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The Measurement of erythrocyte carbonic anhydrase and its use in the diagnosis of thyrotoxicosis.January 1992 (has links)
by Judy, Po-shan Lai. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references(leaves 83-87). / Abbreviations --- p.iii / List of Tables --- p.iv / List of Figures --- p.v / Acknowledgements --- p.vi / SUMMARY --- p.1 / Chapter 1. --- INTRODUCTION --- p.2 / Chapter 2. --- BACKGROUND --- p.4 / Chapter 2.1 --- PROBLEMS ENCOUNTERED IN THYROID FUNCTION TESTS --- p.4 / Chapter 2.2 --- A NEED FOR TISSUE MARKER VERSUS EXISTING THYROID FUNCTION TESTS --- p.8 / Chapter 2.3 --- CHOICE OF TISSUE MARKER AS AN ADJUNCT TO THYROID FUNCTION TESTS --- p.10 / Chapter 2.3.1 --- ERYTHROCYTE ZINC --- p.10 / Chapter 2.3.2 --- ERYTHROCYTE CARBONIC ANHYDRASE ISOENZYME --- p.11 / Chapter 3. --- CARBONIC ANHYDRASE --- p.14 / Chapter 3.1 --- HISTORY AND BACKGROUND --- p.14 / Chapter 3.2 --- CARBONIC ANHYDRASE ISOENZYMES --- p.15 / Chapter 3.2.1 --- STRUCTURE AND DISTRIBUTION --- p.15 / Chapter 3.2.2 --- GENERAL MECHANISMS --- p.18 / Chapter 3.2.3 --- GENETICS AND REGULATION --- p.18 / Chapter 3.2.4 --- PHYSIOLOGICAL AND CLINICAL ASPECTS --- p.20 / Chapter 4. --- ANALYTICAL METHODS FOR MEASUREMENT OF ERYTHROCYTE CARBONIC ANHYDRASE --- p.22 / Chapter 4.1 --- DETERMINATION OF ECAI USING IMMUNOTURBIDIMETRIC ASSAY --- p.23 / Chapter 4.1.1 --- PRINCIPLE OF TECHNIQUE --- p.23 / Chapter 4.1.2 --- COLLECTION AND HANDLING OF BLOOD SPECIMENS --- p.29 / Chapter 4.1.3 --- PREPARATION OF HAEMOLYSATE --- p.31 / Chapter 4.1.4 --- MEASUREMENT OF HAEMOLYSATE CAI --- p.32 / Chapter 4.1.5 --- OPTIMIZATION PROCEDURE --- p.34 / Chapter 4.1.6 --- EVALUATION EXPERIMENTS --- p.36 / Chapter 4.1.7 --- DETERMINATION OF HAEMOGLOBIN CONCENTRATION IN HAEMOLYSATE --- p.39 / Chapter 4.1.8 --- DETERMINATION OF MEAN CELL HAEMOGLOBIN CONCENTRATION --- p.41 / Chapter 4.1.9 --- CALCULATION OF ERYTHROCYTE CARBONIC ANHYDRASE I CONCENTRATION --- p.43 / Chapter 4.2 --- DETERMINATION OF ECAI USING RADIAL IMMUNODIFFUSION METHOD --- p.43 / Chapter 5. --- RESULTS OF OPTIMIZATION AND EVALUATION EXPERIMENTS --- p.46 / Chapter 5.1 --- OPTIMIZATION RESULTS --- p.46 / Chapter 5.1.1 --- WAVELENGTH --- p.46 / Chapter 5.1.2 --- PEG CONCENTRATION --- p.49 / Chapter 5.1.3 --- SAMPLE VOLUME --- p.52 / Chapter 5.1.4 --- ANTIBODY DILUTION --- p.52 / Chapter 5.1.5 --- TEMPERATURE --- p.54 / Chapter 5.1.6 --- TIME OF INCUBATION --- p.54 / Chapter 5.1.7 --- OPTIMIZED TEST PROTOCOL --- p.56 / Chapter 5.2 --- EVALUATION RESULTS --- p.56 / Chapter 5.2.1 --- STABILITY --- p.56 / Chapter 5.2.2 --- LINEARITY --- p.58 / Chapter 5.2.3 --- PRECISION --- p.62 / Chapter 5.2.3.1 --- WITHIN-RUN --- p.62 / Chapter 5.2.3.2. --- BETWEEN-RUN --- p.62 / Chapter 5.2.4 --- CROSS-REACTIVITY --- p.62 / Chapter 5.2.5 --- RECOVERY --- p.62 / Chapter 5.2.6 --- COMPARISON WITH COMPARATIVE METHOD --- p.64 / Chapter 5.3 --- DISCUSSION --- p.64 / Chapter 6. --- ECAI