Semisynthetic aminoglycoside antibiotics : toward biomimetic synthesis, evasion of bacterial resistance and reduced toxicity

Les antibiotiques aminoglycosidiques sont des agents bactéricides de grande valeur et d’efficacité à large spectre contre les pathogènes Gram-positifs et Gram-négatifs, dont plusieurs membres naturels et semisynthétiques sont importants dans l’histoire clinique depuis 1950. Des travaux crystallographiques sur le ribosome, récompensés par le prix Nobel, ont démontré comment leurs diverses structures polyaminées sont adaptées pour cibler une hélice d’ARN dans le centre de codage de la sous-unité 30S du ribosome bactérien. Leur interférence avec l’affinité et la cinétique des étapes de sélection et vérification des tARN induit la synthèse de protéines à basse fidélité, et l’inhibition de la translocation, établissant un cercle vicieux d’accumulation d’antibiotique et de stress sur la membrane. En réponse à ces pressions, les pathogènes bactériens ont évolué et disséminé une panoplie de mécanismes de résistance enzymatiques et d’expulsion : tels que les N acétyltransférases, les O phosphotransférases et les O nucleotidyltransférases qui ciblent les groupements hydroxyle et amino sur le coeur des aminoglycosides; des méthyl-transférases, qui ciblent le site de liaison ribosomale; et des pompes d’expulsion actives pour l’élimination sélective des aminoglycosides, qui sont utilisés par les souches Gram-négatives. Les pathogènes les plus problématiques, qui présentent aujourd’hui une forte résilience envers la majorité des classes d’antibiotiques sur le bord de la pan-résistance ont été nommés des bactéries ESKAPE, une mnémonique pour Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa et Enterobacteriaceae. La distribution globale des souches avec des mécanismes de résistance envers les standards cliniques aminoglycosides, tels que la tobramycine, l’amikacine et la gentamicine, est comprise entre 20 et 60% des isolées cliniques. Ainsi, les aminoglycosides du type 4,6-disubstitués-2-deoxystreptamine sont inadéquats comme thérapies anti-infectieuses à large spectre. Cependant, la famille des aminoglycosides 4,5-disubstitués, incluant la butirosine, la neomycine et la paromomycine, dont la structure plus complexe, pourrait constituter une alternative. Des collègues dans le groupe Hanessian et collaborateurs d’Achaogen Inc. ont démontré que certains analogues de la paraomomycine et neomycine, modifiés par désoxygénation sur les positions 3’ et 4’, et par substitution avec la chaîne N1-α-hydroxy-γ-aminobutyramide (HABA) provenant de la butirosine, pourrait produire des antibiotiques très prometteurs. Le Chapitre 4 de cette dissertation présente la conception et le développement d’une stratégie semi-synthétique pour produire des nouveaux aminoglycosides améliorés du type 4,5 disubstitués, inspiré par des modifications biosynthétiques de la sisomicine, qui frustrent les mécanismes de résistance bactérienne distribuées globalement. Cette voie de synthèse dépend d’une réaction d’hydrogénolyse de type Tsuji catalysée par palladium, d’abord développée sur des modèles monosaccharides puis subséquemment appliquée pour générer un ensemble d’aminoglycosides hybrides entre la neomycine et la sisomicine. Les études structure-activité des divers analogues de cette nouvelle classe ont été évaluées sur une gamme de 26 souches bactériennes exprimant des mécanismes de résistance enzymatique et d’expulsion qui englobe l’ensemble des pathogènes ESKAPE. Deux des antibiotiques hybrides ont une couverture antibacterienne excellente, et cette étude a mis en évidence des candidats prometteurs pour le développement préclinique. La thérapie avec les antibiotiques aminoglycosidiques est toujours associée à une probabilité de complications néphrotoxiques. Le potentiel de toxicité de chaque aminoglycoside peut être largement corrélé avec le nombre de groupements amino et de désoxygénations. Une hypothèse de longue date dans le domaine indique que les interactions principales sont effectuées par des sels des groupements ammonium, donc l’ajustement des paramètres de pKa pourrait provoquer une dissociation plus rapide avec leurs cibles, une clairance plus efficace et globalement des analogues moins néphrotoxiques. Le Chapitre 5 de cette dissertation présente la conception et la synthèse asymétrique de chaînes N1 HABA β substitutées par mono- et bis-fluoration. Des chaînes qui possèdent des γ-N pKa dans l’intervalle entre 10 et 7.5 ont été appliquées sur une neomycine tétra-désoxygénée pour produire des antibiotiques avancés. Malgré la réduction considérable du γ N pKa, le large spectre bactéricide n’a pas été significativement affecté pour les analogues fluorés isosteriques. De plus, des études structure-toxicité évaluées avec une analyse d’apoptose propriétaire d’Achaogen ont démontré que la nouvelle chaîne β,β difluoro-N1-HABA est moins nocive sur un modèle de cellules de rein humain HK2 et elle est prometteuse pour le développement d’antibiotiques du type neomycine avec des propriétés thérapeutiques améliorées. Le chapitre final de cette dissertation présente la proposition et validation d’une synthèse biomimétique par assemblage spontané du aminoglycoside 66-40C, un dimère C2 symétrique bis-imine macrocyclique à 16 membres. La structure proposée du macrocycle a été affinée par spectroscopie nucléaire à un système trans,trans-bis-azadiène anti-parallèle. Des calculs indiquent que l’effet anomérique de la liaison α glycosidique entre les anneaux A et B fournit la pré-organisation pour le monomère 6’ aldéhydo sisomicine et favorise le produit macrocyclique observé. L’assemblage spontané dans l’eau a été étudié par la dimérisation de trois divers analogues et par des expériences d’entre croisement qui ont démontré la généralité et la stabilité du motif macrocyclique de l'aminoglycoside 66-40C.

