The use of matrix assisted laser desorption ionization time-oflight mass spectrometry, Fourier transform infrared spectroscopy, and other analytical means of identifying and differentiating microorganisms hold much promise. These analytical tools have been extensively assessed for their ability to differentiate bacteria and fungi. Most of this research has been coordinated in medically relevant microorganisms, but the technology can work just as well with agriculturally important microorganisms. In this thesis, these technologies were reviewed and then subsequently studied for their ability to differentiate Aspergillus species (that devastate corn and other crops yearly with aflatoxin contamination), as well as Macrophomina phaseolina and Thielaviopsis basicola which limit yields on soybean and other crops yearly. With the use of these technologies, harmful plant pathogens could be identified and subsequently treated to improve crop yields and also help to protect our nation and state’s food supply.

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Le taux de mortalité dû au cancer est en hausse d’année en année. Le cancer du pancréas est l’un des plus mortels. Avec un taux de survie inférieur à 20% un an suivant le diagnostic, il y a une urgence pour développer de nouvelles molécules pour cibler cette maladie. La metformine, un biguanide utilisé cliniquement en tant qu’agent antidiabétique, s’est avérée à avoir des propriétés anticancéreuses. Les patients souffrant de diabète de type 2 prenant la metformine comme traitement sont moins à risque de développer plusieurs cancers dont celui du pancréas. Cependant, la metformine n’étant pas biodisponible, les doses à administrer seraient trop élevées pour la considérer comme thérapie anticancéreuse. Le groupe de recherche Schmitzer a synthétisé de nouveaux analogues de type biguanide plus lipophiles dans le but d’améliorer leur biodisponibilité. Le phényléthynylbenzyle biguanide (PEBB) est un des analogues présentant des propriétés antiprolifératives environ 800 fois plus puissantes que la metformine contre des cellules du cancer du pancréas. L’hexylbiguanide s’est aussi démarqué par sa spécificité pour les cellules cancéreuses et sa faible toxicité pour les cellules saines. Étant de bons candidats, des études in vivo ont été faites sur des souris en leur administrant le PEBB et l’hexylbiguanide afin d’obtenir des informations sur l’absorption et la distribution des composés. Pour ce faire, une méthode par LC-MS en mode multiple reaction monitoring (MRM) a été développée afin de quantifier différents analogues de biguanides dans le plasma de souris. De plus, une méthode par MALDI-TOF a été développée afin de localiser et quantifier les analogues dans les tissus par imagerie couplée à la spectrométrie de masse (IMS). Les expériences réalisées ont permis de suivre les composés dans le plasma et d’établir une cinétique d’absorption révélant que le PEBB atteint sa concentration plasmatique maximale environ à 1h après l’administration et que le composé est éliminé de la circulation sanguine à 80% au bout de 4h. Dans le cas de l’hexylbiguanide, la concentration plasmatique maximale est atteinte environ 30 minutes après l’administration pour être éliminé à plus de 90% après 4h. De plus, les études d’IMS ont révélé que le PEBB se distribue principalement dans le foie et légèrement dans les tumeurs. Aucune accumulation à long terme dans le foie n’a été observée, ce qui signifie que les risques de dommages hépatiques sont faibles. Les deux méthodes développées sont des méthodes puissantes IV et reproductibles afin de quantifier les différents types de biguanides dans les liquides biologiques ainsi que dans les tissus. / The death rate of cancer is increasing every year. Pancreatic cancer is one of the deadliest. With a survival rate of less than 20% one year post diagnosis, there is an emergency to develop new molecules to target this disease. Metformin, a biguanide clinically used as an antidiabetic agent, has been shown to have anticancer properties as well. Patients with type 2 diabetes taking metformin are less likely to develop several cancers including pancreatic cancer. However, due to the poor bioavailability of metformin, the doses would be too high to consider it as an anticancer treatment. The Schmitzer group has synthesized new biguanide analogues that are more lipophilic and thus more bioavailable. Phenylethynylbenzyl biguanide (PEBB) is one of the analogues with about 800 times more effective antiproliferative properties than metformin against pancreatic cancer cells. Hexylbiguanide also stood out for its specificity for cancer cells and its low toxicity for normal cells. In vivo studies were performed on mice by administering PEBB and hexylbiguanide to study the absorption and distribution of the compounds. For this aim, a LC-MS method was developed using Multiple Reaction Monitoring (MRM) mode to quantify different biguanide analogues in mice plasma. Complementarily, a MALDI-TOF method was developed to localize and quantify the analogues in tissues by imaging coupled to mass spectrometry (IMS). The experiments performed allowed to follow the compounds in plasma to establish absorption kinetics. These experiments revealed that PEBB reaches its maximum plasma concentration at 1h after administration and the compound is eliminated from the bloodstream at 80% after 6h. For hexylbiguanide, the maximum plasma concentration is reached about 30 minutes after administration and more than 90% is eliminated after 4 hours. In addition, IMS studies have shown that PEBB is distributed mainly in the liver and slightly in tumors. No accumulation in the liver was observed, which suggests that the risk of liver damage is low. These two methods are powerful and reproducible methods to quantify the different types of biguanides in biological fluids and tissues.