IN NORMAL SUBJECTS --- p.67 / Chapter 6.1 --- SUBJECTS AND METHOD --- p.67 / Chapter 6.2 --- RESULTS --- p.67 / Chapter 6.3 --- DISCUSSION --- p.67 / Chapter 7. --- PATIENT STUDY --- p.69 / Chapter 7.1 --- SUBJECTS AND METHOD --- p.69 / Chapter 7.2 --- RESULTS --- p.70 / Chapter 7.2.1 --- DEMOGRAPHIC AND BIOCHEMICAL PARAMETERS --- p.70 / Chapter 7.2.2 --- CORRELATION BETWEEN ECAI AND FT3 RESULTS OF THYROTOXIC PATIENTS BEFORE TREATMENT --- p.71 / Chapter 7.2.3 --- CORRELATION BETWEEN ECAI AND EZN RESULTS OF THYROTOXIC PATIENTS --- p.71 / Chapter 7.2.4 --- RELATIONSHIP BETWEEN THE CHANGES IN ECAI AND FT3 RESULTS IN THYROTOXIC PATIENT ON TREATMENT --- p.71 / Chapter 7.3 --- DISCUSSION --- p.76 / Chapter 8. --- GENERAL DISCUSSION --- p.79 / Chapter 9. --- REFERENCES --- p.83
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Design, synthesis and pharmacological evaluation of carbonic anhydrase VII and IX inhibitorsThiry, Anne 15 May 2008 (has links)
Carbonic anhydases (CAs) are ubiquitous enzymes present in human under 15 different isozymes. Each active one catalyzes the hydration reaction of carbon dioxide into bicarbonate anion and proton. Some isozymes contribute to basic physiological processes like among other respiration and acid-base homeostasis while other isozymes are involved in pathologies such as epilepsy (CA VII) and cancer (CA IX). Convulsions observed during epileptic seizures are partly attributed to carbonic anhydrase VII which play a role in neuronal excitation phenomenon. Carbonic anhydrase IX (CA IX) is overexpressed in most cancer tissues and is absent from their normal counterparts. It can acidify the extratumoral medium leading to metastatic behavior.
To improve our knowledge on the role of these isozymes, the design of selective CA VII and IX inhibitors is of a great interest. Otherwise, such compounds can potentially be developed as antiepileptic or anticancer agents. A molecular modeling study which combines a direct (homology modelling, docking) and an indirect (pharmacophore, virtual screening) approach of drug design was conducted to create novel and selective inhibitors. In parallel, original indanesulfonamides were designed, synthesized and their inhibitory potencies against the CAs were determined. Docking studies of some derivatives allowed to rationalize the enzymatic data. Then, we evaluated the effect of some indanesulfonamides on a model of cancer cells. The study of the anticonvulsant activity was performed on an in vivo model. Finally, during this work other series of potentially CA inhibitors were also evaluated for their CA inhibitory activities and for one of them for its anticonvulsant effect.
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Molecular kinetic studies I. Sodium lauryl sulfate. II. Carbonic anhydrase /Hakala, Niilo Victor, January 1943 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1943. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves xv-xvii).
