<p>Posmatra se problem optimizacije bez ograničenja. Za re&scaron;avanje<br />problema&nbsp; optimizacije bez ograničenja postoji mno&scaron;tvo raznovrsnih<br />metoda. Istraživanje ovde motivisano je potrebom za metodama koje<br />će brzo konvergirati.<br />Cilj je sistematizacija poznatih rezultata, kao i teorijska i numerička<br />analiza mogućnosti uvođenja parametra u gradijentne metode.<br />Najpre se razmatra problem minimizacije konveksne funkcije vi&scaron;e<br />promenljivih.<br />Problem minimizacije konveksne funkcije vi&scaron;e promenljivih ovde se<br />re&scaron;ava bez izračunavanja matrice hesijana, &scaron;to je naročito aktuelno za<br />sisteme velikih dimenzija, kao i za probleme optimizacije kod kojih<br />ne raspolažemo ni tačnom vredno&scaron;ću funkcije cilja, ni tačnom<br />vredno&scaron;ću gradijenta. Deo motivacije za istraživanjem ovde leži i u<br />postojanju problema kod kojih je funkcija cilja rezultat simulacija.<br />Numerički rezultati, predstavljeni u Glavi 6, pokazuju da uvođenje<br />izvesnog parametra može biti korisno, odnosno, dovodi do ubrzanja<br />određenog metoda optimizacije.<br />Takođe se predstavlja jedan novi hibridni metod konjugovanog<br />gradijenta, kod koga je parametar konjugovanog gradijenta<br />konveksna kombinacija dva poznata parametra konjugovanog<br />gradijenta.<br />U prvoj glavi opisuje se motivacija kao i osnovni pojmovi potrebni za<br />praćenje preostalih glava.<br />U drugoj glavi daje se pregled nekih gradijentnih metoda prvog i<br />drugog reda.<br />Četvrta glava sadrži pregled osnovnih pojmova i nekih rezultata<br />vezanih za metode konjugovanih gradijenata.<br />Pomenute glave su tu radi pregleda nekih poznatih rezultata, dok se<br />originalni doprinos predstavlja u trećoj, petoj i &scaron;estoj glavi.<br />U trećoj glavi se opisuje izvesna modifikacija određenog metoda u<br />kome se koristi multiplikativni parametar, izabran na slučajan način.<br />Dokazuje se linearna konvergencija tako formiranog novog metoda.<br />Peta glava sadrži originalne rezultate koji se odnose na metode<br />konjugovanih gradijenata. Naime, u ovoj glavi predstavlja se novi<br />hibridni metod konjugovanih gradijenata, koji je konveksna<br />kombinacija dva poznata metoda konjugovanih gradijenata.<br />U &scaron;estoj glavi se daju rezultati numeričkih eksperimenata, izvr&scaron;enih<br />na&nbsp; izvesnom skupu test funkcija, koji se odnose na metode iz treće i<br />pete glave. Implementacija svih razmatranih algoritama rađena je u<br />paketu MATHEMATICA. Kriterijum upoređivanja je vreme rada<br />centralne procesorske jedinice.6</p> / <p>The problem under consideration is an unconstrained optimization<br />problem. There are many different methods made in aim to solve the<br />optimization problems.&nbsp; The investigation made here is motivated by<br />the fact that the methods which converge fast are necessary.<br />The main goal is the systematization of some known results and also<br />theoretical and numerical analysis of the possibilities to int roduce<br />some parameters within gradient methods.<br />Firstly, the minimization problem is considered, where the objective<br />function is a convex, multivar iable function. This problem is solved<br />here without the calculation of Hessian, and such solution is very<br />important, for example, when the&nbsp; big dimension systems are solved,<br />and also for solving optimization problems with unknown values of<br />the objective function and its gradient. Partially, this investigation is<br />motivated by the existence of problems where the objective function<br />is the result of simulations.<br />Numerical results, presented in&nbsp; Chapter&nbsp; 6, show that the introduction<br />of a parameter is useful, i.e., such introduction results by the<br />acceleration of the known optimization method.<br />Further, one new hybrid conjugate gradient method is presented, in<br />which the conjugate gradient parameter is a convex combination of<br />two known conjugate gradient parameters.<br />In the first chapter, there is motivation and also the basic co ncepts<br />which are necessary for the other chapters.<br />The second chapter contains the survey of some first order and<br />second order gradient methods.<br />The fourth chapter contains the survey of some basic concepts and<br />results corresponding to conjugate gradient methods.