Master of Science / The role of the microbiology laboratory is (1) to provide infection control information, so that highly transmissible isolates may be identified and appropriate control measures instigated as rapidly as possible and (2) to provide adequate information to the clinician enabling correct antibiotic choices to be made, particularly in the critically ill. Microbiological data is by definition slow as it is culture dependent: this study focused on the development of genetic, culture-independent methods for detection of resistance in nosocomial pathogens that could be introduced into the routine microbiology department and would fit into the routine workflow with a consequent reduction in time to result. Initially a duplex real-time polymerase chain reaction was developed for the rapid identification and detection of S. aureus and methicillin-resistance. This was optimised for immediate as-needs testing of positive blood cultures signalling with “Gram positive cocci, possibly staphylococcus” evident on Gram stain, on a random access real-time PCR platform. This technology, allowing early identification of S. aureus and its susceptibility to methicillin, by simple automated methodology, may soon become the standard for all microbiology laboratories servicing the critically ill. The second part of the study involved the development of a selective broth and multiplex PCR for detection of three important nosocomial isolates at this institution, methicillin-resistant S. aureus (MRSA), carbapenem-resistant Enterobacteriaceae, and multi-resistant Acinetobacter baumannii (MRAB). A multiplex PCR using four primer sets was designed to detect low colonisation levels of these isolates after overnight incubation in selective broth, significantly reducing the time to result and associated costs. This potentially useful epidemiological screening tool is practical, reproducible and sensitive with the potential of moving to an automated test (using real-time PCR, for example) in the future. The availability of early negative results judged by simple visual scanning (or by densitometry), means that the result is less operator-dependent, potentially reducing error rate. The last part of the study dealt with an important resistance phenotype, aminoglycoside resistance. There had been no recent comprehensive local surveys performed to determine the frequency of aminoglycoside resistance amongst the Enterobacteriaceae, or to identify the genetic determinants and their transmissibility. The isolates collected for the study were all resistant to at least one of gentamicin, tobramycin or amikacin. Identification of integron cassette arrays and use of specific internal primers identified at least one genetic determinant for gentamicin and tobramycin resistance in 22 of 23 isolates. Three isolates had two aminoglycoside resistance genes, and three isolates had three aminoglycoside resistance genes identified (Table 6.1). Transferable gentamicin-resistant plasmids were predominant amongst Klebsiella spp., but less so amongst Enterobacter spp. and E. coli. Gentamicin-resistant Klebsiella spp. were often ESBL positive, the genetic determinants of which were typically co-transferred on a conjugative plasmid. The importance of screening at a local level was demonstrated by the unexpected predominance of aac(6')-IIc amongst Enterobacter spp. and the detection of a new gene (aac(6')-LT). This part of the study has provided an understanding of the primary aminoglycoside resistance genes present in the local setting and their association with other resistances. This knowledge will allow development of assays for patient screening (clinical isolates and colonising flora), to better understand the epidemiology of aminoglycoside resistance and to allow better choice of antibiotic therapy related to presence or absence of these genes.

