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Fungal infections of animals have become an increasingly important global issue. White-Nose Syndrome is an ongoing fungal epizootic of North American hibernating bats, caused by epidermal infections of the fungus, Pseudogymnoascus destructans. Infections emerged early in 2006 in New York State and have since spread to 35 US States and seven Canadian Provinces, with rates of mortality exceeding 90% in some bat colonies. As an emerging outbreak in North America, the transmission of P. destructans is assumed to occur in a radial fashion outwards from the point of origin. In addition, the factors that may influence P. destructans transmission have been postulated, but not tested before. Lastly, as reproduction is assumed to be strictly clonal in North America, invasive populations should have low genetic diversity, and may even accumulate deleterious mutations over time. The aim of my PhD research is to test these assumptions regarding the spread, evolution, and adaptation of P. destructans using combination of genotyping methods. My results showed how P. destructans isolates have shifted in terms of phenotypes and physiological capabilities since being introduced. In addition, I describe patterns of connectivity across the landscape, which are more consist with the level of anthropogenic activity than variation in climate. The mutations common to all invasive strains of P. destructans are associated with adaptations that have occurred since being introduced from Europe, some with relevant metabolic functions that fit their pathogenic lifestyle. Together, my results revealed significant phenotypic and genotypic changes during the spread of P. destructans in North America. The factors identified here that influence the phenotypic and genotypic changes should help developing better management strategies against the White-Nose Syndrome pathogen. / Thesis / Doctor of Philosophy (PhD)

New methods and technology are needed to detect biological agents that threaten the health of humans and domestic animals. The bacterium Bacillus anthracis, causal agent of anthrax, has been used as a biological warfare agent. Here, we extend the work of Chinowksy et al. (2007) to the detection of a surrogate of B. anthracis, B. globigii (also known as B. atrophaeus, B. subtilis var. niger, B. subtilis var. subtilis) in a mixed sample containing two different species of Bacillus using a portable surface plasmon resonance (SPR) biosensor (SPIRIT 4.0, Seattle Sensor Systems). Two methods (direct capture and antibody injection) were used to determine the limit of detection for spores of B. globigii and to detect spores of B. globigii in a mixed sample containing at least one other Bacillus spp. Spores of B. globigii were detected on freshly coated sensors (not previously exposed to spores) with direct capture at a minimum concentration of 10^7 spores/mL, and with antibody injection at a concentration of 10^5 spores/mL. Spores of B. globigii were also detected when mixed with B. pumilus spores in the same sample at equal concentrations (107 spores/mL) using antibody injection. An SPR method using synthetic miRNA was adapted to the portable SPR unit (SPIRIT), and preliminary experiments suggested that the target sequence could be detected. SPR methods using nucleic acids have an exciting future in the detection of biological agents, such as B. anthracis. With the availability of portable instrumentation to accurately detect biological warfare agents such as B. anthracis, emergency responders can implement emergency protocols in a timely fashion, limiting the amount of people and domestic animals exposed. / Master of Science

