Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第19092号 / エネ博第316号 / 新制||エネ||64 / 32043 / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 木下 正弘, 教授 森井 孝, 教授 片平 正人 / 学位規則第4条第1項該当

The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. Gram-negative bacteria such as Escherichia coli are intrinsically more resistant to antimicrobials. There are very few drugs either on the market or in the pharmaceutical pipeline targeting Gram-negative pathogens. Two mechanisms, the protection of the outer membrane and the active efflux by the multidrug transporters, play important roles in conferring multidrug resistance to Gram-negative bacteria. My work focuses on two main directions, each aligning with one of the known multidrug resistance mechanisms. The first direction of my research is in the area of the biogenesis of the bacterial outer membrane. The outer membrane serves as a permeability barrier in Gram-negative bacteria. Antibiotics cross the membrane barrier mainly via diffusion into the lipid bilayer or channels formed by outer membrane proteins. Therefore, bacterial drug resistance is closely correlated with the integrity of the outer membrane, which depends on the correct folding of the outer membrane proteins. The folding of the outer membrane proteins has been studied extensively in dilute buffer solution. However, the cell periplasm, where the folding actually occurs, is a crowded environment. In Chapter 2, effects of the macromolecular crowding on the folding mechanisms of two bacterial outer membrane proteins (OmpA and OmpT) were examined. Our results suggested that the periplasmic domain of OmpA improved the efficiency of the OmpA maturation under the crowding condition, while refolding of OmpT was barely affected by the crowding. The second direction of my research focuses on the major multidrug efflux transporter in Gram-negative bacteria, AcrB. AcrB is an obligate trimer, which exists and functions exclusively in a trimeric state. In Chapter 3, the unfolding of the AcrB trimer was investigated. Our results revealed that sodium dodecyl sulfate induced unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild type AcrB. In Chapter 4, the correlation between the AcrB trimer stability and the transporter activity was studied. A non-linear correlation was observed, in which the threshold trimer stability was required to maintain the efflux activity. Finally, in Chapter 5, the stability of another inner membrane protein, AqpZ, was studied. AqpZ was remarkably stable. Several molecular engineering approaches were tested to improve the thermal stability of the protein.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants. Due to their properties, PCBs accumulate in the food-chain and post a threat to the health of human beings and wildlife. Hydroxylated PCBs (OH-PCBs) are oxidative metabolites of PCBs and are more hydrophilic than their parent PCBs. One of the best approaches to break down these contaminants is through bioremediation, which is an environmental friendly process that uses microorganisms to restore natural environment. Towards this goal, we have investigated the toxicity and accumulation of PCBs and OH-PCBs in a Gram-negative bacterium, Escherichia coli. We have also determined the role played by a primary multidrug efflux transporter AcrB on the accumulation of PCBs and OH-PCBs in bacterial cell. We found that one of the PCBs tested was toxic to E. coli, while different OH-PCBs have different levels of toxicity; the acrB knockout strain accumulated significantly more PCBs and OH-PCBs than the wild-type strain, suggesting that these compounds are substrates of the efflux pump; higher cytoplasmic concentrations of OH-PCBs were also observed in the acrB knockout strain using the biosensors. Based on these observations, we conclude that both PCBs and OH-PCBs are substrates of protein AcrB. Therefore the efflux activities of multidrug resistant pumps in Gram-negative bacteria should be considered while designing bioremediation approaches.

Les gliomes malins constituent les tumeurs crbrales primaires les plus agressives et le glioblastome (GBM) est la plus frquente et agressive dentre elles. La survie mdiane associe nest que de 14,6 mois. D au caractre hautement invasif de ces tumeurs, la rsection maximale de la tumeur doit imprativement tre suivie de traitement de radio- et/ou chimiothrapie. Cependant, la prsence de la barrire hmatoencphalique et des mcanismes de chimiorsistance, tels que les pompes efflux, limitent lacheminement et lefficacit des composs aux cellules tumorales. Louverture osmotique de la barrire hmatoencphalique (OBHE) a t dveloppe afin damliorer lacheminement dagents anti-noplasiques au cerveau et la tumeur. Bien que plusieurs tudes aient t effectues afin de caractriser son processus, beaucoup dinformations restent dcouvrir afin dapprofondir nos connaissances sur lOBHE et amliorer son application en clinique. Avec lobjectif ultime de contourner ces deux obstacles, jai caractris le processus dynamique de lOBHE pour deux molcules de tailles diffrentes par imagerie par rsonance magntique dynamique, ainsi que pour une molcule tant un substrat des pompes efflux par tomographie dmission par positron dans le modle murin Fischer-F98. Jai galement tudi lexpression et la localisation de diffrentes pompes efflux par PCR quantitative et immunohistochimie dans des spcimens de gliomes malins. Les rsultats obtenus dmontrent que la barrire hmato-tumorale limite lacheminement la tumeur de composs de diffrent poids molculaire. Lacheminement au parenchyme crbral et la tumeur suite une procdure dOBHE est aussi dpendant du poids molculaire et de la taille de la molcule acheminer. LOBHE moins dtre de qualit excellente, ne semble pas suffisante pour acheminer au parenchyme crbral des substrats des pompes efflux. Les GBM expriment la MRP1, MRP3 et BCRP diffrents niveaux. La PGP, MRP1 et BCRP sont exprimes par les cellules endothliales des microvaisseaux crbraux. Lensemble de ces rsultats suggre que ladministration dagents thrapeutiques suite la procdure dOBHE doit tre optimise selon lagent administr et que linhibition de pompes efflux ou une autre stratgie rendant les agents de chimiothrapie invisibles aux pompes efflux sera bnfique pour amliorer leur acheminement au systme nerveux central et aux cellules tumorales.

