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Saccharomyces cerevisiae V. P. I. Strain No. 1 was found to estimate the growth of Proteus vulgaris V. P. I. Strain No. 3 in a chemically-defined medium when growth curves of the viable counts of the two organisms in pure and in mixed culture were studied. The yeast stimulated the growth of the bacterium without its own numbers being affected, indicating a true commensalism. The stimulation began by the 8th hour of incubation and continued throughout the growth period. The organisms were plated on differential plating media designed to specifically permit the growth of one organism while inhibiting the other. The medium used for P. vulgaris contained bile salts to inhibit the yeast, while the medium used for S. cerevisiae contained penicillin to which the bacterium was sensitive. During the phase of active growth of the yeast, a temporary decrease or dip in viable count occurred to the extent of about 1/2 to 1 log unit, regardless of whether the yeast was in pure of mixed culture. Total counts of the yeast cells (by direct microscopic methods) failed to show any corresponding decrease. A rough correspondence of the appearance of amounts of ethanol detectable by gas chromatography in the medium was observed; however, the factors causing the viable count dip remain obscure. It is possible that clumping of yeast cells is closely connected with this dip. Separation of the yeast and bacterium by a dialysis membrane failed to halt the stimulatory action of the yeast. The ability of P. vulgaris to exhibit a growth response toward nicotinic acid, nicotinamide, or nicotinamide adenine dinucleotide was demonstrated, suggesting that the stimulatory factor formed by the yeast was a compound of this group. The amount of stimulatory factor formed by the yeast in pure culture after 48 hr corresponded to 0.20 µg of nicotinamide adenine dinucleotide/ml. Exhaustive attempts to identify the stimulatory factor by bioautography were unsuccessful; the amount of factor was too slight to be detectable by even this highly sensitive method. Evidence is presented to indicate that the high amount of biotin employed in the defined medium may have resulted in production of only small quantities of nicotinic acid-like factor in comparison to the amounts formed when less biotin was present. / Master of Science

4 Abstract The main goal of my thesis was to create our own proposals for laboratory work in biology of unicellular organisms (Protista). I designed the laboratory tasks, so that they can be mastered by the pupils of sixth year of primary school. Creating the proposals for laboratory exercises was preceded by the work in the laboratory at Charles University in Prague, Faculty of Education, the Department of Biology and Environmental Studies. In the laboratory, I verified the methods of cultivation of selected representatives of ciliates (Paramecium caudatum), amoebae (Amoeba proteus) and euglenids (Euglena gracilis) and selected methods of dyeing their different organels. Key words: protists, Paramecium caudatum, Amoeba proteus, Euglena gracilis, cultivation, dying

Cepas com altas taxas de mutação (mutadoras) foram detectadas em diversos gêneros bacterianos. A alta taxa de mutação está relacionada a defeitos em sistemas de reparo de DNA. Uma alta incidência de isolados clínicos de Proteus mirabilis com altas frequências de mutação foi descrita anteriormente. O fenômeno foi induzido em Escherichia coli, quando transformada com um plasmídeo de P. mirabilis. Com coleção de 77 isolados clínicos de P. mirabilis, medimos a frequência de mutantes espontâneos e verificamos a presença do elemento conjugativo ICE SXT/R391, para desvendar possível relação entre a presença do ICE e a frequência de mutação. 9 isolados clínicos apresentam o ICE. A frequência de mutantes mostrou que não existem mutadores verdadeiros, mas 11 isolados apresentam uma alta frequência de mutantes FosR. Considerando o alto índice de infecções por P. mirabilis, é importante entender a resistência à fosfomicina, já que esta é usada na clínica. Não existe relação entre uma frequência de mutantes espontânea e a presença de ICE SXT/R391 em isolados de P. mirabilis. / Strains with high mutation rates (mutators) were detected in several bacterial genera. The increased mutation rate is related to defects in DNA repair systems. A high incidence of Proteus mirabilis clinical isolates with high mutation frequencies were described previously. The phenomenon was induced in Escherichia coli, when transformed with a plasmid of P. mirabilis. 77 P. mirabilis clinical isolates were tested for the frequency of spontaneous mutants and the presence of a conjugative element found in this species, ICEs SXT/R391, to verify if there is a relation between the element and the mutation frequencies. 9 isolates carry the ICE SXT/R391. The frequency of mutants showed no true mutators among the isolates. 11 isolates show a high frequency of FosR mutants. Considering the high rate of infections by P. mirabilis, it is important to understand the fosfomycin phenomenon, since it is currently used to treat urinary infections. We have seen no relation between a high spontaneous mutation frequency and the presence of ICE SXT/R391 in isolates of P. mirabilis.