Unpacking Japan's 21st century "National Conversation" : images of the future beyond the iron cage of the "Catch Up" model

How does the image of the future operate upon history, and upon national and individual identities? To what extent are possible futures colonized by the image? What are the un-said futurecratic discourses that underlie the image of the future? Such questions inspired the examination of Japan’s futures images in this thesis. The theoretical point of departure for this examination is Polak’s (1973) seminal research into the theory of the ‘image of the future’ and seven contemporary Japanese texts which offer various alternative images for Japan’s futures, selected as representative of a ‘national conversation’ about the futures of that nation. These seven images of the future are: 1. Report of the Prime Minister’s Commission on Japan’s Goals in the 21st Century—The Frontier Within: Individual Empowerment and Better Governance in the New Millennium, compiled by a committee headed by Japan’s preeminent Jungian psychologist Kawai Hayao (1928-2007); 2. Slow Is Beautiful—a publication by Tsuji Shinichi, in which he re-images Japan as a culture represented by the metaphor of the sloth, concerned with slow and quality-oriented livingry as a preferred image of the future to Japan’s current post-bubble cult of speed and economic efficiency; 3. MuRatopia is an image of the future in the form of a microcosmic prototype community and on-going project based on the historically significant island of Awaji, and established by Japanese economist and futures thinker Yamaguchi Kaoru; 4. F.U.C.K, I Love Japan, by author Tanja Yujiro provides this seven text image of the future line-up with a youth oriented sub-culture perspective on that nation’s futures; 5. IMAGINATION / CREATION—a compilation of round table discussions about Japan’s futures seen from the point of view of Japan’s creative vanguard; 6. Visionary People in a Visionless Country: 21 Earth Connecting Human Stories is a collection of twenty one essays compiled by Denmark born Tokyo resident Peter David Pedersen; and, 7. EXODUS to the Land of Hope, authored by Murakami Ryu, one of Japan’s most prolific and influential writers, this novel suggests a future scenario portraying a massive exodus of Japan’s youth, who, literate with state-of-the-art information and communication technologies (ICTs) move en masse to Japan’s northern island of Hokkaido to launch a cyber-revolution from the peripheries. The thesis employs a Futures Triangle Analysis (FTA) as the macro organizing framework and as such examines both pushes of the present and weights from the past before moving to focus on the pulls to the future represented by the seven texts mentioned above. Inayatullah’s (1999) Causal Layered Analysis (CLA) is the analytical framework used in examining the texts. Poststructuralist concepts derived primarily from the work of Michel Foucault are a particular (but not exclusive) reference point for the analytical approach it encompasses. The research questions which reflect the triangulated analytic matrix are: 1. What are the pushes—in terms of current trends—that are affecting Japan’s futures? 2. What are the historical and cultural weights that influence Japan’s futures? 3. What are the emerging transformative Japanese images of the future discourses, as embodied in actual texts, and what potential do they offer for transformative change in Japan? Research questions one and two are discussed in Chapter five and research question three is discussed in Chapter six. The first two research questions should be considered preliminary. The weights outlined in Chapter five indicate that the forces working against change in Japan are formidable, structurally deep-rooted, wide-spread, and under-recognized as change-adverse. Findings and analyses of the push dimension reveal strong forces towards a potentially very different type of Japan. However it is the seven contemporary Japanese images of the future, from which there is hope for transformative potential, which form the analytical heart of the thesis. In analyzing these texts the thesis establishes the richness of Japan’s images of the future and, as