We present the first characterization of K+ optimization of N uptake and metabolism in an NH4+-tolerant species, tropical lowland rice (cv. IR-72). 13N radiotracing showed that increased K+ supply reduces futile NH4+ cycling at the plasma membrane, diminishing the excessive rates of both unidirectional influx and efflux. Pharmacological testing showed that low-affinity NH4+ influx may be mediated by both K+ and non-selective cation channels. Suppression of NH4+ influx by K+ occurred within minutes of increasing K+ supply. Increased K+ reduced free [NH4+] in roots and shoots by 50-75%. Plant biomass was maximized on 10 mM NH4+ and 5 mM K+, with growth 160% higher than 10 mM NO3--grown plants, and 220% higher than plants grown at 10 mM NH4+ and 0.1 mM K+. Unlike in NH4+-sensitive barley, growth optimization was not attributed to a reduced energy cost of futile NH4+ cycling at the plasma membrane. Activities of the key enzymes glutamine synthetase (GS) and phosphoenolpyruvate carboxylase (PEPC) were strongly stimulated by elevated K+, mirroring plant growth and protein content. Improved plant performance through optimization of K+ and NH4+ is likely to be of substantial agronomic significance in the world’s foremost crop species.

We present the first characterization of K+ optimization of N uptake and metabolism in an NH4+-tolerant species, tropical lowland rice (cv. IR-72). 13N radiotracing showed that increased K+ supply reduces futile NH4+ cycling at the plasma membrane, diminishing the excessive rates of both unidirectional influx and efflux. Pharmacological testing showed that low-affinity NH4+ influx may be mediated by both K+ and non-selective cation channels. Suppression of NH4+ influx by K+ occurred within minutes of increasing K+ supply. Increased K+ reduced free [NH4+] in roots and shoots by 50-75%. Plant biomass was maximized on 10 mM NH4+ and 5 mM K+, with growth 160% higher than 10 mM NO3--grown plants, and 220% higher than plants grown at 10 mM NH4+ and 0.1 mM K+. Unlike in NH4+-sensitive barley, growth optimization was not attributed to a reduced energy cost of futile NH4+ cycling at the plasma membrane. Activities of the key enzymes glutamine synthetase (GS) and phosphoenolpyruvate carboxylase (PEPC) were strongly stimulated by elevated K+, mirroring plant growth and protein content. Improved plant performance through optimization of K+ and NH4+ is likely to be of substantial agronomic significance in the world’s foremost crop species.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The goal of this study was to determine the occurrence of two resistance mechanisms described in micro-organisms, but not described in R. equi isolates from horses and environment samples. The first mechanism studied it was the biofilm formation in R. equi isolates from clinical (n=41) and fecal (n=72) equine samples. In order to verify the biofilm formation it was employed the biofilm-culturing assay (with and without glucose supplementation) and the epifluorescence microscopy method. Biofilm-producers R. equi isolates (n=8) were selected and analyzed by two in vitro susceptibility tests with three antimicrobial agents belonging to macrolides group and commonly used in equine rhodococcosis treatment. The biofilm formation was observed in 80.5% of fecal and 63.4% of clinical isolates of R. equi. The methods used in this study were useful to verify the biofilm formation by R. equi isolates. However, the glucose supplementation was an important factor for biofilm formation in fecal samples. Antimicrobial resistance was demonstrated in biofilm antimicrobial susceptibility test. The second mechanism studied included the efflux system in R. equi isolates from clinical (n=30), fecal (n=30) and soil (n=30) sources. The presence of req_39680 gene, encoding a putative efflux system, was determined by PCR. Phenotypic expression of efflux systems was determined in agar containing ethidium bromide. The req_39680 gene was detected in 60% of the isolates and the phenotypic expression in 20%. In clinical isolates was observed high correlation between the presence of this gene and the expression of efflux systems. Both mechanisms studied were demonstrated in R. equi and may be contributing to increase the antimicrobial resistance, as well as to be associated to the survival of R. equi in the environment and host. / O objetivo do presente estudo foi determinar a ocorrência de dois mecanismos de