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Mesoporous silica nanoparticles (MSNPs) possess large surface areas and ample pore space that can be readily modified with specific functional groups for targeted binding of bioactive materials to be transported through cellular barriers. Engineered silica nanoparticles (ESNP) have been used extensively to deliver bio-active materials to target intracellular sites, including as non-viral vectors for nucleic acid (DNA/RNA) delivery such as for siRNA induced interference. The reverse process guided by the same principles is called “nanoharvesting”, where valuable biomolecules are carried out and separated from living and functioning organisms using nano-carriers. This dissertation focuses on ESNP design principles for both applications. To investigate the bioactive materials loading, the adsorption of antioxidant flavonoids was investigated on titania (TiO2) functionalized MSNPs (mean particle diameter ~170 nm). The amount of flavonoid adsorbed onto particle surface was a strong function of active group (TiO2) grafting and a 100-fold increase in the adsorption capacity was observed relative to nonporous particles with similar TiO2 coverage. Active flavonoid was released from the particle surface using citric acid-mediated ligand displacement. Afterwards, nanoharvesting of flavonoids from plant hairy roots is demonstrated using ESNP in which TiO2 and amine functional groups are used as specific binding sites and positive surface charge source, respectively. Isolation of therapeutics was confirmed by increased pharmacological activity of the particles. After nanoharvesting, roots are found to be viable and capable of therapeutic re-synthesis. In order to identify the underlying nanoparticle uptake mechanism, TiO2 content of the plant roots was quantified with exposure to nanoparticles. Temperature (4 or 23 °C) dependent particle recovery, in which time dependent release of ESNP from plant cells showed a similar trend, indicated an energy independent process (passive transport). To achieve the selective separation and nanoharvesting of higher value therapeutics, amine functionalized MSNPs were conjugated with specific functional oligopeptides using a hetero-bifunctional linker. Fluorescence spectroscopy was used to confirm and determine binding efficiency using fluorescently attached peptides. Binding of targeted compounds was confirmed by solution depletion using liquid chromatography–mass spectrometry. The conjugation strategy is generalizable and applicable to harvest the pharmaceuticals produced in plants by selecting a specific oligopeptide that mimic the appropriate binding sites. For related gene delivery applications, the thermodynamic interaction of amine functionalized MSNPs with double-stranded (ds) RNA was investigated by isothermal titration calorimetry (ITC). The heat of interaction was significantly different for particles with larger pore size (3.2 and 7.6 nm) compared to that of small pore particles (1.6 nm) and nonporous particles. Interaction of dsRNA also depended on molecular length, as longer RNA (282 base pair) was unable to load into 1.6 nm particles, consistent with previous confocal microscopy observations. Calculated thermodynamic parameters (enthalpy, entropy and free energy of interaction) are essential to design pore size dependent dsRNA loading, protection and delivery using MSNP carriers. While seemingly diverse, the highly tunable nature of ESNP and their interactions with cells are broadly applicable, and enable facile nano-harvesting and delivery based on a continuous uptake-expulsion mechanism.

(in English): The master's thesis deals with the question of how verb conjugation is presented in selected Czech textbooks for Russian-speaking students. It focuses on the way selected teaching materials classify Czech verbs and introduce their conjugation. The verb classification used in textbooks is compared with that in grammar books for native Czech speakers and that in grammar books for foreigners. and with CEFR recommendations for Czech at A1 and A2 levels. Each of the analyzed textbooks presents a system of Czech verbs in their own way. There is no unified approach to the classification of verbs. The systems used in textbooks differ between those in grammar books for native speakers and in those for foreigners. The aim of the work is to reveal similarities and differences in the classification and presentation of verbs in selected textbooks. The analysis of the gaps in the presentation of verb classes in present tense can be useful in the creation of teaching materials.

