Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Harmattan and Dust (sand) storms together with anthropogenic activities including the use of firewood and kerosene as fuel for cooking, and diesel/petrol generators for electricity generation are potential sources of particulate and gaseous pollutants in homes in Damaturu town, Nigeria. Other activities like the burning of locally produced incense and mosquito coils as well as the use of aerosol sprays are further possible sources of indoor pollution, which may result in exposure of people to a range of pollutants through inhalation, by ingestion of settled dusts as well as dermal contact. Local people associate occurrence of dust events with adverse health effects and hence there is a need for an understanding of the composition of the settled and airborne dusts in order to assess the possible associated health risks. The first phase of the study involved selection and development of methods of dust sampling and analysis. For validation of the methods employed and to establish a broad understanding of the characteristics of the settled dusts, an initial survey study was conducted involving the application of thermal desorption/gas chromatography/mass spectrometry (TD/GC/MS) analysis for organic compound analysis, scanning electron microscopy (SEM), inductively coupled plasma-mass spectrometry (ICP-MS) for analysis of metals, and microbiological analysis. Airborne samples were also collected using sorbent tubes to determine organic compounds in air during activities such as cooking with kerosene, gas, and firewood as well as during electricity generation with fossil fuels. Carbon monoxide (CO) and ultrafine particles (UFPs) monitored simultaneously during some of the household activities. The study involved a novel method of extracting organic chemical emissions from dust by heating of the dusts directly in a micro chamber (μ-CTETM) and collection of emissions on sampling tubes. The method provided a relatively quick way of collecting chemical emissions from dusts that are readily available for release. The sampled tubes were analysed by TD/GC/MS. The conventional solvent extraction of the dusts was also carried out and the extracts were analysed by liquid injection-GC/MS and results of the two methods compared. The study determined a number of constituents (metals, SVOCs, phthalates and physical properties) of dusts collected from households in Damaturu during different weather events and from different indoor/outdoor locations; and compared with some UK samples. The samples investigated include dusts deposited; during two notable dusty-weather events (Harmattan and Storm) as well as when there was no notable dust event; during human activities; and dusts from different types of buildings (modern and traditional homes) as well as inside and outside homes. A standard reference material for organic chemicals (SRM 2585) was also analysed. The physical characterization of the settled house dust samples analysed revealed the various shapes and sizes, and elemental composition of the constituents, which included respirable particles. The microbial analysis also indicated the presence of the spores of a host of fungi and bacterial species; and the possible contributions of household activities to the increased production of pollutants (UFP and CO) ascertained. The μ-CTE extraction of the house dusts by heating with TD/GC/MS analysis of the emissions as well as the solvent extraction-GC/MS revealed the presence of many organic chemical compounds with different analytical retention times and varying concentrations in the dust samples. Chemicals of interest quantified: benzene, hexanal, nonanal, diethyl phthalate (DEP), diisobutylphthalate (DIBP), dibutylphthalate (DBP), and diethylhexylphthalate (DEHP). A host of other chemicals commonly present in the analysed samples identified using the NIST library associated with the MS system software. These chemicals included naphthalene and C10-C16 aliphatic and aromatic hydrocarbons, which would need confirmation by running the pure compound samples. There was an observed higher concentration of the chemicals in the solvent extracts than the μ-CTE extracted dust. The higher concentration of the chemicals in the solvent extracts expected due to the aggressive removal of the chemicals by the organic solvent whereas in the case of thermal extraction only the readily available chemicals (loosely bound to the matrix) released by increases in temperature were removed. Generally, the concentrations of the chemicals found were higher in the indoor than in the outdoor dust samples. In the analysis of the dusts collected during weather events; higher chemical concentrations observed in the samples collected during Harmattan period than the other periods. The Harmattan dust period may pose increased exposures to dust and possible health risks. More exposure is expected to occur in the traditional homes compared with the modern homes due to the higher concentrations of the chemicals in both the indoors and the outdoors and this may be especially important to women and children who spend most of their times at home. Metal analysis involved microwave-assisted digestion of the dust samples followed by ICP-MS analysis. The total quant method of metal analysis for a general profiling indicated the presence of more than 50 elemental contaminants in house dust. The results of the quantitative analysis for six target metals: Cd, Cr, Cu, Ni, Pb, and Zn showed their presence in all indoor and the outdoor dust samples. The mean concentrations showed that the metals were in higher concentrations in the indoor dusts than in the outdoor dusts. The quantitative analysis carried out indicated higher metal contents in the storm dusts than the dusts during the other periods. Results of the dusts collected from modern and traditional homes indicated the presence of the metals in higher concentrations in the dusts from traditional homes than the dusts from the modern homes. The estimated mean concentrations of the metals and phthalates inadvertently ingested as a constituent of dust indicated that some of the pollutants could exceed the tolerable daily intake (TDI) due to high exposures to dust expected to be the case in Damaturu. The results of the investigation of the dust composition, combined with information on exposure to dust and pollutants, show that dusts are a risk to the health of people in the Damataru community. Recommendations are made for more studies to provide a better understanding of dust ingestion and exposure to some phthalates and heavy metals in particular and the possible health risks. To the best of my knowledge, this is the first ever research study of airborne and settled dusts undertaken in North-Eastern Nigeria.

