Les anhydrases carboniques (CAs, EC4.2.1.1) sont une famille ubiquitaire de métalloenzymes à zinc. Ces enzymes catalysent la réaction réversible d'hydratation du dioxyde de carbone en bicarbonate avec la formation d'un proton. Elles jouent ainsi un important rôle dans de nombreux processus physiologiques tels que la respiration, le transport des ions entre les tissus, l´homéostasie et la régulation du pH. Chez l'homme, seize isoformes différents ont été décrits et certains d'entre eux sont impliqués dans divers troubles pathologiques comme le cancer avec les isoformes CA IX et CA XII. Par ailleurs, des deux enzymes, la CA IX est non seulement l'isoforme le plus actif pour la réaction précédemment décrite mais, elle est aussi la plus largement exprimée dans les tumeurs sous hypoxie (carcinome du sein, du colon…). Grâce à son rôle dans l'acidification du microenvironnement tumoral, la CA IX est associée au phénomène de métastases. Il a été démontré que l'inhibition de son activité catalytique permet de réduire non seulement la croissance et la prolifération tumorale mais aussi la résistance de ces tumeurs aux traitements anticancéreux conventionnels. Dans le cadre d'une approche pharmacologique, cette inhibition se fait via des petites molécules possédant en leur sein une fonction liant l'atome de zinc du site actif de l'enzyme. Dans ce manuscrit, nous avons décrit la synthèse de glycoinhibiteurs insaturés inédits à travers le développement d'une nouvelle méthodologie de synthèse, la sulfamidoglycosylation, basée sur le réarrangement de Ferrier puis le développement d'une nouvelle fonction liant l'atome de zinc, l'hydroxylamine-O-sulfonamide, qui a ensuite servi pour la synthèse d'autres glycoinhibiteurs insaturés par sulfonamidoglycosylation. Ces composés ont montré des activités inhibitrices de l'ordre du nanomolaire sur la CA IX et la CA XII. / The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc enzymes. These enzymes catalyse the reversible hydration reaction of carbon dioxide to bicarbonate, releasing a proton in the process. The enzymes are thus key players in numerous physiological processes such as respiration, ion transport between tissues, homeostasis and pH regulation. In humans, sixteen different isoforms have been described and some of them are involved in diverses pathological conditions such as the CA IX and CA XII isoforms in cancer. Furthermore, from the two enzymes, CA IX is not only the most active isoform for the previously described reaction, but also the most widely expressed under hypoxia in hypoxic tumors (breast carcinoma, colon ...). Through its role in the acidification of the tumoral microenvironment, the CA IX is associated with metastases. It has been demonstrated that inhibition of its catalytic activity reduces not only the tumoral growth and proliferation, but also the resistance of these tumors to conventional cancer treatments. As part of a pharmacological approach, the known inhibitors are small molecules bearing a zinc binding function. In this manuscript, we described the synthesis of novel unsaturated glycoinhibitors through the development of a new synthetic methodology, the sulfamidoglycosylation based on the Ferrier rearrangement, and the development of a new zinc binding function, the hydroxylamine-O-sulfonamide which was then used for the synthesis of other unsaturated glycoinhibitors by sulfonamidoglycosylation. These compounds showed nanomolar inhibitory activities against the CA IX and CA XII.

