A obesidade e a cárie dentária são problemas de saúde pública, que atingem a população infantil. O objetivo deste estudo foi identificar a prevalência de cárie dentária e relacioná-la com a concentração da enzima anidrase carbônica VI, do íon cálcio, fluxo salivar e quantidade de biofilme dentário em crianças com sobrepeso/obesidade. Foram avaliadas 112 crianças de 4 a 6 anos de idade, de ambos os gêneros. A análise antropométrica foi realizada (percentil do IMC) e através dessa análise as crianças foram divididas em dois grupos: G1 sobrepesos/obesos (n=41) e G2 normais (n=71). Os exames bucais realizados para a cárie dentária foram os índices ceo-s e ICDAS II, quantidade de biofilme dentário pelo Índice de Placa de Turesky e volume de fluxo salivar estimulado. A concentração do íon Cálcio na saliva foi analisada pelo kit colorimétrico e da enzima Anidrase Carbônica VI pelo kit ELISA. Na sequência, as crianças de cada grupo foram divididas em 3 subgrupos: LC (livres de cárie), LI (com lesões iniciais) e C (com cárie). Os testes Wilcoxon, Mann-Whitney, teste t e correlação de Spearman foram aplicados (p<0,05). Não houve diferença significativa no ceo-s entre os grupos. Houve maior concentração média de cálcio salivar no G1 (G1=2847,96mM; G2=1230,90mM;p=0,001) e maior concentração da Anidrase Carbônica VI no G2 (G1=3455,18 pg/mL; G2=442428,9pg/mL;p=0,000). No G1 houve correlação negativa entre o ceo-s e íon Cálcio (r=-0,444;p=0,010). Já no G2, houve correlação negativa entre placa e a Anidrase Carbônica VI (r=-0,551;p=0,014). Pode-se concluir que o íon cálcio é fator protetor para cárie dentária em crianças. Já a anidrase carbônica VI parece não ser biomarcador para a cárie dentária. / Obesity and dental caries are public health problems that affect the child population. The aim of this study was to identify the prevalence of dental caries and relate it to the concentration of the enzyme carbonic anhydrase VI, calcium ion, salivary flow, and dental plaque in overweight/obesity children. The study was conducted on 112 children aged 4-6, of both genders. Anthropometric analysis was performed (BMI percentile) and by this analysis the children were divided into two groups: G1 - overweight/obese (n=41) and G2 - normal (n=71). The oral examinations performed for dental caries were the dmfs and ICDAS II indexes, measurement of the amount of dental plaque by the Turesky Board Index and volume of stimulated salivary flow. The concentration of calcium ion in saliva was measured by a colorimetric kit and the enzyme carbonic anhydrase VI by an ELISA kit. Then, children from each group were divided into three subgroups: CF (caries-free), IL (initial lesions) and D (decayed teeth). The Wilcoxon test, Mann-Whitney, t test and Spearman correlation (p<0.05) were applied. There was no significant difference in the dmfs between groups. There was higher concentration of salivary calcium in G1 (G1=2847.96mM; G2=1230.90mM; p=0.001), and higher concentration of carbonic anhydrase VI in G2 (G1 = 3455.18 pg/ml; G2 = 442428.9pg/ml; p = 0.000). In G1, there was negative correlation between dmfs and salivary calcium (r = -0.444; p = 0.010). In G2, there was negative correlation between dental plaque and carbonic anhydrase VI (r=-0.551; p=0.014). It can be concluded that the calcium ion is a protective factor for dental caries in children. The carbonic anhydrase VI does not seem to be a biomaker of dental caries.

L'hypoxie est une caractéristique saillante de nombreuses tumeurs solides et provient d'un apport vasculaire inadéquat et immature, entraînant une diminution l'apport d'oxygène et nutriments. Ces régions hypoxiques montrent une résistance aux modalités de traitement classiques telles que la radio et la chimiothérapie et sont associées à une survie insuffisante. Dans des conditions hypoxiques,le HIF-1α améliore l'expression de nombreux gènes cibles,l'un d'eux étant l'anhydrase carbonique IX (CAIX). La CAIX est une enzyme transmembranaire,impliquée dans l'hydratation réversible du dioxyde de carbone au bicarbonate et au proton. L'objectif de cette thèse était de cibler CAIX en utilisant divers médicaments à double cible combinés avec des radiosensibilisateurs, des médicaments cytotoxiques etdes médicaments bio-réductibles. Dans cette thèse,plusieurs approches de ciblage CAIX ont été étudiées.Étant donné que les nitroimidazoles sont de bons sensibilisants cellulaires hypoxiques, nous avons conçu plusieurs composés à double cible existant par combinaison d'un nitroimidazole et d'une fraction inhibitrice d'anhydrase carbonique IX (chapitre 3). Auparavant, notre groupe a montré que le dérivé à base de sulfamide 7 a amélioré l'efficacité thérapeutique de l'irradiation de manière dépendante la CAIX avec un rapport d'amélioration de la sensibilisation (SER) de 1,50, ce qui est plus élevé que plusieurs radiosensibilisateurs testés cliniquement tels que le misonidazole et le nimorazole. L'efficacité de la chimiosensibilisation a été observée lors de la combinaison de 7 avec doxorubicine chez des souris porteurs de tumeur HT29 (chapitre 3).Une approche à double cible similaire peut être exploitable pour délivrer des médicaments cytotoxiques vers des cellules exprimant CAIX, ce qui entraîne une administration ciblée spécifique de la tumeur et par conséquent réduit la toxicité normale des tissus. Le chapitre 4 décrit la conception et la synthèse de nouvelles séries de composés à double cible combinant plusieurs médicaments anti-cancéreux. Un seul composé,c'est-à-dire un dérivé ATRi, a montré une efficacité supérieure en association avec un rayonnement dans des cellules surexprimant CAIX par rapport aux cellules dépourvues d'expression CAIX.Le chapitre 5 décrit la