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Objectives: Since ultrasonic dental scalers contain internal narrow-diameter waterline tubing susceptible to bacterial biofilm growth, this study determined the cultivable prevalence of aerobic mesophilic heterotrophic bacteria in ultrasonic scaler output water subjected to one of two commercial biocides favorably reviewed by the American Dental Association as effective for microbial biofilm control in dental unit waterlines, and assessed the potential antimicrobial effect on output water of an ultrasonic scaler tip activated at a maximum power frequency. Methods: Aseptically-collected water samples were obtained, and neutralized for residual chlorine with sodium thiosulfate, from 12 ultrasonic scalers provided municipal tap water treated with an iodine resin cartridge (Group A), and from eight ultrasonic scalers provided distilled water treated with a 0.78% silver ion-based disinfectant (Group B), all within a dental school clinic setting. The latter eight ultrasonic scalers were additionally sampled after output water was passed through an inserted scaler tip activated to a maximum power setting similar to its potential use in clinical patient care. All water samples were evaluated for aerobic mesophilic heterotrophic bacteria using nationally-accepted analytical procedures detailed in Standard Methods for the Examination of Water and Wastewater, 23rd Edition (2017), with sample aliquots spread-plated onto R2A agar and aerobically incubated at 28°C for seven days. Aerobic mesophilic heterotrophic bacteria were considered elevated when ≥ 500 CFU/ml of the species were recovered per water sample. Predominant cultivable isolates of aerobic mesophilic heterotrophic bacteria in the water samples were identified using matrix- assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry and Bruker MALDI Biotyper analytic software. Results: Aerobic mesophilic heterotrophic bacteria averaged 610 ± 380 (SD) CFU/ml per ultrasonic scaler in Group A ultrasonic scalers, with eight of 12 (66.7%) evaluated ultrasonic scalers yielding elevated counts ≥ 500 CFU/ml. Similar mean values of 667 ± 340 (SD) CFU/ml per ultrasonic scaler were found in Group B ultrasonic scalers, with five of eight (62.5%) evaluated ultrasonic scalers yielding elevated aerobic mesophilic heterotrophic bacteria counts ≥ 500 CFU/ml. Acidovorax temerans, Novosphingobium subterraneum, Cupriavidus metallidurans, and Sphingobium yanoikuyae were the most prevalent cultivable species identified from ultrasonic scaler waterlines where the source water was municipal tap water treated with molecular iodine. In contrast, nearly pure growth of Cupriavidus metallidurans alone dominated cultivable waterline isolates from ultrasonic scalers where the source water was distilled water treated with a 0.78% silver ion disinfectant. No statistically significant impact was found on aerobic mesophilic heterotrophic bacterial counts after ultrasonic scaler output water passed through an inserted scaler tip activated to maximum power (P > 0.05, Wilcoxcon signed-rank test). Conclusions: Approximately two-thirds of ultrasonic scalers in a dental school clinic setting yielded output water that failed to meet potable water regulatory standards in the United States with regard to aerobic mesophilic heterotrophic bacterial counts, even when subjected to disinfection with one of two American Dental Association favorably-reviewed biocide products, or exposed to high-frequency ultrasonic scaler tip oscillations. These findings question the effectiveness of infection control procedures used to manage ultrasonic scaler waterlines, and raise concerns about the safety of ultrasonic scaler output water introduced into the oral cavity of dental patients during dental treatment procedures. / Oral Biology