Master of Science / The role of the microbiology laboratory is (1) to provide infection control information, so that highly transmissible isolates may be identified and appropriate control measures instigated as rapidly as possible and (2) to provide adequate information to the clinician enabling correct antibiotic choices to be made, particularly in the critically ill. Microbiological data is by definition slow as it is culture dependent: this study focused on the development of genetic, culture-independent methods for detection of resistance in nosocomial pathogens that could be introduced into the routine microbiology department and would fit into the routine workflow with a consequent reduction in time to result. Initially a duplex real-time polymerase chain reaction was developed for the rapid identification and detection of S. aureus and methicillin-resistance. This was optimised for immediate as-needs testing of positive blood cultures signalling with “Gram positive cocci, possibly staphylococcus” evident on Gram stain, on a random access real-time PCR platform. This technology, allowing early identification of S. aureus and its susceptibility to methicillin, by simple automated methodology, may soon become the standard for all microbiology laboratories servicing the critically ill. The second part of the study involved the development of a selective broth and multiplex PCR for detection of three important nosocomial isolates at this institution, methicillin-resistant S. aureus (MRSA), carbapenem-resistant Enterobacteriaceae, and multi-resistant Acinetobacter baumannii (MRAB). A multiplex PCR using four primer sets was designed to detect low colonisation levels of these isolates after overnight incubation in selective broth, significantly reducing the time to result and associated costs. This potentially useful epidemiological screening tool is practical, reproducible and sensitive with the potential of moving to an automated test (using real-time PCR, for example) in the future. The availability of early negative results judged by simple visual scanning (or by densitometry), means that the result is less operator-dependent, potentially reducing error rate. The last part of the study dealt with an important resistance phenotype, aminoglycoside resistance. There had been no recent comprehensive local surveys performed to determine the frequency of aminoglycoside resistance amongst the Enterobacteriaceae, or to identify the genetic determinants and their transmissibility. The isolates collected for the study were all resistant to at least one of gentamicin, tobramycin or amikacin. Identification of integron cassette arrays and use of specific internal primers identified at least one genetic determinant for gentamicin and tobramycin resistance in 22 of 23 isolates. Three isolates had two aminoglycoside resistance genes, and three isolates had three aminoglycoside resistance genes identified (Table 6.1). Transferable gentamicin-resistant plasmids were predominant amongst Klebsiella spp., but less so amongst Enterobacter spp. and E. coli. Gentamicin-resistant Klebsiella spp. were often ESBL positive, the genetic determinants of which were typically co-transferred on a conjugative plasmid. The importance of screening at a local level was demonstrated by the unexpected predominance of aac(6')-IIc amongst Enterobacter spp. and the detection of a new gene (aac(6')-LT). This part of the study has provided an understanding of the primary aminoglycoside resistance genes present in the local setting and their association with other resistances. This knowledge will allow development of assays for patient screening (clinical isolates and colonising flora), to better understand the epidemiology of aminoglycoside resistance and to allow better choice of antibiotic therapy related to presence or absence of these genes.

Thesis (Ph.D.) -- Memorial University of Newfoundland. / Typescript. Bibliography: leaves 137-147. Also available online.

Située à la pointe du Médoc, la paroisse de Soulac (communes actuelles de Soulac-sur-Mer et du Verdon-sur-Mer, Gironde) est selon une expression médiévale « à la fin des terres ». Prise en tenaille entre l’Atlantique, à l’ouest, et la Gironde, à l’est, son finage comprend un long liseré littoral (14 km de rivages océaniques et 11 km de rives estuariennes) qui l’expose aux phénomènes d’origine naturelle liés à des dynamiques maritimes et fluviomaritimes. Depuis la fin du XVIe siècle, le sable accumulé le long du littoral sous la forme de dunes est porté par le vent sur les habitations et les cultures, poussant à l’abandon de l’église de Soulac et du bourg primitif au milieu du XVIIIe siècle. Parallèlement, le littoral est fortement attaqué par l’érosion qui se généralise au milieu du Siècle des Lumières. À l’est, les marais salants s’envasent, réduisant en moins d’un siècle la moitié de leur surface. Enfin, les zones basses de la paroisse sont exposées aux submersions marines, qui demeurent cependant peu fréquentes. Ce micro-espace de 50 km² constitue un bon point d’observation pour étudier les transformations d’un territoire littoral et l’adaptation des populations à ces changements. Prenant appui sur des ressources documentaires importantes (archives textuelles et cartographiques) et continues dans le temps (de la fin du XVIe au milieu du XIXe siècle), l’analyse croise les méthodes historiques traditionnelles avec les possibilités offertes par les outils informatiques contemporains tels que les SIG (géolocalisation et extraction