Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

El objetivo del presente estudio es el de identificar las tendencias en la traducción de elementos culturales específicos en traducciones de inglés a croata y, posteriormente, correlacionarlas con los desarrollos que ocurrieron en el contexto general en el que se produjeron las traducciones. El estudio se propuso probar hipótesis que predicen una tendencia creciente por emplear tipos de soluciones de asimilación al traducir elementos culturales específicos y también relacionar esta tendencia con los mayores flujos de traducción. Las hipótesis se evalúan usando un corpus de novelas de detectives traducidas en tres períodos: principios de los 60, finales de los 70 y la década del 2000. Los datos cuantitativos obtenidos por medio de análisis textuales se unen con datos cualitativos obtenidos en entrevistas son los agentes de las traducciones. En la conclusión se discuten estos resultados con el objetivo de establecer posibles correlaciones entre los datos del análisis textual, los datos de las entrevistas y los datos extratextuales con las variables contextuales.

No description available.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Univ., Diss.--Ohio.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 217-233).

The plaque reduction neutralization test (PRNT) is a confirmatory diagnostic assay that is used to confirm a variety of diseases. The performance of PRNT requires the use of infectious wild type viruses, which increases the risk of laboratory acquired infections. For instance, eastern equine encephalitis (EEEV) is a highly virulent pathogen used in PRNT that can result in potentially fatal neurological diseases among humans and equines. Therefore, arboviral PRNT must be performed in Biosafety Level 3 (BSL-3) containment facilities and may require select agent approved scientists, like in the case of EEEV. These stringent requirements restrict the ability of public health laboratories to conduct PRNTs. Chimera viruses, recombinant constructs that have been bio-engineered to express the immunogenic structural proteins from the wild type virus in an attenuated form, can serve as a substitution for infectious viruses when performing PRNT. Since chimera viruses do not require the use of a BSL-3 facility and are not classified as select agents, their use offers advantages over wild type viruses. This study aimed at validating the use of EEE and West Nile chimera viruses as an alternative to the corresponding wild type viruses for diagnostic purposes at the Florida Department of Health (FDOH) Bureau of Public Health Laboratories (BPHL). These evaluations were conducted using human and avian sera. The results illustrate that chimera virus-based PRNT portrays specificity comparable to that of the wild type virus, while a slight reduction in sensitivity was observed when human sera was used. Considering their benefits in increasing safety and reducing regulatory requirements, these chimera viruses are an important alternative to the virulent wild type viruses and could be highly beneficial for diagnostic laboratories.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Ce travail porte sur la neutralization résonnante d’ions Li+ sur des métaux et agrégatsmétalliques supportés. La neutralization sur Pd(100) a été étudié, pour différentsparamètres. La neutralisation s’avère très efficace sur cette surface avec un travail desortie grand et est en contradiction avec ce qu’on attend dans le cadre des modèlestraditionnels. Il est proposé qu’une description du processus RCT modifié, issued’études précédentes sur des métaux nobles, doit être utilisé. Dans celle ci desprocessus de neutralisation survenant à des distances atome- surface faible ont lieu etaussi un comportement adiabatique du système aux faible énergies de collisions mèneà une neutralization efficace.Les résultats de l’étude des processus de transfert d’électrons lors de l’interactiond’ions de Li+ avec des agrégats d’or supporté sur HOPG avec Al2O3 sont présentés etdiscuté. L’imagerie STM pour les agrégats d’or supporté sur un substrat HOPG viergeet aussi un substrat de HOPG bombardé par des ions de AR sont présentés. Lesobservations révèlent que agrégats d’or forment préférentiellement des chaines 1D lelong des marches sur HOPG vierge. Dans le cas de HOPG bombardé, la taille et lahauteur des agrégats sont dépendants des défauts de surface.Nous avons trouvé que la neutralisation est très efficace sur les petits agrégats et engénéral est beaucoup plus grande que sur des surfaces de cristaux d'or. Nous montronsdes effets liés à la nature du substrat, comme dans le cas de l’alumine ou le cas desdifférences observées sur des chaines d’agrégats sur HOPG vierge et les agrégatsformés sur des défauts / The present work investigates the neutralization of Li+ ions on metals and supportedmetal clusters. Neutralization on a transition metal surface Pd (100) for differentparameters was studied. Highly efficient neutralization on this surface with a highwork functions was observed and contradicts our traditional views on resonant chargetransfer (RCT) mechanism. A modified RCT picture involving new neutralizationprocesses occurring at a short atom-surface distance and an adiabatic behaviourleading to efficient neutralization at large distances, that has emerged from previousstudies on noble metal surfaces appears in qualitative agreement with our data.The experimental results on the dependence of the Li neutralization on the Auclusters supported on different substrates are reported and discussed. As acomplement to this, a STM study into the morphology of Au nanoparticles on apristine HOPG substrate as well as Ar+ ions sputtered HOPG substrate has beenperformed. The observations reveal that Au clusters preferentially form onedimensional chains along steps on pristine HOPG. In the case of Ar+ ions sputteredHOPG substrate, the size and height of cluster are dependent on surface defects.We found that neutralization is very efficient on small clusters and in general muchlarger than on surfaces of gold crystals. We demonstrate existence of strongdifferences as a function of cluster support type as in case of alumina supports orcluster chains on HOPG and clusters on defects on HOPG terraces.