<br />The first, the second and the fourth&nbsp; chapters are here to help in<br />considering of some known results, and the original results are<br />presented in the chapters 3,5 and 6.<br />In the third chapter, a modification of one unco nstrained optimization<br />method is presented, in which the randomly chosen multiplicative<br />parameter is used. Also, the linear convergence of such modification<br />is proved.<br />The fifth chapter contains the original results, corresponding to<br />conjugate gradient methods. Namely, one new hybrid conjugate<br />gradient method is presented, and this&nbsp; method is the convex<br />combination of two known conjugate gradient methods.<br />The sixth chapter consists of the numerical results, performed on a set<br />of test functions, corresponding to methods in the chapters 3 and 5.<br />Implementation of all considered algorithms is made in Mathematica.<br />The comparison criterion is CPU time.</p> / <p>The problem under consideration is an unconstrained optimization<br />problem. There are many different methods made in aim to solve the<br />optimization problems.&nbsp; The investigation made here is motivated by<br />the fact that the methods which converge fast are necessary.<br />The main goal is the systematization of some known results and also<br />theoretical and numerical analysis of the possibilities to int roduce<br />some parameters within gradient methods.<br />Firstly, the minimization problem is considered, where the objective<br />function is a convex, multivar iable function. This problem is solved<br />here without the calculation of Hessian, and such solution is very<br />important, for example, when the&nbsp; big dimension systems are solved,<br />and also for solving optimization problems with unknown values of<br />the objective function and its gradient. Partially, this investigation is<br />motivated by the existence of problems where the objective function<br />is the result of simulations.<br />Numerical results, presented in&nbsp; Chapter&nbsp; 6, show that the introduction<br />of a parameter is useful, i.e., such introduction results by the<br />acceleration of the known optimization method.<br />Further, one new hybrid conjugate gradient method is presented, in<br />which the conjugate gradient parameter is a convex combination of<br />two known conjugate gradient parameters.<br />In the first chapter, there is motivation and also the basic co ncepts<br />which are necessary for the other chapters.<br />Key&nbsp; Words Documentation&nbsp; 97<br />The second chapter contains the survey of some first order and<br />second order gradient methods.<br />The fourth chapter contains the survey of some basic concepts and<br />results corresponding to conjugate gradient methods.<br />The first, the second and the fourth&nbsp; chapters are here to help in<br />considering of some known results, and the original results are<br />presented in the chapters 3,5 and 6.<br />In the third chapter, a modification of one unco nstrained optimization<br />method is presented, in which the randomly chosen multiplicative<br />parameter is used. Also, the linear convergence of such modification<br />is proved.<br />The fifth chapter contains the original results, corresponding to<br />conjugate gradient methods. Namely, one new hybrid conjugate<br />gradient method is presented, and this&nbsp; method is the convex<br />combination of two known conjugate gradient methods.<br />The sixth chapter consists of the numerical results, performed on a set<br />of test functions, corresponding to methods in the chapters 3 and 5.<br />Implementation of all considered algorithms is made in Mathematica.<br />The comparison criterion is CPU time</p>

La conjugaison de phase ultrasonore à couplage par l’air basée sur une céramique magnétostrictive et une membrane de filtration poreuse pour la microscopie et la vélocimétrie de micro écoulements a été développée. Dans ce but, dans le cadre de l’acoustique géométrique un système d’équations pour décrire mathématiquement le passage par l’interface entre l’élément actif du système de conjugaison de phase confocale – milieu de propagation a été développé.On a développé et réalisé une technique de codage de phase par m-séquence pour l’enregistrement des faibles signaux conjugués en phase. Cette technique a permis de travailler plus efficacement avec fort bruit et des signaux qui se trouvent sous le niveau de bruit. Aussi cette technique a permis d’améliorer une méthode de vélocimétrie des