Throughout the history of the Annular Core Research Reactor (ACRR), Transient Rod (TR) A has experienced an increased rate of failure versus the other two TRs (B and C). Either by pneumatic force or electric motor, the transient rods remove the poison rods from the ACRR core allowing for the irradiation of experiments. In order to develop causes for why TR A is failing (rod break) more often, a better understanding of the whole TR system and its components is needed. This study aims to provide a foundational understanding of how the TR pneumatic system affects the motion of the TRs and the resulting effects that the TR motion has on the neutronics of the ACRR. Transient rod motion profiles have been generated using both experimentally-obtained pressure data and by thermodynamic theory, and input into Razorback, a SNL-developed point kinetics and thermal hydraulics code, to determine the effects that TR timing and pneumatic pressure have on reactivity addition and reactivity feedback. From this study, accurate and precise TR motion profiles have been developed, along with an increased understanding of the pulse timing sequence. With this information, a safety limit within the ACRR was verified for different TR travel lengths and pneumatic system pressures. In addition, longer reactivity addition times have been correlated to cause larger amounts of reactivity feedback. The added clarity on TR motion and timing from this study will pave the way for further study to determine the cause for the increased failure rate of TR A.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-16T14:40:02Z No. of bitstreams: 1 FernandaNomiyamaFigueiredoDissertacao2018.pdf: 2178476 bytes, checksum: 23f38c14567d00ff189eaef20f904495 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-16T14:40:47Z (GMT) No. of bitstreams: 1 FernandaNomiyamaFigueiredoDissertacao2018.pdf: 2178476 bytes, checksum: 23f38c14567d00ff189eaef20f904495 (MD5) / Made available in DSpace on 2018-04-16T14:40:47Z (GMT). No. of bitstreams: 1 FernandaNomiyamaFigueiredoDissertacao2018.pdf: 2178476 bytes, checksum: 23f38c14567d00ff189eaef20f904495 (MD5) Previous issue date: 2018-03-06 / Klebsiella pneumoniae is one of the main bacterial agents that may cause infections related to health care assistance. K. pneumoniae frequently carries the resistance gene K. pneumoniae carbapenemase (KPC). Currently, tigecycline can be considered one of the last therapeutic options for KPC, but reports of tigecycline-resistant KPC isolates are on the rise, being indicated as most common mechanism the increased AcrAB-TolC efflux pump system expression. However, molecular tigecycline resistance mechanisms associated to AcrAB-TolC still remains obscure. Thus, the main goal of this study was to verify if tigecycline resistance can be related to the presence of mutations in the regulatory genes of AcrAB-TolC, AcrR and RamR. Therefore, 32 K. pneumoniae isolates were used, identification and antibiogram performed using Vitek 2 systems. The minimum inhibitory concentrations were confirmed using E-test. Primers were designed in order to verify mutations within AcrR and RamR genes. PCR analysis showed that the mutations found within these genes were transversions (94% for AcrR and 90% for RamR) and transitions (6% for AcrR and 10% for RamR). Nevertheless among the mutations, no distinction between tigecycline susceptible and resistant isolates was found. Some of the transversions caused change in the amino acid encoding 6 in AcrR and 15 in RamR. Presence of these types of mutations evaluation can be seen as the first bacterial resistance study step, as it may be caused by oxidative damage for bacterial DNA, frequently caused by antibiotic selective pressure. Tigecycline resistance found in this study`s clinical isolates may be associated to alterations in another genes that can trigger mechanisms associated to this antibiotic. / Klebsiella pneumoniae consiste em um dos principais agentes bacterianos causadores de infec????es relacionadas ?? assist??ncia ?? sa??de (IRAS). K. pneumoniae carrega frequentemente o gene de resist??ncia K. pneumoniae carbapenemase (KPC). Atualmente, a tigeciclina pode ser considerada uma das ??ltimas op????es terap??uticas