such, demonstrates the robustness of Japan’s stance vis-à-vis the problem of a perceived map-less and model-less future for Japan. Frontier is a useful image of the future, whose hybrid textuality, consisting of government, business, academia, and creative minority perspectives, demonstrates the earnestness of Japan’s leaders in favour of the creation of innovative futures for that nation. Slow is powerful in its aim to reconceptualize Japan’s philosophies of temporality, and build a new kind of nation founded on the principles of a human-oriented and expanded vision of economy based around the core metaphor of slowness culture. However its viability in Japan, with its post-Meiji historical pushes to an increasingly speed-obsessed social construction of reality, could render it impotent. MuRatopia is compelling in its creative hybridity indicative of an advanced IT society, set in a modern day utopian space based upon principles of a high communicative social paradigm, and sustainability. IMAGINATION / CREATION is less the plan than the platform for a new discussion on Japan’s transformation from an econo-centric social framework to a new Creative Age. It accords with emerging discourses from the Creative Industries, which would re-conceive of Japan as a leading maker of meaning, rather than as the so-called guzu, a term referred to in the book meaning ‘laggard’. In total, Love Japan is still the most idiosyncratic of all the images of the future discussed. Its communication style, which appeals to Japan’s youth cohort, establishes it as a potentially formidable change agent in a competitive market of futures images. Visionary People is a compelling image for its revolutionary and subversive stance against Japan’s vision-less political leadership, showing that it is the people, not the futures-making elite or aristocracy who must take the lead and create a new vanguard for the nation. Finally, Murakami’s Exodus cannot be ruled out as a compelling image of the future. Sharing the appeal of Tanja’s Love Japan to an increasingly disenfranchised youth, Exodus portrays a near-term future that is achievable in the here and now, by Japan’s teenagers, using information and communications technologies (ICTs) to subvert leadership, and create utopianist communities based on alternative social principles. The principal contribution from this investigation in terms of theory belongs to that of developing the Japanese image of the future. In this respect, the literature reviews represent a significant compilation, specifically about Japanese futures thinking, the Japanese image of the future, and the Japanese utopia. Though not exhaustive, this compilation will hopefully serve as a useful starting point for future research, not only for the Japanese image of the future, but also for all image of the future research. Many of the sources are in Japanese and their English summations are an added reason to respect this achievement. Secondly, the seven images of the future analysed in Chapter six represent the first time that Japanese image of the future texts have been systematically organized and analysed. Their translation from Japanese to English can be claimed as a significant secondary contribution. What is more, they have been analysed according to current futures methodologies that reveal a layeredness, depth, and overall richness existing in Japanese futures images. Revealing this image-richness has been one of the most significant findings of this investigation, suggesting that there is fertile research to be found from this still under-explored field, whose implications go beyond domestic Japanese concerns, and may offer fertile material for futures thinkers and researchers, Japanologists, social planners, and policy makers.

Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin CDNA

A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)(i)], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)(i)] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells al all stages of development exhibit a sharp transient increase in [(Ca2+)(i)] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)(i)] and show three times as large a response to cAMP stimulation as prespore cells.