Clostridium difficile is a Gram positive pathogen of significant importance in the UK, Europe and the USA. No vaccine has been developed and current treatments are focused on hospital management and the use of antibiotics. The disease is spread in hospitals in the spore form and the role of spores in C. difficile infecton is poorly understood. In this project spores of C. difficile have been characterised. The proteins from the outermost layers of the spore were identified and the genes cloned. Three of these surface proteins have unique enzymatic properties that maybe important for symptoms of disease. The ability of C. difficile spores to adhere to intestinal cells was found to be far greater than with live cells and through this we have identified that the spore may play an important role in colonisation. The regulation of spore coat gene expression during sporulation was also examined and temporal phases of genes expression identified. A major part of this project was to develop a mucosal vaccine to C. difficile. The approach used was to clone the C-terminus of toxin A onto the surface of Bacillus subtilis spores and use these recombinant spores to immunise mice and hamsters. We found that oral delivery of these spores conferred 75% protection to C. difficile infection in a hamster model of infection. Further, parenteral immunisation of the same antigens (toxin A and B) failed to generate mucosal responses and this showed that mucosal immunisation is critical for good protection. Finally, we found that antibodies to the C-terminus of toxin A were cross reactive to the C-terminus of toxin B. This showed that mucosal delivery of just the C-terminus of toxin A is sufficient to confer protection in an animal model of infection. The outcome of this work is that we have shown the parameters for successful immunisation and vaccination against C. difficile.

Advanced biomaterials and sophisticated processing technologies aim to fabricate tissue-engineering scaffolds that can predictably interact within a biological environment at a cellular level. Sterilization of such scaffolds is at the core of patient safety and is an important regulatory issue that needs to be addressed prior to clinical translation. In addition, it is crucial that meticulously engineered micro- and nano- structures are preserved after sterilization. Conventional sterilization methods involving heat, steam and radiation are not compatible with engineered polymeric systems because of scaffold degradation and loss of architecture. Using electrospun scaffolds made from polycaprolactone (PCL), a low melting polymer, and employing spores of Bacillus atrophaeus as biological indicators, we compared ethylene oxide, autoclaving and 80% ethanol to a known chemical sterilant, peracetic acid (PAA), for their ability to sterilize as well as their effects on scaffold properties. PAA diluted in 20% ethanol to 1000 ppm or above, sterilized electrospun scaffolds in 15 min at room temperature while maintaining nano-architecture and mechanical properties. Scaffolds treated with PAA at 5000 ppm were rendered hydrophilic, with contact angles reduced to zero degrees. Therefore, PAA can provide economical, rapid and effective sterilization of heat-sensitive polymeric electrospun scaffolds used in tissue-engineering.

The crayfish plague pathogen, the oomycete Aphanomyces astaci, has been decimating populations of European crayfish species for more than 150 years, and is therefore considered one of the 100 worst world's invasive species. A. astaci is highly specialised for a parasitic life, but it can be isolated from moribund crayfish and grown on synthetic media, as it is the case also for several other oomycetes (chapter 7). The life of A. astaci includes three basic forms: mycelium in host's tissues, and the infective units occurring in water, zoospores and cysts. All North American crayfish species tested so far have shown some resistance to A. astaci, i.e., they could carry the infection for long, serving as vectors of the pathogen. Massive sporulation from infected North American crayfish starts when the host is moulting, stressed, or dying (chapter 4). However, I could show in my experiments that some sporulation occurs even from apparently healthy and non-moulting American crayfish hosting A. astaci, so infected North American crayfish must be considered a permanent source of the infection (chapter 4). Five genotype groups of A. astaci have already been distinguished. Strains from a particular genotype group probably share the same original host crayfish species of North American origin. Nevertheless, they can...