de données qualitatives et quantitatives de cartes anciennes). L’analyse spatio-temporelle permet la reconstitution géohistorique des paysages de Soulac sur un temps long, en mettant en avant les dynamiques naturelles, les transformations importantes et les réponses apportées par les habitants. Ces derniers, d’abord impuissants et livrés à eux-mêmes par les autorités, tentent de s’adapter aux changements en adoptant plusieurs stratégies au cours du XVIIIe siècle. De la simple retenue des sables par des palissades à l’assèchement des marais doux et salés, ils essayent de répondre aux contraintes exercées sur eux par leur environnement. Mais ce sont les actions de l’État au début du XIXe siècle qui conditionnent la réussite des opérations de lutte et le début d’une nouvelle ère. Les dunes fixées par des pins et l’érosion enrayée par la construction d’ouvrages donnent naissance au paysage actuel de la pointe du Médoc. Plus qu’une simple monographie paroissiale, cette étude de cas permet de s’interroger sur notre environnement et les interactions que son exploitation et son peuplement influencent. Reconstruire sur un temps long les transformations permet une meilleure compréhension des risques et des phases de construction d’un espace géographique, et offre un recul historique permettant de mieux comprendre les événements récents. / Situated at the pointe du Medoc region, the parish of Soulac (modern day communes of Soulac-sur-Mer and Verdon-sur-Mer) is according to a medieval expression “at the end of the lands”. Squeezed between the Atlantic to the west, and the Gironde estuary to the east, the area includes a long coastline (14km of ocean coastline and 11km of estuary banks) which exposes it to phenomenon of natural origin linked to these maritime and fluvial-maritime movements. Since the end of the sixteenth century, the sand accumulated along the length of the coastline in the forms of dunes and carried by the wind onto habitations and cultures, causing the abandonment of the Soulac church and the early initial village in the mid eighteenth century. Simultaneously, the coastline was strongly attacked by erosion that spread in the middle of the Age of Enlightenment. To the east, the flat salt marshes were getting wider, reducing half their surface in less than a century. Finally, the low-lying areas of the parish are exposed to marine submersions, despite beginning with little frequency. This micro space, of 50km2, encompasses a good observation point in order to study the transformations of coastal land and the adaptations of populations to these changes. Taking support from important documentary resources (textual archives and cartographic maps) and continuous in the time (from the end of the 16th century to the middle of the 19th century), the analysis combines traditional historic methods with the possibilities offered by the contemporary computer tools, such as GIS (geolocation, extraction of qualitative data and quantitative old maps). The spatiotemporal analysis allows for the geohistoric reconstruction of the Soulac landscape over a long period, reflecting the natural dynamics, the important transformations and the responses from the inhabitants. The inhabitants, firstly made powerless by the authorities and had given up on themselves, have adapted to the changes by adopting many strategies throughout the course of the 18th century. From the simple control of the sand, using boundaries that dry up the soft, dirty marshes, they are trying to respond to the constraints forced upon them by their environment. However, the actions of the government at the beginning of the nineteenth century which allow for the success of the preventative actions and the start of a new era. The dunes fixed by pine trees and the erosion, stopped by the construction of structures, has given birth to the current landscape at the point of Medoc. More than a simple parish monograph, this case study allows us to ask questions about our environment and the affect that the interactions of cultivation and people have. Reconstructing over a long period of time the transformations offers a better understanding of the risks and the phases of construction of a geographical area offering a historical reviews which allows for a better understanding recent events.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Simona Suchá Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study of ABC drug efflux transporter inhibition by selected tyrosine kinase inhibitors using accumulation methods with cytostatic substrates ATP-binding cassette (ABC) drug efflux transporters are transmembrane proteins that utilize the energy from ATP hydrolysis to drive transport of endogenous and exogenous compounds out of the cell. The overexpression of ABC transporters plays a crucial role in the development of multidrug resistance (MDR), a phenomenon responsible for the failure of chemotherapy. Tyrosine kinase inhibitors (TKI) represent novel beneficial therapeutic approach in cancer treatment. TKI block tyrosine kinases which regulate important cellular processes. Deregulation of these enzymes can lead to various types of cancers. In the present work, we investigated interaction potential of selected TKI (alectinib, brivanib, osimertinib, selumetinib) in MDCKII parent cell line and those transduced with human efflux transporters ABCB1, ABCC1 and ABCG2. Using the accumulation studies, we determined the amount of accumulated model substrates (daunorubicin, mitoxantrone) and evaluated the inhibitory effect of...