écoulements gazeux.On a développé et réalisé une technique d’adaptation d’impédance acoustique basée sur la membrane de filtration poreuse imprégnée par de l’huile. Cette technique a permis d’optimiser les conditions de transmission de l’onde à l’interface air–ferrite aux fréquences basse dans bande du MHz.Les applications possibles de l’effet de conjugaison de phase paramétrique à la vélocimétrie des écoulements gazeux et à la microscopie à couplage par l’air ont été présentées. L’application de l’effet de conjugaison de phase permet d’améliorer les performances des méthodes de vélocimétrie et de microscopie ultrasonores à couplage par l’air. Les méthodes élaborées a repoussé les limites d’applications pratiques de l’effet de conjugaison de phase et peuvent être utilisées pour le développement des dispositifs en vélocimétrie, microscopie et tomographie ultrasonore des écoulements gazeux / Air-coupled wave phase conjugation technique, based on magneto-acoustic interaction and porous membrane filters, for microscopy and velocity measurements of gas micro flows is under investigation. For this reason in the frame of ray acoustics the base system of equations for mathematical model of phase conjugate wave passage through the interface active element of con-focal WPC system – medium of propagation is developed. The phase coding technique by pseudonoise M-sequence was used for registration of weak acoustical phase conjugate signals. This method has allowed to work more effectively with strong noisy and being under noise level phase conjugate signals. Also this method has allowed improving a method of gas flow velocimetry.It is developed and realized the technology of acoustical matching on base of thin polycarbonate porous membrane filters impregnated by oil. This technology has allowed optimizing the conditions of wave transmission through the interface air–ferrite in the low megahertz frequency range.Possible applications of phase conjugate waves in air are shown. Results of investigations of air-coupled wave phase conjugation technics can serve for drawing up of new methods ultrasonic velocimetry and microscopy in technical industrial applications. The elaborated methods expand limits of application and can be used for development of devices of ultrasonic microscopy, tomography and velocimetry of gas micro flows

There are two general ways to introduce functionalities into a polymeric structure: functionalization of the monomeric units before polymerization and postfunctionalization of the preformed polymer. Building libraries of polymers with different functionalities can be completed with significantly less effort by the second method, as each postfunctionalization of a single batch of polymeric backbone can involve as little as one synthetic step. One method of building a polymeric backbone for postfunctionalization involves the synthesis of hyperbranched conjugated polymers (HCPs) from AB2 monomeric units. A polymer formed from n AB2 monomeric units should contain n reactive B groups, which act as sites of functionalization. Utilizing this principle, two different hyperbranched poly(phenylene vinylene-phenylene ethynylene) scaffolds were synthesized and studied in both their inherent properties and functionalization. The first HCP synthesized was compared against a monomeric cruciform model and a linear polymer with a similar structure. The hyperbranched polymer has red-shifted absorption and emission in comparison to the cruciform model and linear polymer. The HCP quenches paraquat more efficiently than the linear polymer by a factor of about two, suggesting a greater rate of energy transfer. The functionalization of HCPs was studied; iodine groups decorating the HCPs were replaced with terminal alkynes by Pd-catalyzed coupling, providing a library of 24 differently functionalized HCPs. Elemental analyses of the postfunctionalized polymers show nearly complete substitution of the iodine groups. The postfunctionalized polymers show increased fluorescence compared to the original iodine decorated polymers, due to the loss of the heavy atom effect inducing iodine groups. The emissions of the postfunctionalized polymers in solution show a strong dependence on the groups attached to the conjugated structures, with emission maxima ranging from 505 nm to 602 nm; quantum yields range from 0.7% to 25%. Solid-state emission studies show stronger and more red-shifted spectra compared to emissions observed in solution.