Cyanobacteria are a model organism for photosynthesis and the Calvin cycle, and a promising chassis for 4th generation biofuel production. There are many ongoing efforts to improve the performance of cyanobacteria, in terms of CO2 fixation and production rate of biofuels. One straightforward way to improve CO2 fixation could be to overexpress the genes of limiting enzymes. In this project, we used a high-throughput method to test the overexpression of thousands of genes in cyanobacteria and measure the effect on growth rate. We created barcoded overexpression libraries, consisting of gene fragments from different cyanobacteria strains and transformed them into a model cyanobacterium, Synechocystis PCC 6803. We then cultivated the transformed cyanobacteria libraries and screened for effects of increased gene copy number on both maximum growth rate and robustness of growth under stress conditions. The cell populations were cultivated in a turbidostat, resulting in competitive growth between transformants. The relative abundance of each mutant was estimated using deep sequencing. Fitness scores, for each gene show how expression of that gene affects growth rate. This method, competitive growth and tracking of mutant populations with deep sequencing, is a high throughput method for screening a large proportion of genes from several organism at once, allowing the identification of trans-species effects as well as the effect of single genes on the metabolism of the host cell. / Cyanobakterier är en modellorganism för fotosyntes och Calvin-cykeln och ett lovande chassi för fjärde generationens biobränsleproduktion. Det finns många pågående ansträngningar för att förbättra cyanobakteriens prestanda med avseende på CO2-fixering och produktionshastighet för biobränslen. Ett enkelt sätt att förbättra CO2-fixering kan vara att överuttrycka generna för begränsande enzymer. I detta projekt använde vi en metod med hög kapacitet för att testa överuttryck av tusentals gener i cyanobakterier och mäta effekten på tillväxthastigheten. Vi skapade streckkodade överuttrycksbibliotek, bestående av genfragment från olika arter av cyanobakterier och transformerade in dem i en modellorganism för cyanobakterium, Synechocystis PCC 6803. Vi odlade sedan de transformerade biblioteken och screenade efter effekten av ökade antal genkopior på både maximal tillväxthastighet och robusthet hos tillväxt under stressförhållanden. Cellpopulationerna odlades i en Turbidostat, vilket resulterade i konkurrerande tillväxt mellan transformanter. Den relativa mängden av varje mutant uppskattades med användning av djup sekvensering. "Fitnesspoäng" för varje gen visar hur uttrycket av den genen påverkar tillväxthastigheten. Denna metod, konkurrerande tillväxt och spårning av mutantpopulationer med djup sekvensering, är en metod med hög genomströmning för att screena en stor andel gener från flera organismer samtidigt, vilket möjliggör identifiering av trans-art effekter såväl som effekten av enstaka gener på värdcellens metabolism.

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Les entérocoques font partie de la flore normale intestinale des animaux et des humains. Plusieurs études ont démontré que les entérocoques d’origine animale pouvaient représenter un réservoir de gènes de résistance aux antibiotiques pour la communauté humaine et animale. Les espèces Enterococcus faecalis et Enterococcus faecium sont importantes en santé publique; elles sont responsables d’environ 12% de toutes les infections nosocomiales aux États-Unis. Au Canada, les cas de colonisation et/ou d’infections à entérocoques résistants à la vancomycine ont plus que triplé de 2005 à 2009. Un total de 387 isolats E. faecalis et E. faecium aviaires, et 124 isolats E. faecalis porcins ont été identifiés et analysés pour leur susceptibilité aux antibiotiques. De hauts pourcentages de résistance envers les macrolides et les tétracyclines ont été observés tant chez les isolats aviaires que porcins. Deux profils phénotypiques prédominants ont été déterminés et analysés par PCR et séquençage pour la présence de gènes de résistance aux antibiotiques. Différentes combinaisons de gènes de résistance ont été identifiées dont erm(B) et tet(M) étant les plus prévalents. Des extractions plasmidiques et des analyses par hybridation ont permis de déterminer, pour la première fois, la