Spontaneous expulsion of various forms and types of matter from their solid substrates has always been an integral part of interfacial physics problems. A thorough understanding of such interactions between a solid surface and different soft materials not only expands our theoretical knowledge, but also has applications in self-cleaning, omniphobic surfaces and phase-change heat transfer. Although there is a renewed interest in the design of robust functional surfaces which can passively remove highly viscous liquids or dew, or retard ice accretion or frost formation, the physics of several dewetting and/or deicing mechanisms are yet to be fully understood. Even though we know how jumping-droplet condensation offers significantly better heat transfer performance than regular dropwise condensation and can liberate foreign particles, fundamental questions on the effect of surface orientation on jumping-droplet condensation or how it helps in large-scale fungal disease epidemic in plants are still unanswered. Thus, we first try to fill the knowledge gap in jumping-droplet condensation by characterizing their orientation-dependence and their role in a large-scale pathogenic rust disease dissemination among wheat. Unfortunately, understanding of such dewetting mechanisms does not necessarily translates to prevention or removal of ice and frost on subzero surfaces. Use of superhydrophobic structures or hygroscopic materials to retard the growth of frost was found to be limiting. Therefore the search for an efficient, inexpensive, and environmentally favorable anti-icing or de-icing mechanism is still underway. Here we give a framework for making a novel de-icing construct by analyzing a peculiar jumping frost phenomena where frost particles spontaneously jump off the surface when a polar liquid is brought above. Lastly, we demonstrate a simple and cost-effective technique to design a slippery liquid-infused surface from low-density hydrocarbon-based polymers, which is able to effectively remove a wide variety of soft materials. The main all-encompassing theme of this dissertation is to enhance our understanding of several dewetting phenomena, which might enable better design and/or mitigation strategies to control the expulsion of various forms of matter from a wide variety of surfaces. / Doctor of Philosophy / A few years back, a laundry detergent company in India came up with a famous ad campaign; it showed kids coming home from school with dirt all over their clothes to face the wrath of their parents. Rather than casually disparaging their mischievousness, the ad would make us think with their tagline: "Agar daag (Lit. stain, Fig. mess) lagne se kuch achha hota hain, toh daag achhe hain na? (Fig. If something good comes out of a mess, is it a mess?)". While this presents to us an excellent philosophical conundrum, in reality, we always find ways to get rid of foreign materials from surfaces of everyday use. Using water or dirt-repellent coatings on our shoes/clothes/car windshields or in worst case, spending hours trying to clean frost off our cars is something we are all familiar with. Finding innovative ways to remove unwanted materials from surfaces is not limited to humans, but also exhibited by various natural organisms. The excellent water repellency of lotus leaves, antifogging abilities of mosquito eyes or cicada wings, and slipperiness of pitcher plants are just few examples of natural self-cleaning surfaces designed to keep foreign materials or dew droplets off the surface. Sometimes we take a leaf or two out of these natural designs to help our cause. Surfaces with extreme water repellency are called superhydrophobic (hydro: water, phobos: fear). For a long time, gravity was considered to be the only passive droplet removal mechanism on these surfaces. About ten years ago, researchers found out that when two or more small dew droplets come together on these surfaces, they jump off the surface. Compared to the gravity removal, much smaller droplets can be removed via this method resulting in better anti-fogging qualities and heat transfer performance on the surface. As the jumping droplet event itself is independent of gravity, it was long assumed that the performance of these surfaces would not be dependent on their orientation. These jumped droplets can also take off with contaminating particles by partially or fully engulfing them. A recent study has brilliantly showed how rust spores are liberated from the superhydrophobic wheat leaves via jumping dew droplets. This fundamentally new mode of pathogen transport is yet to be fully understood at the same scale as we know wind or rain-induced fungal spore transport. In this work, we try to fill the knowledge gap by answering questions such as whether the surfaces with the abilities of gravity-independent jumping-induced droplet removal ironically fail to gravity and how far can spore(s) travel engulfed in a jumped droplet. But it is not just water droplets (or particles collected by water droplets) on a surface that we want to get rid off. The solid phase of water, i.e., ice or frost, when formed on regular surfaces, is actually harder to remove. The common ice-preventing surfaces are generally unable to stop complete frost formation and forces us to use salt or other moisture attracting chemicals to remove ice from a surface, knowing very well what is the economic and environmental cost of these chemicals. Here, we have introduced a novel de-icing mechanism by holding only a drop of water over a sheet of frost. The simplicity of our experimental setup may remind you the home physics experiments we all did in our childhood. We finish our discussion by designing a slippery surface from regular polymer films used in food packaging. Although the idea behind these slippery surfaces has been around since 2011, polyethylene films have never been used to make such surfaces before. Here, we show through extensive characterization that by choosing a suitable lubricating oil and a polyethylene-based film, we can finally get all of our ketchup to slide out of their packets, without struggle. If the future design of superhydrophobic condensers, de-icing constructs, or slippery surfaces benefit from the work reported here, may be I can finally say with certainty, "Daag Achhe Hain (Dirt is good.)."

Bacterial spore peptidoglycan (PG) is very loosely cross-linked relative to vegetative PG. Theories suggest that loosely cross-linked spore PG may have a flexibility which contributes to the attainment of spore core dehydration. The structure of the PG found in fully dormant spores has previously been examined in wild type and many mutant strains. These analyses showed little correlation between the degree of spore PG cross-linking and core dehydration. However, these studies only examined the structure of PG from dormant spores and did not allow for the structural analysis of spore PG during sporulation when actual spore PG synthesis and core dehydration occur. Structural analyses of developing spore PG from wild type Bacillus subtilis and eight mutant strains are included in this study. Structural analyses of developing spore PG suggest the following: a) the germ cell wall PG is synthesized first next to the inner forespore membrane; b) cross-linking is relatively high in the first 10% of spore PG synthesized; b) a rapid decrease in cross-linking is observed during synthesis of the next 20% of the spore PG; and c) this decrease is followed by an eightfold rise in the degree of cross-linking during synthesis of the final 70% of the spore PG. This increasing gradient of cross-linking was previously predicted to contribute to the attainment of spore core dehydration. However, analyses of mutant strains indicate this cross-linking gradient is not required for the attainment of spore dehydration. / Master of Science