Cepas com altas taxas de mutação (mutadoras) foram detectadas em diversos gêneros bacterianos. A alta taxa de mutação está relacionada a defeitos em sistemas de reparo de DNA. Uma alta incidência de isolados clínicos de Proteus mirabilis com altas frequências de mutação foi descrita anteriormente. O fenômeno foi induzido em Escherichia coli, quando transformada com um plasmídeo de P. mirabilis. Com coleção de 77 isolados clínicos de P. mirabilis, medimos a frequência de mutantes espontâneos e verificamos a presença do elemento conjugativo ICE SXT/R391, para desvendar possível relação entre a presença do ICE e a frequência de mutação. 9 isolados clínicos apresentam o ICE. A frequência de mutantes mostrou que não existem mutadores verdadeiros, mas 11 isolados apresentam uma alta frequência de mutantes FosR. Considerando o alto índice de infecções por P. mirabilis, é importante entender a resistência à fosfomicina, já que esta é usada na clínica. Não existe relação entre uma frequência de mutantes espontânea e a presença de ICE SXT/R391 em isolados de P. mirabilis. / Strains with high mutation rates (mutators) were detected in several bacterial genera. The increased mutation rate is related to defects in DNA repair systems. A high incidence of Proteus mirabilis clinical isolates with high mutation frequencies were described previously. The phenomenon was induced in Escherichia coli, when transformed with a plasmid of P. mirabilis. 77 P. mirabilis clinical isolates were tested for the frequency of spontaneous mutants and the presence of a conjugative element found in this species, ICEs SXT/R391, to verify if there is a relation between the element and the mutation frequencies. 9 isolates carry the ICE SXT/R391. The frequency of mutants showed no true mutators among the isolates. 11 isolates show a high frequency of FosR mutants. Considering the high rate of infections by P. mirabilis, it is important to understand the fosfomycin phenomenon, since it is currently used to treat urinary infections. We have seen no relation between a high spontaneous mutation frequency and the presence of ICE SXT/R391 in isolates of P. mirabilis.