Les anhydrases carboniques (AC) sont des enzymes qui catalysent les réactions d'hydratation du CO2 et d'hydrolyse de l’OCS. L’AC présente dans les plantes et les microorganismes du sol influence le bilan atmosphérique d'OCS ainsi que celui du CO18O car les isotopes de l’oxygène sont échangés avec ceux des pools d'eau pendant l'hydratation duCO2. L’utilisation de l’OCS et du CO18O comme traceurs du cycle du C global ouvre une nouvelle voie pour estimer les contributions de la photosynthèse et de la respiration à grande échelle. Ceci requiert néanmoins une meilleure compréhension des facteurs contrôlant l'activité de l’AC des sols. Nous avons étudié le rôle du pH du sol et des communautés microbiennes sur l'activité de l’AC. Nous avons testé l’hypothèse que l'activité de l’AC serait (H1) inhibée dans les sols acides, et que (H2) les échanges isotopiques CO2-H2O seraient réduits dans les sols alcalins. Nous avons également présumé que l'activité de l’AC serait (H3) positivement corrélée à l'abondance des microorganismes phototrophes, et que (H4) la structure des communautés affecterait différemment les flux de CO18O et d’OCS. Nos résultats valident H1 et H2. Ils montrent aussi que les flux de CO2 dans le sol et l'activité d’AC associée sont positivement corrélés à l'abondance des microorganismes phototrophes (H3), tandis que le dépôt d'OCS dans les sols dépend de l'abondance des champignons (H4). Ces résultats sont en cours d’intégration dans un modèle de l'activité de l’AC des sols mondiaux, ce qui permettra une estimation robuste des flux globaux de photosynthèse et de respiration à partir de bilans atmosphériques de COS et CO18O. / Carbonic anhydrases (CA) are a group of enzymes that catalyse CO2 hydration and OCS hydrolysis. The presence of CA in plants and soil microorganisms is responsible for the largest atmosphere-biosphere exchange of OCS but also CO18O, because oxygen isotopes are exchanged with soil and plant water pools during CO2 hydration. Consequently, CO18O and OCS atmospheric mixing ratios have been proposed as complementary tracers of the global C cycle that could open avenues to estimate the contribution of photosynthesis and respiration at global scales. However, a mechanistic understanding of the drivers of CA activity is required. We investigated the role of soil pH and microbial community on soil CA activity. We hypothesised that CA activity should be(H1) inhibited in acidic soils but that (H2) the associated CO2-H2O exchange would also be reduced in alkaline soils. We further assumed that (H3) soil CA activity would be enhanced by an increase in soil phototrophs abundance, but that (H4) soil community structure would affect differently CO18O and OCS fluxes. Our results confirmed H1 and H2. We also confirmed that soil CO2 fluxes and the associated CA activity were positively correlated with phototrophic communities abundance (H3), while soil OCS uptake and the associated CA activity seemed driven by fungal abundance (H4). These findings are now being incorporated into a model of soil CA activity worldwide that will allow robust estimates of photosynthesis and respiration at large scales from the atmospheric budgets of OCS and CO18O.

Small cell lung cancer (SCLC) is an extremely aggressive disease characterized by early metastasis and acquired resistance to therapy. SCLC is distinguished by its neuroendocrine (NE) component; the role of which is not fully understood in metastasis and response to therapy. Patients respond exceptionally well to first round chemotherapy; however, relapse with therapy-resistant tumours is virtually inevitable. Hypoxic regions within tumours can contribute towards metastasis and therapy resistance, highlighting hypoxia-targeted therapy as a novel approach for improving treatment for SCLC patients. Tumours are highly phenotypically heterogeneous, raising debate over the roles played by each cell type. Analysis of NE and epithelial markers in SCLC cell lines highlighted this inter-tumour heterogeneity. Further heterogeneity is displayed in SCLC xenograft tumours that show areas of dual epithelial and NE marker expression as well as regions negative for both markers. Irradiating xenograft tumours enhanced heterogeneity of the NE marker, pro-opiomelanocortin (POMC), which is ectopically secreted by a subset of SCLC tumours. Examining changes in marker expression post-therapy could provide vital information regarding transitions that can serve to guide therapy. SCLC is a highly metastatic disease. The role of the NE phenotype in human SCLC is not fully understood, but is considered essential for metastasis in murine models. Sub-cutaneous, intravenous and intra-splenic injection were carried out and resulted in no metastasis, spontaneous tumour generation and peripheral liver tumour growth, respectively. POMC expression was present and extremely heterogeneous within the liver, suggesting that NE properties are maintained in metastases; however, further work is necessary to develop a more consistent metastatic model that can be used to assess responses to therapy in a more clinically relevant setting. SCLC tumours proliferate rapidly and outgrow their nutrient and oxygen supplies, resulting in hypoxic conditions. Here, carbonic anhydrase IX (CA IX) becomes up-regulated in order to maintain pH levels suitable for survival. The specific CA IX inhibitor, S4, induces hypoxia-specific cell death in vitro and impairs tumour growth in vivo. This response is further accentuated by combining S4 with single or repeated cisplatin doses. Combination treatment reduced gene expression of S-phase kinase-associated protein (Skp2), associated with cisplatin resistance. CA IX inhibition combined with cisplatin chemotherapy therefore presents a novel treatment for SCLC tumours that could reduce therapy resistance. In summary, heterogeneity is extremely important when choosing treatment options for SCLC and must be considered when basing treatment on single biopsies. NE and epithelial markers are present within sub-cutaneous and liver tumours; however, a reliable multi-organ metastatic model is necessary to fully appreciate the role of these markers in the spread of SCLC. Hypoxic regions within sub-cutaneous xenograft tumours upregulate CA IX. Inhibition of this enzyme resulted in impaired tumour growth, particularly when used together with cisplatin. Combining CA IX inhibition with cisplatin presents a much-needed novel therapy for SCLC.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第22087号 / エネ博第395号 / 新制||エネ||76(附属図書館) / 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 森井 孝, 教授 木下 正弘, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