Penicillin-binding proteins (PBP's) are membrane-associated enzymes involved in the polymerization of peptidoglycan. PBP's are divided into three classes based upon their molecular weights and functional domains. Gene expression is regulated in the two differentiated cells in Bacillus subtilis, the mother cell and the forespore, by coordinated expression of different sigma factors that recognize specific promoters in each compartment. The functional and compartmental specificity of individual penicillin-binding proteins from the different classes of PBP's were examined during sporulation in B. subtilis. Analyses of three class A high molecular weight PBP's indicated that pbpF and pbpG must be expressed in the forespore to carry out their specific role during spore peptidoglycan synthesis. Expressing pbpD in either the forespore or the mother cell could not complement for the loss of pbpF and pbpG, suggesting that there must be additional sequence information in PBP2c and PBP2d that allows them to carry out their specific role during germ cell wall synthesis. Analyses of a low molecular weight PBP, PBP5*, suggested that expressing dacB in either the mother cell or in the forespore could regulate the level of spore peptidoglycan cross-linking to what is typical of wild type spore peptidoglycan. / Master of Science

Components of the bacterial spore germination apparatus are crucial for survival and for initiation of infection by some pathogens. While some components of the germination apparatus are well conserved in spore-forming species, such as the spoVA operon, each species may possess a different and possibly unique germinant recognition mechanism. The significance of several individual proteins in the germination process has been characterized. However, the mechanisms of how these proteins perform their functions and the network connecting these proteins in the complete germination process are still a mystery. In this study, we characterized a Bacillus subtilis superdormant spore population and investigated the abundance of 11 germination-related proteins. The relative quantities of these proteins in dormant, germinating and superdormant spores suggested that variation in the levels of proteins, other than germinant receptor proteins may result in superdormancy. Specifically, variation in the abundance of the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Spore membrane proteomes of Bacillus anthracis and B. subtilis were characterized to generate a candidate protein list that can be further investigated. Proteins that were not previously known to be spore-associated were identified, and many of these proteins shared great similarity in both Bacillus species. A significant number of these proteins are implicated in functions that play major roles in spore formation and germination, such as amino acid or inorganic ion transport and protein fate determination. By analyzing the in vivo and in vitro activity of HtrC, we proved that the protease is responsible for YpeB proteolytic processing at specific sites during germination. However, without HtrC present in the spore, other proteases appear to degrade YpeB at a reduced rate. The activity of purified HtrC in vitro was stimulated by a relatively high concentration of Mn²⁺ or Ca²⁺ ions, but the mechanism behind the stimulation is not clear. We also demonstrated that YpeB and SleB, in the absence of their partner protein, were degraded by unknown proteases other than HtrC during spore formation. Identification and characterization of these unknown proteases would be a future direction for revealing the roles of proteases in spore germination. / Ph. D.

Notre thèse s’inscrit dans le cadre de la compréhension des mécanismes d’inactivation de microorganismes, principalement des spores bactériennes, suite à un traitement à l’ozone. Les objectifs généraux, au départ, étaient de mettre en place une stratégie expérimentale dans le but de déterminer les espèces et les phénomènes impliqués dans les mécanismes d’inactivation des spores en combinant diverses techniques pluridisciplinaires. Rappelons que les récentes avancées en stérilisation par plasma sont principalement dues aux études menées sur les mécanismes d’inactivation des microorganismes. Dans ce contexte, en nous appuyant sur une synthèse des études publiées dans la littérature scientifique, nous avons élaboré une étude expérimentale en deux étapes : l’étude de la stérilisation à l’ozone en milieu sec puis en milieu humide. Au cours de ces études, nous avons principalement étudié la cinétique d’inactivation des microorganismes, les dommages occasionnés à ces derniers par de tels traitements et nous avons identifié les espèces responsables de l’inactivation pour, enfin, proposer un mécanisme d’inactivation en spécifiant le rôle et l’importance des différents phénomènes en jeu. L’originalité de notre approche s’appuie sur la corrélation faite entre ces deux types de traitement (ozone sec et humidifié) ainsi que la diversité et la complémentarité des moyens de caractérisation utilisés. Dans un second volet, nous avons mis en évidence pour la première fois la possibilité de conférer à une surface de Pétri de polystyrène (PS) une importante activité biocide suite à un traitement à l’ozone. Nous avons mené une étude de caractérisation des propriétés de ces surfaces démontrant, principalement, leur grande efficacité biocide sur une large gamme de microorganismes, l’importance de l’interaction microorganismes/surface traitée, l’existence de la rémanence biocide, les modifications physico-chimiques induites en surface… L’étude des modifications physico-chimiques et microbiologiques nous a permis d’approcher les mécanismes d’inactivation en identifiant, de manière non exhaustive, les phénomènes impliqués. Enfin, nous avons examiné l’applicabilité de cette méthode à divers types de polymères montrant que le degré d’activité biocide induite dépend de la nature du substrat. / The thesis deals with the inactivation mechanisms of microorganisms (mainly endospores) after an ozone treatment. The general objective of this work was to provide an experimental strategy to identify the phenomena and chemical species involved in microorganism inactivation mechanism using multidisciplinary techniques. Recall that the recent advances in plasma sterilization are mainly due to comprehensive studies dealing with the inactivation mechanisms of microorganisms. In this context, based on a review of published studies, we proposed a two-step experimental study of ozone sterilization in both dry and humid media. In the course of this work, we have examined the inactivation kinetics of some microorganisms, their damage after treatments; we have also identified the chemical species responsible for inactivation, and proposed an inactivation mechanism (for each type of ozone treatment) by describing the importance and role of the different implicated phenomena. The originality of our approach rests on the correlation achieved between the dry and humid ozone treatment, and by the diversity and complementarity of the characterization techniques used. In a second part, we show for the first time the possibility to confer biocide activity to surfaces of polystyrene (PS) Petri dishes after their exposure to ozone. A characterization study of these treated surfaces mainly shows a high inactivation efficacy on various microorganisms, the importance of the microorganisms/treated-surface interaction, the biocide persistence of the treated surfaces and physico-chemical modifications. The study on physico-chemical and microbiological changes gives us elements for identifying the phenomena involved in the inactivation mechanisms. Finally, we have shown the possibility to confer biocide properties to polymeric surfaces in general with the same experimental process observing that the biocide efficacy depends on the nature of the polymer. / Une partie du travail a mené a un dépôt de brevet.