Investigations detailed in this thesis constitute a part of the continuing programme of research undertaken in our laboratory on the riboflavin carrier protein (RCP) with partic­ular reference to identification and synthesis of neutralizing antigenic determinants, design of relevant epitope mimetics with improved immunogenic characteristics and relationship between their secondary structures and immunological properties. The riboflavin carrier protein is elaborated as a reproductive stratagem to ensure ade­quate vitamin deposition in the developing oocyte in the chickens. The protein is scrupu­lously conserved through evolution in terms of physico chemical and immunological char­acteristics from fish through birds to mammals, including primates. In rodents and sub­human primates immunization with the heteroantigen viz., chicken egg white RCP leads to functional neutralization of the endogenous maternal protein resulting in curtailment of early pregnancy. Thus, the crucial role of RCP in maintenance of pregnancy is established and the protein identified as a potential candidate vaccine for immimocontraception. Fur­ther studies with the reduced and carboxymethylated (RCM) RCP as the immunogen re­veal that antibodies induced by RCM-RCP are equally effective in bioneutralization of the endogenous protein. So it can be surmised that the native folded structure of RCP is not obligatory for eliciting bioneutralizing antibodies. In an attempt to identify functionally relevant regions of the protein, a panel of monoclonal antibodies (MAbs) have been raised and characterized. One of the MAbs viz., 6J32Ci2 could bring about early fetal resorp-tion when injected to mice with confirmed pregnancies. These results prompted a detail molecular immunological approach to understand underlying mechanisms. The principal aims of the present investigations include: (1) identification of neutralizing epitopes; (2) synthesis of peptidyl sequences incorporating these determinants; (3) an understanding of the structure, antigenic and immunogenic characteristics of these peptides; (4) correlation of conformational and antigenic characteristics; (5) rational design and synthesis of peptide analogs with greater propensity to assume predicted secondary structures; (6) analysis of conformation dependency of peptide antigens and the importance of such conformation in generating an optimal B-cell response; (7) the efficacy of the antibodies elicited by these Peptide antigens in neutralizing endogenous protein with the ultimate aim of designing synthetic vaccines. Chapter 1 of this thesis deals with a general introduction summarizing the current status of knowledge regarding the chemistry and biology of RCP as well as synthetic pep­tides as potential immunogens. Chapter 2 outlines details of the experimental procedures adopted. Chapter 3 describes the results of investigations on the C-terminal fragment (residues 200-219) of cRCR The main consideration in selecting this sequence for the design of a potential peptide-based vaccine relied on the epitopic specificity of the neu­tralizing MAb 6S2C12. Epitope mapping using the Pepscan method revealed that the monoclonal antibody recognizes a core sequence corresponding to residues 203-210 of the cRCP. A 21-residue synthetic peptide (C-21) comprising this epitope was synthesized and antibodies elicited to the peptide conjugated to two different carriers, namely diphtheria toxoid and purified protein derivative (PPD) for T-cell help. In both active and passive immunoneutralization experiments, the peptide specific neutralizing antibodies interfered with the biological function of the protein and hence either protected from pregnancy or caused early fetal resorption in rodents as well as in sub-human primates. The conforma-tional properties of the peptide in aqueous buffers were analyzed from circular dichroism which revealed the absence of any ordered structure in the native C-21 peptide. Theoreti­cal predictions of secondary structure suggested a propensity for an t*-helical structure for this fragment in the native protein. Therefore, influence of the helix-promoting solvent, vizM 2,2,2,trifluroethanol (TFE) on the C-21 peptide was investigated. Addition of TFE resulted in spectral changes with negative bands at 208 and 222 nm and a positive band at 190 rim which are typical of an a-helix. To gain more information on the conformational characteristics of this peptide, it was considered worthwhile to stabilize the native peptide in an a-helical conformation based on simple rational design principles. Towards this end, four analogs of the parent peptide were synthesized and helix stabilization was sought to be achieved by introducing either salt bridges or back-bone conformational constraints such as by incorporating a-amino isobutyric acid at appropriate positions. In all the analogs, the core sequence, recognised