colocalisation des gènes erm(B) et tet(M) sur un plasmide d’environ 9 kb chez des isolats E. faecalis porcins, et des gènes erm(B) et tet(O) sur un plasmide de faible poids moléculaire d’environ 11 kb chez des isolats E. faecalis aviaires. De plus, nous avons démontré, grâce à des essais conjugatifs, que ces plasmides pouvaient être transférés. Les résultats ont révélé que les entérocoques intestinaux aviaires et porcins, lesquels peuvent contaminer la viande à l’abattoir, pouvaient représenter un réservoir de gènes de résistance envers la quinupristine-dalfopristine, la bacitracine, la tétracycline et les macrolides. Afin d’évaluer l’utilisation d’un antisérum polyclonal SA dans l’interférence de la résistance à de fortes concentrations de bacitracine (gènes bcrRAB), lors d’un transfert conjugatif répondant aux phéromones, un isolat multirésistant E. faecalis aviaire a été sélectionné. Après induction avec des phéromones produites par la souche réceptrice E. faecalis JH2-2, l’agrégation de la souche donatrice E. faecalis 543 a été observée ainsi que des fréquences de transfert élevées en bouillon lors d’une courte période de conjugaison. Le transfert conjugatif des gènes asa1, traB et bcrRAB ainsi que leur colocalisation a été démontré chez le donneur et un transconjugant T543-1 sur un plasmide de 115 kb par électrophorèse à champs pulsé (PFGE) et hybridation. Une CMI de > 2 048 µg/ml envers la bacitracine a été obtenue tant chez le donneur que le transconjuguant tandis que la souche réceptrice JH2-2 démontrait une CMI de 32 µg/ml. Le séquençage des gènes asa1, codant pour la substance agrégative, et traB, une protéine régulant négativement la réponse aux phéromones, a révélé une association de cet élément génétique avec le plasmide pJM01. De plus, cette étude présente qu’un antisérum polyclonal SA peut interférer significativement dans le transfert horizontal d’un plasmide répondant aux phéromones codant pour de la résistance à de fortes doses de bacitracine d’une souche E. faecalis aviaire multirésistante. Des isolats cliniques E. faecium d’origine humaine et canine ont été analysés et comparés. Cette étude rapporte, pour la première fois, la caractérisation d’isolats cliniques E. faecium résistants à l’ampicilline (EFRA) d’origine canine associés à CC17 (ST17) au Canada. Ces isolats étaient résistants à la ciprofloxacine et à la lincomycine. Leur résistance envers la ciprofloxacine a été confirmée par la présence de substitutions dans la séquence en acides aminés des gènes de l’ADN gyrase (gyrA/gyrB) et de la topoisomérase IV (parC/parE). Des résistances élevées envers la gentamicine, la kanamycine et la streptomycine, et de la résistance envers les macrolides et les lincosamides a également été observées. La fréquence de résistance envers la tétracycline était élevée tandis que celle envers la vancomycine n’a pas été détectée. De plus, aucune résistance n’a été observée envers le linézolide et la quinupristine-dalfopristine. Les données ont démontré une absence complète des gènes esp (protéine de surface des entérocoques) et hyl (hyaluronidase) chez les isolats canins EFRA testés tandis qu’ils possédaient tous le gène acm (adhésine de liaison au collagène d’E. faecium). Aucune activité reliée à la formation de biofilm ou la présence d’éléments CRISPR (loci de courtes répétitions palindromiques à interespaces réguliers) n’a été identifiée chez les isolats canins EFRA. Les familles de plasmide rep6 and rep11 ont significativement été associées aux isolats d’origine canine. Les profils PFGE des isolats d’origine humaine et canine n'ont révélé aucune relation (≤ 80%). Ces résultats illustrent l'importance d'une utilisation judicieuse des antibiotiques en médecine vétérinaire afin d’éviter la dissémination zoonotique des isolats EFRA canins. Nous pensons que ces résultats contribueront à une meilleure compréhension des mécanismes de résistance aux antibiotiques et de leurs éléments mobiles ainsi qu’à de nouvelles stratégies afin de réduire le transfert horizontal de la résistance aux antibiotiques et des facteurs de virulence. / Enterococci are part of normal intestinal gut flora of animals and humans. Many studies have shown that enterococci from animal origin could represent an antimicrobial resistance genes reservoir for the human community. The two species Enterococcus faecalis and