Le « Computational protein design » ou CPD est la recherche des séquences d’acides aminés compatibles avec une structure protéique ciblée. L’objectif est de concevoir une fonction nouvelle et/ou d’ajouter un nouveau comportement. Le CPD est en développement dans de notre laboratoire depuis plusieurs années, avec le logiciel Proteus qui a plusieurs succès à son actif.Notre approche utilise un modèle énergétique basé sur la physique et s’appuie sur la différence d’énergie entre l’état plié et l’état déplié de la protéine. Au cours de cette thèse, nous avons enrichi Proteus sur plusieurs points, avec notamment l’ajout d’une méthode d’exploration Monte Carlo avec échange de répliques ou REMC. Nous avons comparé trois méthodes stochastiques pour l’exploration de l’espace de la séquence : le REMC, le Monte Carlo simple et une heuristique conçue pour le CPD, le «Multistart Steepest Descent » ou MSD. Ces comparaisons portent sur neuf protéines de trois familles de structures : SH2, SH3 et PDZ. En utilisant les techniques d’exploration ci-dessus, nous avons été en mesure d’identifier la conformation du minimum global d’énergie ou GMEC pour presque tous les tests dans lesquels jusqu’à 10 positions de la chaîne polypeptidique étaient libres de muter (les autres conservant leurs types natifs). Pour les tests avec 20 positions libres de muter, le GMEC a été identifié dans 2/3 des cas. Globalement, le REMC et le MSD donnent de très bonnes séquences en termes d’énergie, souvent identiques ou très proches du GMEC. Le MSD a obtenu les meilleurs résultats sur les tests à 30 positions mutables. Le REMC avec huit répliques et des paramètres optimisés a donné le plus souvent le meilleur résultat lorsque toutes les positions peuvent muter. De plus, comparé à une énumération exacte des séquences de faible énergie, le REMC fournit un échantillon de séquences de grande diversité.Dans la seconde partie de ce travail, nous avons testé notre modèle pour la conception de domaines PDZ. Pour l’état plié,nous avons utilisé deux variantes d’un modèle de solvant GB. La première utilise une frontière diélectrique protéine/solvant effective moyenne ; la seconde, plus rigoureuse, utilise une frontière exacte qui fluctue le long de la trajectoire MC. Pour caractériser l’état déplié, nous utilisons un ensemble de potentiels chimiques d’acide aminé ou énergies de références. Ces énergies de références sont déterminées par maximisation d’une fonction de vraisemblance afin de reproduire les fréquences d’acides aminés des domaines PDZ naturels. Les séquences conçues par Proteus ont été comparées aux séquences naturelles. Nos séquences sont globalement similaires aux séquences Pfam, au sens des scoresBLOSUM40, avec des scores particulièrement élevés pour les résidus au cœur de la protéine. La variante de GB la plus rigoureuse donne toujours des séquences similaires à des homologues naturels modérément éloignés et l’outil de reconnaissance de plis Super family appliqué à ces séquences donne une reconnaissance parfaite. Nos séquences ont également été comparées à celles du logiciel Rosetta. La qualité, selon les mêmes critères que précédemment, est très comparable, mais les séquences Rosetta présentent moins de mutations que les séquences Proteus. / Computational Protein Design, or CPD is the search for the amino acid sequences compatible with a targeted protein structure. The goal is to design a new function and/or add a new behavior. CPD has been developed in our laboratory for several years, with the software Proteus which has several successes to its credit. Our approach uses a physics-based energy model, and relies on the energy difference between the folded and unfolded states of the protein. During this thesis, we enriched Proteus on several points, including the addition of a Monte Carlo exploration method with Replica Exchange or REMC. We compared extensively three stochastic methods for the exploration of sequence space: REMC, plain Monte Carlo and a heuristic designed for CPD: Multistart Steepest Descent or MSD.These comparisons concerned nine proteins from three structural families: SH2, SH3 and PDZ. Using the exploration techniques above, we were able to identify the Global Minimum EnergyConformation, or GMEC for nearly all the test cases where up to10 positions of the polypeptide chain were free to mutate (the others retaining their native types). For the tests where 20positions were free to mutate, the GMEC was identified in 2/3 of the cases. Overall, REMC and MSD give very good sequences in terms of energy, often identical or very close to the GMEC. MSDperformed best in the tests with 30 mutating positions. REMCwith eight replicas and optimized parameters often gave the best result when all positions could mutate. Moreover, compared to an exact enumeration of the low energy sequences, REMC provided a sample of sequences with a high sequence diversity.In the second part of this work, we tested our CPD model forPDZ domain design. For the folded state, we used two variants ofa GB solvent model. The first used a mean, effective protein/solvent dielectric boundary; the second one, more rigorous, used an exact boundary that flucutated over the MCtrajectory. To characterize the unfolded state, we used a set of amino acid chemical potentials or reference energies. These reference energies were determined by maximizing a likelihoodfunction so as to reproduce the amino acid frequencies in naturalPDZ domains. The sequences designed by Proteus were compared to the natural sequences. Our sequences are globally similar to the Pfam sequences, in the sense of the BLOSUM40scores, with especially high scores for the residues in the core ofthe protein. The more rigorous GB variant always gives sequences similar to moderately distant natural homologues and perfect recognition by the the Super family fold recognition tool.Our sequences were also compared to those produced by the Rosetta software. The quality, according to the same criteria as before, was very similar, but the Rosetta sequences exhibit fewer mutations than the Proteus sequences.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Herbal teas and fruit juices used in our regular diet may have importance in the protective treatment of some infectious diseases. In this study, selected dietary beverages were investigated for their antioxidant capacities and antimicrobial activities against Proteus mirabilis, a well known bacteria in urinary tract infections. Herbal teas / sage (Salvia fruticosa Mill), anise (Pimpinella anisum L.), rosehip (Rosa canina L.), camomile (Anthemis arvensis L.) and fruit juices / grape (Vitis vinifera L.), orange (Citrus sinensis L.), peach (Prunus persica L.), and pomegranate (Punica granatum L.) were chosen as samples of regular diets. Selected fruit juices and aqueous infusion tea extracts, lyophilised to dryness, were used throughout this study. Antioxidant capacities of the extracts were carried out by using 2,2&rsquo / -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH) methods along with the determination of total phenolic compounds in the extracts. Antimicrobial activities of extracts were determined by disc diffusion test, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods. Among the herbal teas, sage infusion extract has displayed the highest radical scavenging capacity with ABTS EC50 value of 5.152 mg/mL, DPPH EC50 value of 0.072 mg/mL and with its high phenolic content of 0.411 mg/mg gallic acid equivalence. Among the fruit juices pomegranate has revealed significantly high DPPH EC50 and TEAC values 0.924 mg/mL and 0.552 mmol/g, respectively. Peach juice has been found with the highest total phenolic amount of 0.067 mg/mg gallic acid equivalent. Antimicrobial activities of herbal teas were correlating with antioxidant capacity studies, whereas sage infusion tea extract exhibited 3 mg/mL of minimum inhibitory concentration (MIC) and 6 mg/mL of minimum bactericidal concentration (MBC). Rosehip was also found as an effective antimicrobial agent with a minimum inhibitory concentration value of 3 mg/mL. In the meantime, there was no significant difference in the zone inhibition of herbal tea infusion extracts. In case of fruit juices grape and pomegranate may be effective antimicrobials in P. mirabilis infections with 0.75 mg/mL MIC and 6 mg/mL MBC, respectively at the same time both juices revealed significantly high inhibition zones with 11 mm.

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 78-86). Also available on the World Wide Web.

Thèse (M.Sc.)--Université Laval, 2007. / Bibliogr.: f. 105-109. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.