Concern regarding the health effects of indoor air quality has grown in recent years, due to the increased prevalence of many diseases, as well as the fact that many people now spend most of their time indoors. While numerous studies have reported on the dynamics of aerosols indoors, the dynamics of bioaerosols in indoor environments are still poorly understood and very few studies have focused on fungal spore dynamics in indoor environments. Consequently, this work investigated the dynamics of fungal spores in indoor air, including fungal spore release and deposition, as well as investigating the mechanisms involved in the fungal spore fragmentation process. In relation to the investigation of fungal spore dynamics, it was found that the deposition rates of the bioaerosols (fungal propagules) were in the same range as the deposition rates of nonbiological particles and that they were a function of their aerodynamic diameters. It was also found that fungal particle deposition rates increased with increasing ventilation rates. These results (which are reported for the first time) are important for developing an understanding of the dynamics of fungal spores in the air. In relation to the process of fungal spore fragmentation, important information was generated concerning the airborne dynamics of the spores, as well as the part/s of the fungi which undergo fragmentation. The results obtained from these investigations into the dynamics of fungal propagules in indoor air significantly advance knowledge about the fate of fungal propagules in indoor air, as well as their deposition in the respiratory tract. The need to develop an advanced, real-time method for monitoring bioaerosols has become increasingly important in recent years, particularly as a result of the increased threat from biological weapons and bioterrorism. However, to date, the Ultraviolet Aerodynamic Particle Sizer (UVAPS, Model 3312, TSI, St Paul, MN) is the only commercially available instrument capable of monitoring and measuring viable airborne micro-organisms in real-time. Therefore (for the first time), this work also investigated the ability of the UVAPS to measure and characterise fungal spores in indoor air. The UVAPS was found to be sufficiently sensitive for detecting and measuring fungal propagules. Based on fungal spore size distributions, together with fluorescent percentages and intensities, it was also found to be capable of discriminating between two fungal spore species, under controlled laboratory conditions. In the field, however, it would not be possible to use the UVAPS to differentiate between different fungal spore species because the different micro-organisms present in the air may not only vary in age, but may have also been subjected to different environmental conditions. In addition, while the real-time UVAPS was found to be a good tool for the investigation of fungal particles under controlled conditions, it was not found to be selective for bioaerosols only (as per design specifications). In conclusion, the UVAPS is not recommended for use in the direct measurement of airborne viable bioaerosols in the field, including fungal particles, and further investigations into the nature of the micro-organisms, the UVAPS itself and/or its use in conjunction with other conventional biosamplers, are necessary in order to obtain more realistic results. Overall, the results obtained from this work on airborne fungal particle dynamics will contribute towards improving the detection capabilities of the UVAPS, so that it is capable of selectively monitoring and measuring bioaerosols, for which it was originally designed. This work will assist in finding and/or improving other technologies capable of the real-time monitoring of bioaerosols. The knowledge obtained from this work will also be of benefit in various other bioaerosol applications, such as understanding the transport of bioaerosols indoors.