Enterococcus faecium are important in public health; they are responsible for approximately 12% of all nosocomial infections in the United States. In Canada, cases of colonization and/or infections to vancomycin resistant enterococci have more than tripled from 2005 to 2009. A total of 387 poultry E. faecalis and E. faecium isolates, and 124 porcine E. faecalis isolates were identified and analyzed for their antibiotic susceptibilities. High percentages of resistance to macrolides and tetracyclines were found in both avian and porcine isolates. Two predominant phenotypic profiles were determined and analyzed by PCR and sequencing for the presence of antimicrobial resistance genes. Various combinations of antibiotic resistance genes were detected; erm(B) and tet(M) were the most common genes. For the first time, plasmid extraction and hybridization revealed colocalization of erm(B) and tet(M) on a plasmid of ~9 kb in porcine E. faecalis isolates, and of erm(B) and tet(O) on a low-molecular-weight plasmid of ~11 kb in poultry E. faecalis isolates. Furthermore, we demonstrated, through mating experiments, these plasmids could be transferred. Results indicate that the intestinal enterococci of healthy pigs and poultry, which can contaminate meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes. To assess the use of a polyclonal antiserum AS on the contact interference of a high level bacitracin resistant (bcrRAB genes) pheromone-responsive plasmid, a multiresistant E. faecalis isolate of poultry origin was selected. After induction with pheromones produced by the recipient strain E. faecalis JH2-2, clumping of the donor E. faecalis strain 543 was demonstrated as well as high transfer frequencies in short time broth mating. Conjugative transfer of asa1, traB and bcrRAB genes and their co-localization were also demonstrated in the donor strain and a transconjugant T543-1 on a plasmid band of 115 kb by PFGE and Southern blotting. A MIC to bacitracin of > 2 048 µg/ml was obtained for both strains 543 and T543-1 whereas the recipient strain JH2-2 demonstrated a MIC of 32 µg/ml. Sequencing of the asa1 gene encoding for an AS, and traB for a pheromone shutdown protein, confirmed the association of this genetic element to the pheromone-responsive plasmid related to pJM01. More significantly, this study presents the evidence that a polyclonal antiserum AS can significantly interfere with the horizontal transfer of a pheromone-responsive plasmid encoding high-level bacitracin resistance of a poultry multidrug resistant E. faecalis strain. Clinical isolates of E. faecium of human and canine origin were analyzed and compared. This report describes for the first time the characterization of canine clinical ampicillin-resistant E. faecium (AREF) isolates related to CC17 (ST17) in Canada. These isolates were resistant to ciprofloxacin and lincomycin. Resistance to ciprofloxacin was confirmed by amino acid substitutions in DNA gyrase (gyrA/gyrB) and topoisomerase IV (parC/parE) genes. High-level gentamicin, -kanamycin and -streptomycin resistances and macrolides resistance were also observed. The frequency of tetracycline resistance was high whereas vancomycin resistance was not detected. Also, no resistance was observed to linezolid and quinupristin-dalfopristin antibiotics. Data demonstrated the complete absence of enterococcal surface protein (esp) and hyaluronidase (hyl) genes among the canine AREF isolates tested while all were acm (collagen adhesin from E. faecium) positive. However, most of them were shown to harbor efaAfm gene, encoding for a cell wall adhesin. No biofilm formation or clustered regularly interspaced short palindromic repeats (CRISPR) elements were identified in these canine AREF isolates. rep6 and rep11 families of plasmids were significantly associated with isolates from dogs. The PFGE patterns of human and dog isolates were considered unrelated (≤ 80%). These findings also support the importance of prudent use of antibiotics in veterinary medicine to avoid zoonotic spread of canine AREF isolates. We are confident that our results may help to better understand the mechanisms of antibiotic resistance and mobile element carrying them as well as new strategies to reduce the horizontal transfer of antibiotic resistance and virulence traits.

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