by the neutralizing MAb 6B2C12 was maintained intact to ensure induction of antibodies capable of recognizing the native protein. CD spectral analysis of the analog peptides indicated that all the engineered peptides had varying degrees of enhanced helicities as compared to the parent peptide. The immunogenicity of each analog was studied by to the relevant peptide-diphtheria toxoid conjugates and analyzing their reactivities with the native protein by direct and competitive ELISA. The results revealed that these engi­neered conformational analogs axe highly immunogenic eliciting high titers of anti-protein antibodies. The relative affinities of these antibodies to bind cRCP were investigated. The antibodies to peptide analogs had higher affinities for the native protein and a positive correlation was found between the helical content of the peptide antigen in question and the relative affinity of corresponding antibody. The antibodies directed to all the peptide analogs could block the function of RCP resulting in early embryonic resorption when ad­ministered to pregnant mice. An interesting pattern of immunological cross-reactivity has been observed with the native and designed peptides. Antibodies raised to constrained helical analogs could bind the C-21 peptide which is structurally flexible. In contrast, the antibodies raised to the flexible native peptide antigen were inefficient in recognizing the structured peptides. The ability of all the peptide antibody to bind the native protein has been interpreted in terms of a conformationally flexible C-terminus region in cRCP. Chapter 4 details investigations on a 21-residue peptide (N- 21) from the N-terminiis (4-24) of the protein. Selection of this peptidyl sequence relied on theoretical prediction of potential sequential determinants on RCP other than at C-terminus as well as on the outcome of immunoneutralisation experiments using antibodies to egg yolk RCP which lacks the relevant C-terminal determinants. The structure of this peptide in solution was analyzed by two dimensional NMR and CD. NMR experiments revealed the presence of two structured regions in the peptide. Diagnostic nuclear Overhauser effects characteristics of reverse turns or short frayed helical segments over residues 3-9 and 18-21 of the peptide were obtained. CD spectra showed the presence of a strong, negative band at 204 nm over a wide range of solvent conditions, a feature which has been interpreted in terms of a "polyproline Il-like" segment encompassing residues 11-16 which corresponds to an interesting (X-Pro)^ repeat in the N-21 sequence. Specific antibodies were generated to this peptide as a conjugate with diphtheria tox­oid. Administration of the antipeptide antibodies could neutralize the protein in vivo as demonstrated by early embryonic loss in pregnant mice. In limited experiments the anti­peptide antibodies showed propensity to protect bonnet monkeys from pregnancy over a few consecutive ovulatory cycles when titres are maintained elevated by periodic boosting. To address the relationship between peptide structures and antigenicity, epitope mapping of this antipeptide antibodies as well- as the polyclonal antibodies to native RCP was undertaken using the Pepscan method. The results reveal that antigenic regions correspond well to conformationally well-defined elements of structure with the polyproline II-like seg­ment being a common antigenic determinant on both the peptide and the native protein. These observations are suggestive of the involvement of both the N and C-terminal regions of RCP in terms of its binding to putative plasma membrane receptors.

Protein therapeutics offer numerous advantages over small molecule drugs and are rapidly becoming one of the most prominent classes of therapeutics. Unfortunately, they are delivered almost exclusively by injection due to biological obstacles preventing high bioavailability via the oral route. In this work, numerous approaches to overcoming these barriers are explored. PH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) (P(IA-co-NVP)) hydrogels were synthesized, and the effects of monomer ratios, crosslinking density, microparticle size, protein size, and loading conditions were systematically evaluated using in vitro tests. P(IA-co-NVP) hydrogels demonstrated up to 69% greater equilibrium swelling at neutral conditions than previously-studied poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels and a 10-fold improvement in time-sensitive swelling experiments. Furthermore, P(IA-co-NVP) hydrogel microparticles demonstrated up to a 2.7-fold improvement in delivery of salmon calcitonin (sCT) compared to methacrylic acid-based systems, with a formulation comprised of a 1:2 ratio of itaconic acid to N-vinylpyrrolidone demonstrating the greatest delivery capability. Vast improvement in delivery capability was