Length-frequency estimation for yellowfin tuna (Thunnus albacares) caught by commercial fishing gear in the Eastern Pacific Ocean

ENGLISH: Methods of collecting samples for the purpose of estimating the numbers and weights of fish caught, by length interval, are described. Several models for two-stage sampling are described, and the equations for the estimators and their variances are given. The results from a brief simulation study are used to show the differences between estimates made with the different models. Estimators for the average weights of fish in the catch and their variances are also described. These average weights are used to provide improved estimates of the total annual catches of yellowfin taken from the eastern Pacific Ocean, east of 150°W, between 1955 and 1990. SPANISH: Se describen los métodos de recoger de muestreo para estimar el número o peso de peces capturados, por intervalo de talla. Se describen varios modelos para el muestreo de dos etapas, y se presentan las ecuaciones para los estimadores y sus varianzas. Se usan los resultados de un breve estudio de simulación para indicar las diferencias entre estimaciones realizadas con los distintosmodelos. También se describe un estimador para el peso promedio de peces en la captura y su varianza. Se usan estos estimadores para calcular estimaciones mejoradas de las capturas anuales totales de aleta amarilla tomadas del Océano Pacífico oriental, al este de 150°W, entre 1955 y 1990. (PDF contains 41 pages.)

Migration patterns of young Pacific bluefin tuna (Thunnus orientalis) determined with archival tags

We investigated the migration and behavior of young Pacific bluefin tuna (Thunnus orientalis) using archival tags that measure environmental variables, record them in memory, and estimate daily geographical locations using measured light levels. Swimming depth, ambient water temperature, and feeding are described in a companion paper. Errors of the tag location estimates that could be checked were –0.54° ±0.75° (mean ±SD) in longitude and –0.12° ±3.06° in latitude. Latitude, estimated automatically by the tag, was problematic, but latitude, estimated by comparing recorded sea-surface temperatures with a map of sea-surface temperature, was satisfactory. We concluded that the archival tag is a reliable tool for estimating location on a scale of about one degree, which is sufficient for a bluefin tuna migration study. After release, tagged fish showed a normal swimming behavioral pattern within one day and normal feeding frequency within one month. In addition, fish with an archival tag maintained weight-at-length similar to that of wild fish; however, their growth rate was less than that of wild fish. Of 166 fish released in the East China Sea with implanted archival tags, 30 were recovered, including one that migrated across the Pacific Ocean. Migration of young Pacific bluefin tuna appears to consist of two phases: a residency phase comprising more than 80% of all days, and a traveling phase. An individual young Pacific bluefin tuna was observed to cover 7600 km in one traveling phase that lasted more than two months (part of this phase was a trans-Pacific migration completed within two months). Many features of behavior in the traveling phase were similar to those in the residency phase; however the temperature difference between viscera and ambient temperature was larger, feeding was slightly more frequent, and dives to deeper water were more frequent.

Swimming depth, ambient water temperature preference, and feeding frequency of young Pacific bluefin tuna (Thunnus orientalis) determined with archival tags

We investigated the migration and behavior of young Pacific Bluefin tuna (Thunnus orientalis) using archival tags. The archival tag measures environmental variables, records them in its memory, and estimates daily geographical locations based on measured light levels. Of 166 archival tags implanted in Pacific bluefin tuna that were released at the northeastern end of the East China Sea from 1995 to 1997, 30 tags were recovered, including one from a fish that migrated across the Pacific. This article describes swimming depth, ambient water temperature, and feeding frequency of young Pacific bluefin tuna based on retrieved data. Tag performance, effect of the tag on the fish, and horizontal movements of the species are described in another paper. Young Pacific bluefin tuna swim mainly in the mixed layer, usually near the sea surface, and swim in deeper water in daytime than at nighttime. They also exhibit a pattern of depth changes, corresponding to sunrise and sunset, apparently to avoid a specific low light level. The archival tags recorded temperature changes in viscera that appear to be caused by feeding, and those changes indicate that young Pacific bluefin tuna commonly feed at dawn and in the daytime, but rarely at dusk or at night. Water temperature restricts their distribution, as indicated by changes in their vertical distribution with the seasonal change in depth of the thermocline and by the fact that their horizontal distribution is in most cases confined to water in the temperature range of 14−20°C.