conception et la synthèse de divers dérivés de nitroimidazole bio-réductibles,des agents alkylants de moutarde d'azote et des dérivés de N-oxyde combinés avec une fraction inhibitrice de l'anhydrase carbonique IX. Tous ces composés présentent des profils d'inhibition faibles à modérés vers plusieurs isoformes CA testées. Nous avons observé que différentes substitutions et liens dans la même famille de composés influencent la capacité de liaison à CAIX.Pour élargir notre étude sur les médicaments bio-réductibles, le chapitre 6 décrit la conception et la synthèse de 2, 5-nitroimdazole et moutarde d'azote combinées avec des inhibiteurs de l'anhydrase carbonique IX par un agent de liaison de carbamate. Le dérivé 2-nitroimidazole 1b a révélé une cytotoxicité dans les lignées cellulaires HT29 et HCT116 et pourrait être expliqué par le potentiel de réduction des 2-nitroimidazoles par rapport aux 5-nitroimidazoles, car nos résultats n'ont montré aucune cytotoxicité des dérivés du 5-nitroimidazole. Nos futures études visent à optimiser l'efficacité radiosensibilisante de 2b et 2c explorent davantage les propriétés cytotoxiques de 1b.En conclusion, cette thèse a montré que le double nitroimidazole cible combiné à la CAIXi augmente l'efficacité des modalités de traitement standard telles que la chimiothérapie et la radiothérapie. Le ciblage de la CAIX avec la combinaison de médicaments cytotoxiques continue d'être une approche intéressante pour les tumeurs hypoxiques cibles à l'avenir. Les médicaments bio-réductibles avec des potentiels de réduction plus élevés serviraient d'agents cytotoxiques potentiels aux tumeurs hypoxiques, ce qui réduirait la toxicité tissulaire normale. / Hypoxia is a salient feature in many solid tumors and arises due to an inadequate and immature vascular supply resulting in a decreased delivery of oxygen and nutrients. These hypoxic regions show resistance towards conventional treatment modalities such as radio- and chemotherapy and are associated with poor survival. Under hypoxic conditions HIF-1α enhances the expression of many target genes, one of them being carbonic anhydrase IX (CAIX).CAIX is a transmembrane enzyme, which is involved in reversible hydration of carbon dioxide to bicarbonate and a proton.The aim of this thesis was to target CAIX using various dual target drugs combined with radiosensitizers, cytotoxic drugs and bio-reducible drugs.In this thesis several CAIX targeting approaches have been investigated.Since nitroimidazoles are good hypoxic cell sensitizers,we have designed several dual target compounds existing out of a combination of a nitroimidazole and a carbonic anhydrase IX inhibitory moiety (Chapter 3). It has been shown that extracellular acidity limits the uptake of weak basic chemotherapeutic drugs, such as doxorubicin, and thereby decreases its efficacy. We hypothesized that combining these nitroimidazole moieties with a sulfonamide/sulfamide/sulfamate to target CAIX results in a decrease in extracellular acidosis and sensitizes hypoxic tumors to chemo- and radiotherapy. Previously, our group has shown that the sulfamide-based derivative 7 enhanced the therapeutic efficacy of irradiation in a CAIX dependent manner with a sensitization enhancement ratio of 1.50,which is higher than several clinically tested radiosensitizers such as misonidazole and nimorazole. Chemosensitization efficacy was observed upon combination of 7 with doxorubicin in HT29 tumor-bearing mice (Chapter 3).A similar dual target approach may be exploitable to deliver cytotoxic drugs towards CAIX expressing cells, resulting in a specific tumor targeted delivery and consequently reduced normal tissue toxicity. Chapter 4 describes the design and synthesis of new series of dual target compounds combining several anti-cancer drugs, Only one compound, i.e. an ATRi derivative, showed a higher efficacy in combination with radiation in CAIX overexpressing cells as compared to cells lacking CAIX expression.Chapter 5 describes the design and synthesis of various bio-reducible nitroimidazole derivatives, nitrogen mustard alkylating agents and N-oxide derivatives combined with a carbonic anhydrase IX inhibiting moiety. All these compounds showed weak to moderate inhibition profiles towards several tested CA isoforms. We have observed that different substitutions and linkers within the same family of compounds influence the binding capacity to CAIX. For example derivatives 17 and 20 belong to same family, but have a different linker and substitution of the aromatic ring, leading to a different binding capacity towards CAIX.To expand our study on bio-reducible drugs, Chapter 6 describes the design and synthesis of 2-, 5-nitroimdazole and nitrogen mustards combined with carbonic anhydrase IX inhibitors by a carbamate linker. The 2-nitroimidazole derivative 1b revealed cytotoxicity in HT29 and HCT116 cell lines and might be explain by the higher reduction potential of 2-nitroimidazoles compared to 5-nitroimidazoles, since our results did show no cytotoxicity of the 5-nitroimidazole derivatives. Our future studies aim to optimize the radiosensitizing efficacy of 2b and 2c and further explore the cytotoxic properties of 1b.In conclusion, this thesis showed that the dual target nitroimidazole combined with CAIXi increases the efficacy of standard treatment modalities such as chemo and radiotherapy. Targeting CAIX with combination of cytotoxic drugs continues to be an interesting approach to target hypoxic tumors in future. Bio-reducible drugs with higher reduction potentials would serve as potential cytotoxic agents to hypoxic tumors thereby decreasing the normal tissue toxicity.

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.