achieved using reduced ionic strength conditions during drug loading. Use of a 1.50 mM PBS buffer during loading yielded an 83-fold improvement in delivery of sCT compared to a standard 150 mM buffer. With this improvement, a daily dose of sCT could be provided using P(IA-co-NVP) microparticles in one standard-sized gel capsule. P(IA-co-NVP) was also tested with larger proteins urokinase and Rituxan. Crosslinking density provided a facile method for tuning hydrogels to accommodate a wide range of protein sizes. The effects of protein PEGylation were also explored. PEGylated sCT displayed lower release from P(IA-co-NVP) microparticles, but displayed increased apparent permeability across a Caco-2 monolayer by two orders of magnitude. Therefore, PEG-containing systems could yield high bioavailability of orally delivered proteins. Finally, a modified SELEX protocol for cellular selection of transcellular transport-initiating aptamers was developed and used to identify aptamer sequences showing enhanced intestinal perfusion. Over three selection cycles, the selected aptamer library showed significant increases in absorption, and from an initial library of 1.1 trillion sequences, 5-10 sequences were selected that demonstrated up to 10-fold amplification compared to the naïve library. These sequences could provide a means of overcoming the significant final barrier of intestinal absorption. / text

The synthesis of benzo[e]dimethyldihydropyrene (BDHP) photoswitches with ter-27, quarter-36, and quinque-28 thiophene oligomers attached on the same side of the switch was achieved using Stille coupling reactions. BDHP photoswitches with bi-75, ter-76 and quinque-77 thiophene oligomers attached directly to the switch on one side, and via a carbonyl spacer on the opposite side of the switch were also synthesized. Dimethyldihydropyrene (DHP) photoswitches with a naphthoyl functional group in the 2 position were synthesized using a Friedel Crafts reaction, and ter-96, quinque-97 and septi-98 thiophene oligomers were attached on opposite sides of the switch using Stille coupling reactions. All compounds were characterized by NMR, IR UV-vis spectroscopy and mass spectrometry. The relative rates of the photo-opening reactions under excess light conditions and the UV closing reactions versus BDHP were measured. Improvements in the photo-opening properties of the oligothiophene functionalized switches compared to BDHP were observed. The most dramatic photo-opening improvement was found for the quinquethienyl substituted DHP switch 97 which photo-opened when irradiated with visible light over 100 times faster than BDHP. UV closing rates were virtually the same as that of BDHP. However the addition of oligothiophenes led to an increase in the thermal closing reaction rates. Compounds with the naphthoyl functional group in the 2 position of DHP were found to have dramatically increased thermal closing rates. The electrochemical properties of oligothiophene functionalized BDHP and naphthoyl functionalized DHP switches in the closed form were studied by cyclic voltammetry and spectroelectrochemistry. During the oxidation cycle, a closing reaction from the cyclophanediene (CPD) form to the DHP form of the switches occurred which prevented the study of the electrochemical properties of the switches in the open form. Conductivity testing was performed on the quinquethienyl substituted DHP switch 97 using a gold interdigitated micro electrode array. The conductivity of undoped 97 was greater in the closed DHP isomer than in the open CPD isomer. Irradiation with red or blue light allowed for repetitive switching between the more highly conducting closed form and the less conducting open form. When electrochemically doped, 97 showed improved conductivity over the undoped form but only the conductivity of the closed doped form could be measured due to electrochemically induced closing.

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.

This master thesis deals with possibilities of using nanoparticles of different metals as markers for immune labeling in the low voltage electron microscope. It presents the low voltage elektron microscope LVEM 5 (Delong Instruments, Brno, Czech Republic) as a new type of a table-size microscope specially designed for observation of specimens composed from elements with low atomic numbers.

