamental properties of a drug to be altered, creating a product with improved reactivity and bioavailability, leading to more targeted treatments and reduced dosage. In the present study, the genotoxic effects of three commonly used anti-inflammatory drugs; aspirin, ibuprofen and hydrocortisone, in their bulk and nano forms were evaluated on peripheral blood lymphocytes of healthy donors using the comet assay and the micronucleus assay. In order to determine any anti-cancer effects, these agents were also tested in peripheral blood lymphocytes in patients with haematological cancers. The glucocorticoid hydrocortisone was also evaluated for anti-oxidant capacity. Our results demonstrate that the nano versions of each drug produced a different response than the bulk counterpart, indicating that a reduction in particle size had an impact on the reactivity of the drug. Our results also indicate that the nano versions of each drug were less genotoxic than the bulk formulation, further emphasising the potential of nanoparticles as an improvement to current treatment options. We also found an anti-oxidant effect with hydrocortisone, with a more profound effect seen with the nano formulation.

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Les quantum dots (QDs) sont des cristaux semi-conducteurs de dimensions nanométriques. Ils peuvent être employés comme des marqueurs photosensibles du métabolisme cellulaire et peuvent être utiles dans différents domaines notamment en médecine mais il s’est rapidement avéré nécessaire de démontrer leur innocuité avant leur utilisation à grande échelle et leur diffusion dans l’environnement. Nous proposons un projet de thèse de doctorat sur le thème : La génotoxicité des quantum dots et le rôle du stress oxydant, implications sur l’environnement et la santé humaine. Il s’organise suivant trois axes: L’étude in vitro des propriétés génotoxiques et mutagènes des QDs Les QDs induisent des lésions primaires de l’ADN sur cellules CHO-K1 par le test des comètes qui sont associées à un stress oxydant. Ils sont plus actifs après irradiation par le spectre solaire. Ils induisent des mutations chromosomiques. L’étude in vivo des propriétés génotoxiques et mutagènes des QDs Les QDs induisent une augmentation significative des lésions de l'ADN chez le rat qui varie selon l’organe considéré (foie, rein, poumon, cerveau et testicule). Ils induisent une augmentation significative et une réponse dose-dépendante des micronoyaux indiquant nettement leur pouvoir clastogène/aneugène. Aucune variation significative des variables biochimiques mesurées n’est apparue. La mise en évidence de leurs effets sur l’environnement L'exposition aux QDs et au CdCl2 a entraîné une augmentation significative des lésions de l'ADN chez E. fetida et N. diversicolor. / Quantum dots (QDs) are semiconductor nanocrystals which can be employed as sensitive biomarkers of cellular metabolism and thus show their usefulness in various fields, including medicine and it soon became necessary to prove their safety before their widespread use and their distribution in the environment. The thesis project targeted on: Genotoxicity of quantum dots and the role of oxidative stress implications for the environment and human health. This study was organized in three main parts The in vitro study of the genotoxic and mutagenic properties of QDs QDs induced primary DNA lesions in CHO-K1 cells using the comet assay and were associated with oxidative stress. We demonstrated that the QDs were more active after irradiation by the solar spectrum. We showed the ability of QDs to induce chromosomal mutations. The main mechanism was probably that of the production of free radicals. The in vivo study of the genotoxic and mutagenic properties of QDs The comet assay shows that QDs induced an overall significant increase in DNA lesions of different organs (liver, kidneys, lungs, brain and testes). However, each organ had a specific susceptibility. QDs induced a significant increase in a dose-dependent manner of micronuclei. These results clearly indicated the in vivo clastogenic / aneugenic properties of QDs. No significant variation in the measured biochemical variables. The evidence of their effects on the environment Evaluation of genotoxicity was performed on coelomocytes of E. fetida and N. diversicolor resulting in a significant increase in DNA damage.

Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...