Wie in anderen Zellen sind auch bei Säugerspermien spezifische Lipide und Proteine der Zellmembran aufgrund ihrer heterogenen lateralen Verteilung in speziellen Domänen angereichert, die in unterschiedlichen räumlichen und zeitlichen Dimensionen existieren und der Zelle funktionale Variabilität ermöglichen. Aufgrund der fehlenden aktiven Proteinbiosynthese bietet dies den Spermien eine Möglichkeit, auf unterschiedliche Anforderungen zu reagieren. In der vorliegenden Arbeit wurden daher sogenannte detergenzresistente Membrandomänen (DRMs) aus Eberspermien unterschiedlicher Reifestadien präpariert und untersucht. Dabei stieg bereits in den Dichtegradienten mit zunehmender Reife die Dichte, bei der die opaleszenten Banden auftraten. Eine Analyse dieser mittels 31P-NMR zeigte mit zunehmender Reife eine Anreicherung an Glycerophosphatidylethanolamin und Phosphatidylinositol bei den Glycerophospholipiden, der Gehalt an Sphingomyelin hingegen nahm während der Nebenhodenreifung und auch nach der Ejakulation ab. Diese Veränderungen könnten auf eine Destabilisierung von Membrandomänen hindeuten, um eine Zusammenlagerung zu größeren Domänenclustern zu erleichtern, möglicherweise in Vorbereitung auf Kapazitation und Akrosomenreaktion. Zunächst werden die destabilisierten Membrandomänen jedoch durch die Anlagerung von Seminalplasmaproteinen geschützt, was vermutlich für das verringerte Lipid- zu Proteinverhältnis der DRMs bei Ejakulatspermien sorgt. Aufgrund der generellen Kälteempfindlichkeit von Eberspermien findet ihre Lagerung üblicherweise bei 16°C statt. Dies ist aus mikrobiologischer Sicht nachteilig gegenüber einer kälteren Lagerungstemperatur. Eine Untersuchung der Spermien von 64 Ebern zeigte jedoch bei 10% der Ejakulate eine individuum-spezifische Resistenz gegenüber der Lagerung bei 4°C. Die DRMs der kälteresistenten Spermien hatten einen erhöhten Anteil an langkettigen, mehrfach ungesättigten Fettsäuren, wie 31P-NMR und MALDI-TOF MS Analysen zeigten. / The lateral distribution of lipids and proteins in the plasma membrane is heterogeneous. Therefore specific lipids and proteins in membranes of mammalian spermatozoa are enriched in special domains of varying size and different time scales enabling the cell’s membrane functional variability. Being transcriptional inactive this is especially relevant for spermatozoa in responding to multiple challenges on their way to fertilization. Therefore so called detergent resistant membrane domains (DRMs) from boar spermatozoa of different developmental stages were investigated. Already in the sucrose density gradients differences were visible, so the opalescent bands of more maturated sperm had a higher density. An analysis of these bands by 31P-NMR showed an enrichment of glycerophosphatidylethanolamine and phosphatidylinositol during maturation and a decrease of sphingomyelin during maturation in the epididymis and even after ejaculation. This suggests destabilization of DRMs and hence of putative membrane domains. This could enable clustering to bigger membrane domain platforms in preparation for capacitation and acrosome reaction. First, however, seminal fluid proteins cover the spermatozoa protecting the membrane with the destabilized membrane domains. This could have led to the detected decrease of the lipid to protein ratio in DRMs of ejaculated sperm. Boar spermatozoa are sensitive to storage at cold temperatures and are therefore usually stored at 16°C, which is especially disadvantageous with regard to growing of bacteria. A screening of sperm from 64 boars showed a ratio of 10% individuals with cold resistant sperm which could be stored at 4°C without quality loss. The DRMs of cold resistant sperm had a higher proportion of longchained, polyunsaturated fatty acids, as shown by analysis with 31P-NMR und MALDI-TOF MS.