Bacillus subtilis é uma bactéria gram positiva de solo, não patogênica, não colonizadora de tecidos, naturalmente transformável e formadora de esporos utilizada como modelo de estudo de bactérias gram-positivas. Essas características acarretam em vantagens para a produção de proteases de interesse industrial e para utilização como veículo de antígenos vacinais, porém a falta de vetores induzíveis torna sua utilização como ferramenta biológica pouco explorada. No presente trabalho descrevemos a construção de diferentes vetores capazes de expressar os antígenos subunidade B da toxina termo-lábil (LTB) e subunidade estrutural da fímbria CFA/I (CFAB) de Escherichia coli enterotoxigênica (ETEC) e avaliamos seu potencial vacinal. Foi avaliada a imunogenicidade de linhagens capazes de expressar LTB sob o controle de diferentes promotores: PgsiB (induzido em condições de estresse), PlepA (promotor constitutivo) e Pspac (induzido pela adição de IPTG) e em diferentes locais da célula (ancorada à parede celular ou secretada para o meio externo). Avaliamos ainda a imunogenicidade de linhagens capazes de co-expressar LTB e a listeriolisina O (LLO) de Listeria monocytogenes. O antígeno CFAB foi produzido no citoplasma ou ancorado à parede celular de B. subtilis em condições de estresse e as linhagens bacterianas administradas sozinhas ou conjuntamente com a toxina termo-lábil (LT) como adjuvante de mucosa. Camundongos imunizados com células ou esporos de B. subtilis recombinantes desencadearam respostas de anticorpos sistêmicos e secretados específicos para os antígenos (LTB e CFAB), não alterados pela adição do adjuvante. A expressão de LLO causou a supressão da resposta de anticorpos específicos para o antígeno LTB. Os resultados obtidos demonstram a viabilidade do uso de B. subtilis como veículo vacinal. / Bacillus subtilis is a gram positive, generally regarded as safe and spore forming soil bacteria used as a model for genetic and phisiological studies. This safety status allow its use as host for production of industrial protases and its application as vaccine vehicles, however the lack of epissomal inducible expression systems disable the exploration of this organism as a biotechnologic tool. In this work we describe the construction of epissomal vectors able to express the B subunit of the heat-labile toxin (LTB) and the structural subunit of the CFA/I fimbrae (CFAB) from the enterotoxigenic Escherichia coli (ETEC). We evaluate strains able to express LTB under the control of three promoters: PgsiB (stress inducible), PlepA (constitutive) e Pspac (IPTG inducible) and allowing the expression of LTB secreted or anchored to the cell wall We also evaluate the immunogenicity of strains able to co-express LTB and the listeriolysin O (LLO) from Listeria monocytogenes. CFAB was expressed in the cytoplasm or anchored to the cell wall and administred alone or with the mucosal adjuvant LT. Mice immunized both with cells or spores elicited secreted and systemic specific antibodies responses, which were not altered by the addition of the adjuvant LT. LLO expression suppressed the antibodies responses against LTB. The data shows the ability of B. subtilis to be used as vaccine vehicle.