This work deals with carborane and metallacarborane clusters, in terms of their fundamental chemistry and complexation with cyclodextrins, and in the context of emerging pharmacophores applicable in medicinal chemistry. Arguably, the most important part of this work is the preparation of cobalt bis(dicarbollide) sulfamide derivatives. The sulfamido group is attached to the metallacarborane carbon vertex by an alkyl chain that may be modified in its length. This was accomplished by, firstly, the abstraction of the acidic hydrogen, located on the {CH}-vertex from the metallacarborane, by reaction with lithium base, followed by, secondly, reaction with electrophilic agents (PFA, oxirane and oxetane), which leads to a cascade of reactions to give the desired sulfamide derivatives. These compounds were then tested by collaborators in other institutes for in vitro and in vivo activity towards Carbonic Anhydrase IX (CA IX), which is an enzyme associated with tumour growth. In vivo tests on mice have shown that these types of substances are able to effectively reduce tumour size by 30%. The synthetic research continued with the preparation of sulfonamide compounds of the isomers of the carborane series. The reactions began exclusively with propylhydroxy carborane starting materials, which provide optimum...

This work deals with carborane and metallacarborane clusters, in terms of their fundamental chemistry and complexation with cyclodextrins, and in the context of emerging pharmacophores applicable in medicinal chemistry. Arguably, the most important part of this work is the preparation of cobalt bis(dicarbollide) sulfamide derivatives. The sulfamido group is attached to the metallacarborane carbon vertex by an alkyl chain that may be modified in its length. This was accomplished by, firstly, the abstraction of the acidic hydrogen, located on the {CH}-vertex from the metallacarborane, by reaction with lithium base, followed by, secondly, reaction with electrophilic agents (PFA, oxirane and oxetane), which leads to a cascade of reactions to give the desired sulfamide derivatives. These compounds were then tested by collaborators in other institutes for in vitro and in vivo activity towards Carbonic Anhydrase IX (CA IX), which is an enzyme associated with tumour growth. In vivo tests on mice have shown that these types of substances are able to effectively reduce tumour size by 30%. The synthetic research continued with the preparation of sulfonamide compounds of the isomers of the carborane series. The reactions began exclusively with propylhydroxy carborane starting materials, which provide optimum...

Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the interconversion of carbon dioxide and carbonate and has been found to play a wide range of roles in animals, plants and bacteria. Cotton genomic and cDNA libraries were screened for the plastidial isoform of carbonic anhydrase. The nucleotide sequences of two 1.2 Kb partial cDNA clones were determined. These clones exhibit high homology to carbonic anhydrases from other dicot plants and possess all the expected peptide motifs. For example, serine and threonine rich chloroplastic targeting peptide and conserved zinc binding residues are both present. These clones were utilized to isolate two carbonic anhydrase genes that were shown to encode different isoforms by PCR and RFLP analysis.

Concrete is the most widely used construction material in the world and is responsible for 7% of global carbon emissions. It is inherently brittle, and it requires frequent repair or replacement which is economically expensive and further generates large volumes of carbon dioxide. Current methods of repair by agents such as mortar, epoxies, and bacteria result in structures with reduced strength and resiliency. Recent advances in the design of structural composites often mimic natural microstructures. Specifically, the structure of abalone nacre with its high stiffness, tensile strength, and toughness is a source of inspiration from the process of evolution. The inspiration from nacre can lead to design of a new class of architected structural materials with superb mechanical properties. This body of work first presents a method to reinforce concrete with an architected polymer phase. Second is presented how a ubiquitous enzyme, Carbonic anhydrase (CA), can be used to repair and strengthen cracked concrete, and how it can be used as an additive in fresh concrete. The first study presents an experimental and computational study on a set of bioinspired architected composites created using a cement mortar cast with brick-and-mortar and auxetic polymer phases. The impact of this unit-cell architected polymer phase on the flexural and compressive strengths, resilience, and toughness is studied as a function of microstructural geometry. All mechanical properties of the architected composite samples are found to be greater than those of control samples due to prevention of localized deformation and failure, resulting in higher strength. The microstructurally designed composites showed more layer shear sliding during fracture, whereas the control samples showed more diagonal shear failure. After initial cracking, the microstructurally designed composites gradually deformed plastically due to interlocking elements and achieved high stresses and strains before failure. Results also show that microstructurally designed composites with the architected polymer phase outperform control samples with equal volume fraction of a randomly oriented polymer fiber phase. Computational studies of the proposed unit cells are also performed, and the results suggest that the orientation of cells during loading is critical to achieve maximum performance of a cementitious composite. The implications of these results are immense for future development of high performing construction materials. The second study outlines methods for repair of concrete and lays the groundwork to develop a self-healing concrete that uses trace amounts of the CA enzyme. The CA catalyzes the reaction between calcium ions and carbon dioxide to create calcium carbonate that naturally incorporates into concrete structures with similar thermomechanical properties as concrete. The reaction is safe, actively consumes carbon dioxide, generates low amounts of heat, and avoids using unhealthy reagents, resulting in a strong structure. This repair method results in concrete samples with similar strength and water permeability as the intact materials. These results offer an inexpensive, safe, and efficient method to create self-healing concrete structures. The science underlying the creation of self-healing concrete is described, producing a material intrinsically identical to the original using the CA enzyme. Using this strategy, a preliminary self-healing concrete mix is able to self-repair fractures via hydration. This body of work addresses a major issue: Is there an efficient and ecological repair for decaying concrete infrastructure? These methods propose alternative reinforcement, alleviates high monetary and energy costs associated with concrete replacement, and consume the greenhouse gas, carbon dioxide.