para KPC, mas os relatos de isolados de KPC resistentes a tigecilina est??o em ascens??o, sendo a hiperexpress??o da bomba de efluxo AcrAB-TolC indicado como mecanismo mais comum. No entanto, os mecanismos moleculares de resist??ncia ?? tigeciclina associada ao AcrAB-TolC permanecem obscuros. Desta forma o objetivo deste trabalho foi verificar se a resist??ncia a tigeciclina pode estar relacionada ?? presen??a de muta????es nos genes ArcR e RamR, reguladores de AcrAB-TolC. Para tanto, 32 isolados de K. pneumoniae foram utilizados, sendo a identifica????o e o antibiograma feitos utilizando o sistema Vitek 2. A confirma????o das concentra????es inibit??rias m??nimas (CIMs) foram realizadas por E-test. Iniciadores foram desenhados para verifica????o de muta????es nos genes (AcrR e RamR). As an??lises por PCR mostraram que as muta????es encontradas nos genes AcrR e RamR foram substitui????es por transvers??o (94% e 90% para AcrR e RamR respectivamente) e transi????o (6% e 10% para AcrR e RamR respectivamente), por??m n??o foi identificada distin????o da presen??a de muta????es entre isolados sens??veis e resistentes a tigeciclina. Algumas tranvers??es ocasionaram mudan??a na codifica????o do amino??cido, sendo 6 em AcrR e 15 em RamR. A avalia????o da presen??a desses tipos de muta????es consiste em um primeiro passo para o estudo da resist??ncia bacteriana, j?? que pode ser causada por dano oxidativo ao DNA bacteriano, frequentemente ocasionado por press??o seletiva dos antibi??ticos. A resist??ncia a tigeciclina encontrada nos isolados cl??nicos do presente estudo, provavelmente pode estar associada a altera????es em outros genes desencadeadores de mecanismos de resist??ncia a tigeciclina.

The crucial role of antibiotics in modern medicine, in curing infections and enabling advanced medical procedures, is being threatened by the increasing frequency of resistant bacteria. Better understanding of the forces selecting resistance mutations could help develop strategies to optimize the use of antibiotics and slow the spread of resistance. Resistance to ciprofloxacin, a clinically important antibiotic, almost always involves target mutations in DNA gyrase and Topoisomerase IV. Because ciprofloxacin is a substrate of the AcrAB-TolC efflux pump, mutations causing pump up-regulation are also common. Studying the role of efflux pump-regulatory mutations in the development of ciprofloxacin resistance, we found a strong bias against gene-inactivating mutations in marR and acrR in clinical isolates. MIC and fitness measurements revealed that amino acid substitutions conferred smaller susceptibility reductions and smaller fitness costs than gene-inactivating mutations, suggesting that resistance mutations in clinical isolates are selected for high fitness rather than high resistance (Paper I and II). We asked whether the high fitness costs of marR-inactivating mutations could be ameliorated without affecting the resistance phenotype. Multiple independent lineages were experimentally evolved to select for improved growth fitness. Whole genome sequencing revealed mutations affecting marA, lon and arcA as potential compensatory pathways. For the marA and lon mutations the improved growth rate was associated with an increased susceptibility (arcA is being investigated). (Paper III). An evolution experiment selecting for ciprofloxacin resistance revealed upon whole genome sequencing the expected mutations in drug target and efflux-regulatory genes, but also in genes encoding aminoacyl-tRNA synthetases. We investigated two independently selected leuS mutations, and concluded that they contributed to ciprofloxacin resistance by activating the stringent response that in turn caused up-regulation of genes involved in efflux. However, these leuS mutations incur a high fitness cost (Paper IV). To summarize, the research findings in this thesis suggest that the potential ciprofloxacin resistome may include more genes than previously thought, but a strong selection for high fitness selectively purifies many resistance mutations from clinical isolates. In conclusion, selection for high relative fitness constrains the spectrum of mutations that survive and get fixed in clinical populations of bacteria.