Ziel der Arbeit war es, die Besonderheiten des Gärungsverlaufes bei der Silierung von nitratarmem Grünfutter aufzuklären. Dazu wurden sechs Silierversuche zur Untersuchung des Gärungsverlaufes mit unterschiedlichem Clostridiensporenbesatz durchgeführt. Dabei wurde auch die Wirkung eines Zusatzes von 0,05 bzw. 0,1 % N / TS als Nitrat und Nitrit sowie Zusätze von Inoculantien und Ameisensäure geprüft. Weiterhin wurde untersucht, ob die unter Laborbedingungen gefundenen Auswirkungen des Fehlens von Nitrat ebenso bei Grünfutter, das unter praxisnahen Bedingungen geerntet wurde, auftreten. Zur Fragestellung, welche Siliermittel bei nitratarmem Grünfutter eingesetzt werden können, wurde auch hier der Zusatz von zwei MSB-Präparaten und Ameisensäure, sowie eines nitrithaltigen Siliermittels bei geringem und erhöhtem Clostridiensporengehalt des Siliergutes geprüft. Als Ergebnis dieser mehrjährigen, umfangreichen Untersuchungen mit verschiedenen Futterpflanzen und unterschiedlicher Clostridiensporenbelastung hatte sich gezeigt, daß der Verlauf der Stoffumsetzungen und das Gärproduktmuster am Ende der Gärung in Abhängigkeit vom Nitratgehalt wesentlich unterschiedlich ist. In Silagen aus nitratarmem Grünfutter trat Buttersäure bereits von Gärbeginn und parallel zur Milchsäuregärung auf. Die Essigsäuregehalte waren stets sehr niedrig. Anaerob stabile und instabile Silagen aus nitratarmem Grünfutter weisen in allen Stadien des Gärungsprozesses ein völlig anderes Verhältnis zwischen Buttersäure und den übrigen Merkmalen des unerwünschten Stoffabbaus - Essigsäure, Ammoniak, pH - auf als Silagen aus nitrathaltigem Grünfutter. Für die Einschätzung der Vergärbarkeit sind außer TS und Z/PK auch der Nitratgehalt sowie epiphytischer Keimbesatz und Clostridiensporengehalt des AM zu berücksichtigen. Bei Fehlen von Nitrat besteht, unabhängig von der nach TS und Z/PK vorhergesagten Vergärbarkeit, ein besonderes Risiko für das Auftreten von Buttersäure. In Abhängigkeit vom Aufwuchs war die Einschätzung der Vergärbarkeit des Grünfutters verschieden und es trat eine unterschiedliche Gärqualität der Silagen auf. Bei nitratarmem Grünfutter ist der strategische Einsatz von MSB-Präparaten zu empfehlen. Das nitrithaltige Siliermittel hatte sich insbesondere bei erhöhter Clostridiensporenbelastung und/oder niedrigem TS-Gehalt des Grünfutters als sehr wirksam erwiesen. Bei Anwendung des derzeit gültigen, für Silagen aus nitratreichem Grünfutter entwickelten DLG-Beurteilungsschlüssels auf Silagen aus nitratarmem Ausgangsmaterial ist mit einer Fehlbewertung zu rechnen. / The object of this work is to explain the distinctive feature of ensilage of green forage low in nitrate. The fermentation process of ensiling green forage low in nitrate was proved in 6 experiments with different content of spores of clostridia. In all experiments the green forage was ensiled with following treatments: without additives (control), with 0,05 and 0,1% N / DM as nitrate or nitrite, with lactic acid bacteria and formic acid. Furthermore the effects of absence of nitrate, proved under laboratory conditions, has been investigated in experiments with green forage produced under practical conditions. The treatments were the same as mentional above. As a result of this several years and extensive investigations with different green fodder and different content of spores of clostridia it was shown that metabolism during fermentation process and pattern of fermentation products in the end of fermentation are significant different depending on content of nitrate in green forage. In ensiling material low in nitrate butyric acid was formed already at the beginning of the fermentation process, parallel to the lactic acid fermentation. The content of acetic acid was always extremely low. In comparison with silages from green forage high in nitrate anaerobe stable or unstable silages show a different ratio between butyric acid and other characteristics of undesirable decomposition during fermentation - acetic acid, ammonia, pH - in equal fermentation stages. In summary, one can say that epiphytic lactic acid bacteria, content of clostridia spores and nitrate of herbage take into account to judge the fermentability more than previous, together with DM and WSC/BC. Furthermore the judgement of herbage fermentability and the fermentation quality of silages were different depending on number of growth. In silages low in nitrate is an especially risk for occurrence of butyric acid, independing on judgement of fermentability on the basis of DM and ratio of watersoluble carbohydrates to buffering capacity. It`s advisable to use lactic acid bacteria additives (inoculants) always for ensiling green forage low in nitrate. The additive with nitrite has proved as most effectively especially for green forage high in clostridia spores and/ or low content of dry matter of green forage. It is very probably, that the use of current DLG- evaluation system to estimate the fermentation quality, developed for silages from green forage high in nitrate, is not correct and leads to error of judgement of silages from green forage low in nitrate.