Efficient field emission from triode-type 1D arrays of carbon nanotubes.

Carbon nanotube (CNT) emitters were formed on line-patterned cathodes in microtrenches through a thermal CVD process. Single-walled carbon nanotubes (SWCNTs) self-organized along the trench lines with a submicron inter-CNT spacing. Excellent field emission (FE) properties were obtained: current densities at the anode (J(a)) of 1 microA cm(-2), 10 mA cm(-2) and 100 mA cm(-2) were recorded at gate voltages (V(g)) of 16, 25 and 36 V, respectively. The required voltage difference to gain a 1:10 000 contrast of the anode current was as low as 9 V, indicating that a very low operating voltage is possible for these devices. Not only a large number of emission sites but also the optimal combination of trench structure and emitter morphology are crucial to achieve the full FE potential of thin CNTs with a practical lifetime. The FE properties of 1D arrays of CNT emitters and their optimal design are discussed. Self-organization of thin CNTs is an attractive prospect to tailor preferable emitter designs in FE devices.

Field emission properties of single-walled carbon nanotubes with a variety of emitter morphologies

Field emission properties of single-walled carbon nanotubes (SWCNTs), which were prepared through alcohol catalytic chemical vapor deposition for 10-60s, were characterized in a diode configuration. Protrusive bundles at the top surface of samples act selectively as emission sites. The number of emission sites was controlled by emitter morphologies combined with texturing of Si substrates. SWCNTs grown on a textured Si substrate exhibited a turn-on field as low as 2.4 V/μm at a field emission current density of 1 μA/cm 2. Uniform spatial luminescence (0.5 cm2) from the rear surface of the anode was revealed for SWCNTs prepared on the textured Si substrate. Deterioration of field emission properties through repetitive measurements was reduced for the textured samples in comparison with vertically aligned SWCNTs and a random network of SWCNTs prepared on flat Si substrates. Emitter morphology resulting in improved field emission properties is a crucial factor for the fabrication of SWCNT-electron sources. Morphologically controlled SWCNTs with promising emitter performance are expected to be practical electron sources. © 2008 The Japan Society of Applied Physics.

Tailoring the morphology of carbon nanotube assemblies using microgradients in the catalyst thickness

In addition to the structural control of individual carbon nanotubes (CNTs), the morphological control of their assemblies is crucial to realize miniaturized CNT devices. Microgradients in the thickness of catalyst are used to enrich the variety of available self-organized morphologies of CNTs. Microtrenches were fabricated in gate/spacer/cathode trilayers using a conventional self-aligned top-down process and catalyst exhibiting a microgradient in its thickness was formed on the cathode by sputter deposition through gate slits. CNTs, including single-walled CNTs, of up to 1μm in length were grown within 5-15 s by chemical vapor deposition. The tendency of thin CNTs to aggregate caused interactions between CNTs with different growth rates, yielding various morphologies dependent on the thickness of the catalyst. The field emission properties of several types of CNT assemblies were evaluated. The ability to produce CNTs with tailored morphologies by engineering the spatial distribution of catalysts will enhance their performance in devices. © 2011 The Japan Society of Applied Physics.

Conformal oxide coating of carbon nanotubes

The International Roadmap for Ferroelectric Memories requires three-dimensional integration of high-dielectric materials onto metal interconnects or bottom electrodes by 2010. Here, we demonstrate the possibility of conformally coating carbon nanotubes with high-dielectric oxide as a first step toward ultrahigh integration density of three-dimensional ferroelectric random access memories.

Liquid source misted chemical deposition process of three-dimensional nano-ferroelectrics with substrate heating

A modification of liquid source misted chemical deposition process (LSMCD) with heating mist and substrate has developed, and this enabled to control mist penetrability and fluidity on sidewalls of three-dimensional structures and ensure step coverage. A modified LSMCD process allowed a combinatorial approach of Pb(Zr,Ti)O-3 (PZT) thin films and carbon nanotubes (CNTs) toward ultrahigh integration density of ferroelectric random access memories (FeRAMs). The CNTs templates were survived during the crystallization process of deposited PZT film onto CNTs annealed at 650 degrees C in oxygen ambient due to a matter of minute process, so that the thermal budget is quite small. The modified LSMCD process opens up the possibility to realize the nanoscale capacitor structure of ferroelectric PZT film with CNTs electrodes toward ultrahigh integration density FeRAMs.