Made available in DSpace on 2016-06-02T19:55:29Z (GMT). No. of bitstreams: 1 3391.pdf: 9156638 bytes, checksum: 0de8724bcb52a60114f5d3639d17f212 (MD5) Previous issue date: 2010-08-11 / Financiadora de Estudos e Projetos / Molecular biology and genetic engineering have been deployed widely in recent years, several protocols have been established, presenting new methodologies capable of altering the genetics of bacterial strains industrial interest. This research had as main objectives: (1) the construction of the following plasmids: p&#61636;lat, pAMB4 and pAMB3RpoZneo, (2) the production of Streptomyces clavuligerus mutants by gene disruption, by insertion of plasmid integrative, and by replication of multi-copy plasmid in Streptomyces clavuligerus by conjugation and (3) study the effect of these mutations on clavulanic acid production and lipolytic activity. In Spain (University of Leon), six mutants Streptomyces clauligerus &#61636;lat::apr, Streptomyces clavuligerus cvm7P::neo, Streptomyces clauligerus &#61636;Lat::apr &#61636;cvm7P::neo, Streptomyces clavuligerus pRAneoZ, Streptomyces clavuligerus pAMB4 and Streptomyces clavuligerus pAMB3RpoZneo were produced. In Leon, the fermentations were performed with the SA culture medium and only with the mutants Streptomyces clavuligerus &#61636;lat::apr and Streptomyces clavuligerus pRAneoZ. In this case, there was no statistically significant difference at 5% probability by analysis of variance (ANOVA). In Brazil, the fermentations with all mutants in the culture medium-based oil and soybean meal, showed a different pattern in the production of clavulanic acid. The mutants Streptomyces clavuligerus pAMB3RpoZneo and Streptomyces clavuligerus pRAneoZ (&#61665;&#61472;= 5%) showed clavulanic acid titles higher when compared with the wild type. The double mutant Streptomyces clavuligerus &#61636;lat::apr &#61636;cvm7P::neo, contrary to expectations, showed the lowest levels of clavulanic acid in relation to its parental strain. Streptomyces clavuligerus pRAneoZ mutant and the mutant Streptomyces clavuligerus pAMB4 control, showed the highest lipolytic activity at 5% probability. The double mutant in turn, had the lowest lipolytic activities. A direct relationship between levels of clavulanic acid and lipase production was observed. All mutants produced in this work could be fermented into bioreactor to assess production levels of clavulanic acid and lipase. The construction of a new double mutant named Streptomyces clavuligerus &#61636;lat::apr RpoZneo from existing ones mutant could be of great interest to investigate this new combination of mutations on clavulanic acid production and lipolytic activity. / A biologia molecular e a engenharia genética têm se desenvolvido muito nos últimos anos e vários protocolos foram estabelecidos, apresentando novas metodologias capazes de alterar a genética de linhagens bacterianas de interesse industrial. O presente estudo teve por objetivos principais: (1) a construção dos seguintes plasmídeos: p&#61636;lat, p&#61636;cmv7P, pAMB4 e pAMB3RpoZneo; (2) a produção de mutantes de Streptomyces clavuligerus por disrupção gênica, por inserção de plasmídeo integrativo, e por replicação de plasmídeo multi-cópia em Streptomyces clavuligerus por conjugação bacteriana; (3) o estudo do efeito destas mutações na produção de ácido clavulânico e na atividade lipolítica. Na Espanha (Universidade de León), seis mutantes foram produzidos: Streptomyces clavuligerus &#61636;lat::apr, Streptomyces clavuligerus &#61636;cvm7P::neo, Streptomyces clavuligerus &#61636;lat::apr &#61636;cvm7P::neo, Streptomyces clavuligerus pRAneoZ, Streptomyces clavuligerus pAMB4 e Streptomyces clavuligerus pAMB3RpoZneo. Em León, as fermentações foram realizadas com o meio de cultura SA e somente com os mutantes Streptomyces clavuligerus &#61636;lat::apr e Streptomyces clavuligerus pRAneoZ. Nestas fermentações não houve diferença estatística significativa ao nível de 5% de significância, pela análise de variânica (ANOVA). No Brasil, as fermentações com todos os mutantes no meio de cultura a base de óleo e farinha de soja, mostraram um padrão diferenciado na produção de ácido clavulânico. Os mutantes Streptomyces clavuligerus pRAneoZ e Streptomyces clavuligerus pAMB3RpoZneo apresentaram títulos de ácido clavulânico superiores quando comparados com a linhagem selvagem (&#61665;&#61472;= 5%). O duplo mutante Streptomyces clavuligerus &#61636;lat::apr &#61636;cvm7P::neo, ao contrário do esperado, apresentou os níveis mais baixos de ácido clavulânico e em relação a sua linhagem parental. Os mutantes Streptomyces clavuligerus pRAneoZ e o mutante controle Streptomyces clavuligerus pAMB4, apresentaram a maior atividade lipolítica ao nível de 5% de significância. O duplo mutante por sua vez, apresentou as menores atividades lipolíticas. Uma relação direta entre os níveis de ácido clavulânico e a produção de lipase foi observada. Todos os mutantes produzidos neste trabalho poderiam ser fermentados em biorreator de bancada para se avaliar os níveis de produção de ácido clavulânico e de lipase. A construção de um novo duplo mutante denominado, Streptomyces clavuligerus p&#61636;lat::apr RpoZneo, a partir dos existentes, poderia ser de grande interesse para investigar esta nova combinação de mutação na produção de ácido clavulânico e na atividade lipolítica.

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