Gammaproteobacterium Thiomicrospira crunogena thrives at deep-sea vents despite extreme oscillations in the environmental supply of dissolved inorganic carbon (DIC; =CO2 + HCO3- + CO3-2). Survival in this habitat is likely aided by the presence of a carbon concentrating mechanism (CCM). Though CCMs are well-documented in cyanobacteria, based on this study T. crunogena is the first chemolithoautotroph to have a physiologically characterized CCM. T. crunogena is capable of rapid growth in the presence of 20 micrometers DIC, has the ability to use both extracellular HCO3- and CO2, and generates intracellular DIC concentrations 100-fold greater than extracellular, all of which are consistent with a CCM analogous to those present in cyanobacteria. Interestingly, however, the T.crunogena genome lacks apparent orthologs of many of the components of the cyanobacteria CCM (e.g., HCO3- transporters). However, despite this lack, several candidate genes were identified during genome annotation as likely to play a role in DIC uptake and fixation (three carbonic anhydrase genes: alpha-CA, beta-CA, and csoSCA, as well as genes encoding three RubisCO enzymes: cbbLS, CScbbLS, and cbbM, which encode a cytoplasmic form I RubisCO, a carboxysomal form I RubisCO, and a form II RubisCO, respectively). In order to clarify their possible roles in DIC uptake and fixation, alpha-CA, beta-CA and csoSCA transcription by low-DIC and high-DIC T. crunogena were assayed by qRT PCR, heterologous expression in E. coli, and potentiometric assays of low-DIC and high-DIC T. crunogena. Transcription of alpha-CA and beta-CA were not sensitive to the DIC concentration available during growth. When overexpressed in E.coli, carbonic anhydrase activity was detectable, and it was possible to measure the effects of the classical carbonic anhydrase inhibitors ethoxyzolamide and acetazolamide, as well as dithiothreitol (DTT; recently determined to be a carboxysomal CA inhibitor). The alpha-CA was sensitive to both of the classical inhibitors, but not DTT. Beta-CA was insensitive to all inhibitors tested, and the carboxysomal carbonic anhydrase was sensitive to both ethoxyzolamide and DTT. The observation that the CA activity measureable potentiometrically with intact T. crunogena cells is sensitive to classical inhibitors, but not DTT, strongly suggests the alpha-CA is extracellular. The presence of carbonic anhydrase activity in crude extracts of high-DIC cells that was resistant to classical inhibitors suggests that beta-CA may be more active in high-DIC cells. Incubating cells with ethoxyzolamide (which permeates cells rapidly) resulted in inhibition of carbon fixation, but not DIC uptake, while incubation with acetazolamide (which does not permeate cells rapidly) had no apparent effect on either carbon fixation or DIC uptake. The observations that inhibition of alpha-CA has no effect on DIC uptake and fixation, and that the beta-CA is not transcribed more frequently under low-DIC conditions, make it unlikely that either play a role in DIC uptake and fixation in low-DIC cells. Further studies are underway to determine the roles of alpha-CA and beta-CA in T. crunogena. To assay the entire genome for genes transcribed more frequently under low-DIC conditions, and therefore likely to play a role in the T. crunogena CCM, oligonucleotide arrays were fabricated using the T. crunogena genome sequence. RNA was isolated from cultures grown in the presence of both high (50 mM) and low (0.05 mM) concentrations of DIC, directly labeled with cy5 fluorophore, and hybridized to microarrays. Genes encoding the three RubisCO enzymes present in this organism demonstrated differential patterns of transcription consistent with what had been observed previously in Hydrogenovibrio marinus. Genes encoding two conserved hypothetical proteins were also found to be transcribed more frequently under low-DIC conditions, and this transcription pattern was verified by qRT-PCR. Knockout mutants are currently being generated to determine whether either gene is necessary for growth under low-DIC conditions. Identifying CCM genes and function in autotrophs beyond cyanobacteria will serve as a window into the physiology required to flourish in microbiallydominated ecosystems where noncyanobacterial primary producers dominate.