Direct functionalization of heterocyclic and non-heterocyclic arenes

L’application des métaux de transition à la fonctionnalisation directe a ouvert la voie à une nouvelle classe de réactions pour la formation de liens carbone-carbone. De par l'omniprésence des liaisons C–H, l’introduction de nouvelles fonctionnalités chimiques par voie directe et pré-activation minimale s’impose comme une stratégie de synthèse très attrayante. Ainsi, il est envisageable de préparer de manière rapide et efficace des supports complexes menant à des molécules complexes, qui pourraient être utiles dans divers domaines de la chimie. L'objectif principal de la présente thèse vise la fonctionnalisation directe des arènes hétérocycliques et non hétérocycliques et, plus précisément, les techniques d’arylation. Dans un premier temps, nous allons aborder le thème de l’arylation directe tout en mettant l’accent sur les pyridines (Chapitre 1). Ces molécules sont à la base d'une multitude de composés biologiquement actifs et jouent un rôle important dans le domaine des sciences des matériaux, de l’agrochimie et de la synthèse des produits naturels. Dans un deuxième temps, nous discuterons de nos travaux sur l’arylation directe catalysé par un complex de palladium sur des ylures de N-iminopyridinium en soulignant la dérivatisation du sel de pyridinium après une phénylation sp2 (Chapitre 2). L’étude de ce procédé nous a permis de mettre en lumière plusieurs découvertes importantes, que nous expliquerons en détails une à une : l’arylation benzylique directe lorsque des ylures N-iminopyridinium substituées avec un groupement alkyl à la position 2 sont utilisés comme partenaires dans la réaction; les allylations Tsuji-Trost catalysée par un complex de palladium; et l’alkylation directe et sans métal via une catalyse par transfert de phase. Plusieurs défis restent à relever pour le développement de procédés directs utilisant des métaux de transition peu coûteux, d’autant plus que la synthèse par transformation directe des pyridines 2-alcényles, lesquelles sont pertinentes sur le plan pharmacologique, n’a pas encore été rapportée à ce jour. Avec cette problématique en tête, nous avons réussi à mettre au point une alcénylation directe catalysé par un complex de cuivre sur des ylures de N-iminopyridinium. Nous discuterons également d’une nouvelle méthode pour la préparation des iodures de vinyle utilisés dans les couplages. Ces réactions sont non seulement remarquablement chimiosélectives, mais sont aussi applicables à plusieurs substrats (Chapitre 3). En optimisant ce procédé direct, nous avons découvert une façon unique de synthétiser les pyrazolo[1,5-a]pyridines 2-substituées (Chapitre 4). Le mécanisme global met en jeu une séquence tandem de fonctionnalisation-cyclisation directe et un procédé direct en cascade, qui n’avais jamais été rapporté. Cela simplifie ansi la synthèse autrement compliquée de ces substrats en y apportant une solution à un problème de longue date. Dans les deux derniers chapitres, nous examinerons en détail les techniques d’arylation directe qui n'impliquent pas les partenaires de couplage hétérocycliques. Entre autres, au Chapitre 5, nous soulignerons notre découverte d’un umpolung dirigé et catalysé par un complexe de palladium du benzène et de quelques autres dérivés arènes. Il s’agit là du premier cas de fonctionnalisation directe dans laquelle le groupe directeur se trouve sur le partenaire halogène et il s’ajoute à la courte liste d’exemples connus dans la littérature rapportant une arylation directe du benzène. Finalement, au Chapitre 6, nous passerons en revue une nouvelle arylation directe catalysée au fer, qui se veut un procédé peu coûteux, durable et présentant une économie d’atomes. Nous discutons des substrats possibles ainsi des études mécanistiques réalisés.