resistência descritos em micro-organismos, porém, ainda não verificados em isolados de R. equi provenientes de amostras de equinos e de solo. O primeiro mecanismo estudado foi a formação de biofilmes em isolados de R. equi de amostras clínicas (n=41) e fecais (n=72) de equinos. Para verificação da formação de biofilmes empregaram-se as técnicas de formação de biofilme em cultura (com e sem a suplementação de glicose) e a microscopia de epifluorescência. Oito isolados formadores de biofilme foram selecionados e submetidos a testes de susceptibilidade frente a três antimicrobianos da classe dos macrolídeos, comumente utilizados no tratamento da rodococose equina. A formação de biofilmes foi observada em 80,5% dos isolados fecais e em 63,4% dos clínicos. Os métodos utilizados neste estudo foram adequados para verificar a formação de biofilmes nos isolados de R. equi. No entanto, foi constatado que a glicose é um substrato importante para formação de biofilme em isolados de origem fecal. Nos testes de susceptibilidade observou-se resistência aos antimicrobianos testados apenas quando os isolados de R. equi foram desafiados na forma de biofilme. O segundo mecanismo explorado envolveu a pesquisa de sistema de efluxo em isolados de R. equi de origem clínica (n=30) fecal (n=30) e de solo (n=30). Neste estudo buscou-se identificar a presença de um gene denominado req_39680, o qual foi descrito como codificador de um possível sistema de efluxo, Por fim, avaliou-se a expressão fenotípica de mecanismo de efluxo nos isolados analisados. A presença do gene req_39680, foi determinada por meio da técnica de reação em cadeia da polimerase (PCR) e a análise da expressão fenotípica de mecanismo de efluxo foi visualizada em ágar contendo brometo de etídeo. Foi evidenciada a presença do gene req_39680 em 60% dos isolados e a expressão fenotípica em 20%. Nos isolados de origem clínica, um alto índice de correlação entre a presença do gene estudado e a expressão de mecanismo de efluxo foi observada. Ambos os mecanismos estudados estão presentes em R. equi e podem estar contribuindo para a crescente resistência aos antimicrobianos, bem como, estar relacionados à sobrevivência de R. equi tanto no ambiente quanto no hospedeiro.

Forests of the northeastern U.S. help maintain water and air quality by reducing losses of nitrogen (N) into nearby waterways and removing carbon dioxide (CO2) from the atmosphere. However, carbon (C) and N retention in northeastern forests may decrease in response to projected changes in climate, including reductions in winter snowpack and increased soil freezing. Together, these climatic changes may damage tree roots and alter soil processes. Few studies have investigated the extent to which snowpack and soil frost drive C and N fluxes during spring snowmelt, a biogeochemically important period. Similarly, little is known about how changes in winter climate affect above- and belowground CO2 fluxes to the atmosphere. My dissertation combines laboratory and field experiments to quantify the effects of reduced snowpack and increased soil freezing on C and N cycling in northeastern forests. I conducted a laboratory experiment to study the effects of soil freezing on C and N losses during snowmelt. Organic horizon soils collected from mixed Acer saccharum-Fagus grandifolia and Picea rubens-Abies balsamea forests were incubated in severe, mild, and no soil frost conditions prior to snowmelt. Results show that losses of N in leachate, as well as total C and N fluxes (gases + leachate), were reduced following severe soil frost, indicating the response to winter climate depends on both the presence and severity of soil frost. I also implemented a snow removal experiment in a mixed Quercus rubra-Acer rubrum forest at Harvard Forest, MA to quantify the effects of depth and duration of snowpack and soil frost on CO2 losses from tree stems and soils. This study provides evidence that reduced snowpack and increased soil freezing may increase annual soil CO2 efflux, but have no significant effect on tree stem CO2 efflux. Taken together, results from my dissertation highlight the importance of winter climate as a driver of C and N fluxes in northeastern forests and suggest that while soil frost reduces C and N losses during snowmelt, annual losses of CO2 may increase Future studies investigating controls on C and N cycling in northeastern forests should account for changes in winter climate.