A transmissão de parasitas da malária por mosquitos vetores é dependente do desenvolvimento bem sucedido das formas infectantes de Plasmodium sp., especialmente os esporozoítas, que são as formas que infectam o hospedeiro vertebrado. A manipulação genética de mosquitos vetores tem sido uma estratégia alternativa na tentativa de controle da malária. Um componente extremamente importante desta estratégia é a escolha de uma molécula efetora capaz de reduzir a transmissão do patógeno. Microplusina é um peptídeo antimicrobiano rico em cisteína, originalmente descrito como um componente antimicrobiano da hemolinfa e dos ovos de carrapato bovino Rhipicephalus (Boophilus) microplus. Testes anteriores utilizando o modelo experimental mosquito Aedes aegypti infectado por Plasmodium gallinaceum mostraram que a microplusina é altamente tóxico para esporozoítas de Plasmodium gallinaceum em concentração relativamente baixa, sem apresentar toxicidade aos mosquitos vetores Aedes aegypti. Nosso objetivo foi analisar a expressão da microplusina e seu efeito na infecção de P. gallinaceum em mosquitos transgênicos. Obtivemos quatro linhagens através da integração de um transgene contendo a região promotora do gene da vitelogenina de Ae. aegypti, peptídeo sinal maltase-like I de Ae. aegypti e a sequência codificadora da microplusina (PMOS [3xP3-EGFP-AeVg Micro]). A atividade anti esporozoítas da microplusina expressa pelos mosquitos transgênicos mostrou diferença significante as linhagens. O desenho de novas moléculas utilizando como molde moléculas efetoras existentes e testadas, possibilitará o aperfeiçoamento da expressão de genes exógenos em mosquitos transgênicos, tornando-os refratários ao parasita. / Transmission of malaria parasites by mosquito vectors is dependent on the successful development of Plasmodium sp. infective forms, particularly the sporozoites, which are the forms that enter the vertebrate host. The genetic manipulation of mosquito vectors has been a strategy for malaria control. An extremely important component of this strategy is the effector molecule of choice which reduces parasite transmission. Microplusin is a cysteine-rich antimicrobial peptide originally described as an hemolymph and eggs antimicrobial component of the cattle tick Boophilus microplus. Previous tests using the experimental model Plasmodium gallinaceum infected Aedes aegypti showed that microplusin is highly toxic to P. gallinaceum sporozoites in relatively low concentration, without showing toxicity to the mosquito vector A. aegypti. Our goal was to analyze transgenic mosquitoes expressing microplusin and its effect on infection of P. gallinaceum. We obtained four lines through the integration of transgene that containing the promoter region of the A. aegypti vitelogenin gene, the maltase-like I signal peptide of A. aegypti and microplusin coding sequence (pMos[3xP3-EGFPAeVg-Micro]). The activity anti sporozoites microplusin expressed by transgenic mosquitoes showed significant differences between strains. The design of effector molecules using information from existing and tested molecules as template will enable the improvement of the expression of foreign genes in transgenic mosquitoes, making them resistant to the parasite.

The initial aim of this work was to evaluate processing conditions for extended shelf life (ESL) milk to have a shelf life at refrigeration temperature of 60 d. Milk was processed on a pilot-scale ultra-high-temperature processing plant and evaluated for microbial and sensory quality over 60 d at 7°C storage. Results of this study showed that lower process temperatures were preferable to minimize cooked flavors and that the minimum safe processing temperature was 134°C for 4 s as determined by the destruction of bacterial spores in the processed milk. Consumer preference panel results indicated that consumers preferred milk processed at 134°C for 4 s (those recommended in this study for ESL processing) to commercial UHT milk although there was a slight preference for pasteurized milk. The critical sensory characteristic of the processed milk was a cooked flavor, which decreased with lower processing temperature and shorter storage time; however, a significant increase in flavors that could be associated with lipolytic activity was also noted. This study highlighted deficiencies in existing methods for determining heat-stable bacterial products in thermal-processed foods. No rapid, sensitive assay for detection of heat-stable spores or lipases in milk exists. If such assays were available, it would allow processors to determine Lipase activity and bacterial spore counts before processing and direct raw milk with low spore counts and low lipolytic activity into long-shelf-life products. To this end, assays to rapidly quantitate spores and lipolytic activity in milk were developed. The lipase assay relies on the hydrolysis of p-nitrophenyl caprylate liberating a yellow color that is detected using reflectance colorimetry. The assay is sensitive to 5 mUnits/ml and is linearly correlated to spectrophotometry (r2 = 0.93) and release of titratable free fatty acids (r2 = 0.92 to 0.97). An immunocapture, enzyme-linked immunoassay coupled with a fluorescent detection system was developed for and resulted in a prototype spore assay using Bacillus stearothermophilus spores. This organism was selected because it is extremely heat resistant, is commonly found in milk, and is associated with spoilage of milk and milk products. The assay was able to quantitate spores down to 103 cfu/ml in milk and other products in about 1.5 h. Other detection limits could be set if needed.