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Carbonic anhydrases in the reproductive system:with special emphasis on isoenzymes VI, IX, XII, and a novel nuclear nonclassical formKarhumaa, P. (Pepe) 17 May 2002 (has links)
Abstract
Carbonic anhydrases (CAs) are a group of zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate (CO2 + H2O ⇔ HCO3- + H+). They are present in almost all organs and are implicated in various biological functions, the most important of which is participation in the regulation of ion, water, and acid-base balance. Recently, some members of the CA gene family have been suggested to promote cell proliferation and to act as trophic growth factors.
The present study was undertaken to examine the distribution of CA isoenzymes in the reproductive system, to attain a more detailed view on their linkage to the reproductive processes and to neonatal development.
The expression of membrane-bound CA IX and CA XII was studied in the female and male reproductive tracts by immunohistochemistry and western blotting. CA XII was found to be expressed in the basolateral plasma membrane of luminal and glandular epithelia in human uterus. In human efferent ducts, it was located in the basolateral plasma membrane of luminal epithelium, where it coexpressed with Aquaporin-1. In epididymal duct, CA XII was only expressed in occasional epithelial cells. These cells coexpressed CA II, suggesting that they represent apical mitochondria-rich cells (AMRC). CA IX was also expressed in the basolateral plasma membrane of luminal epithelium in human efferent ducts, but its expression was not uniform among the tubules. These findings suggest that basolateral plasma membrane-associated CA IX and CA XII contribute, along with CA II and CA IV, to the regulation of acid-base balance and water transport in the reproductive tract.
Western blotting of rat Leydig tumor cells and testis for CA II revealed an unidentified 66-kDa polypeptide band. The polypeptide was successfully purified from several rat tissues using CA inhibitor affinity chromatography. The amino acid sequence of the polypeptide showed it to be identical to NonO/p54nrb, a non-POU domain-containing octamer-binding protein previously implicated in transcriptional regulation. The recombinant NonO/p54nrb was shown to display CA activity, and the antibody to it predominantly immunostained the nuclei in lymphocytes, where CA activity was also detected histochemically. Accordingly, the nuclear Leydig cell CA immunoreactivity represents NonO/p54nrb. It is classified as a novel, nonclassical CA, and it may participate in pH-related events in the nucleus.
Human and rat milk was found to contain CA VI by immunohistochemistry and western blotting. The enzyme purified from human milk by CA inhibitor affinity chromatography was confirmed by PNGase F digestion and amino acid sequence as CA VI. The CA VI concentrations in human colostral milk were approximately eight times higher than those in mature milk (34.7 mg/l vs. 4.5 mg/l). Secretion of CA VI into milk is suggested by its localization in the alveolar epithelium of the rat mammary gland. The structural and functional stability of CA VI in an acidic milieu, its suggested growth-supporting function in taste bud stem cells, and its high concentration in colostrum suggest that it is an essential factor for the growth and development of the newborn alimentary canal.
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Synthèse d'inhibiteurs multivalents des anhydrases carboniques / Multivalent inhibitors of carbonics anhydrasesKanfar, Nasreddine 20 October 2017 (has links)
Les anhydrases carboniques (CA, CE. 4.2.1.1) sont des métalloenzymes de zinc, ubiquitaires, qui catalysent l'hydratation réversible du CO2, avec la formation de bicarbonate et de la libération d'un proton. Sur les 13 isoformes actifs présents chez l'homme, certains d'entre eux sont impliqués dans les processus pathologiques. Les CA sont connues depuis plus de 50 ans en tant que cibles thérapeutiques et certains inhibiteurs sont actuellement en phase clinique ou dans des études pré-cliniques pour le traitement du glaucome, de l'épilepsie et de cancer. Néanmoins, le manque de sélectivité contre les différents isoformes responsables des effets secondaires nécessite le développement de nouvelles stratégies. Le but de ce travail est de développer une nouvelle façon pour inhiber les CAs en tirant parti de l'interaction multivalente pour inhiber sélectivement et efficacement les isoformes de l'CA. En effet, les clusters multivalents représentent une classe émergente de composés pour l'inhibition d'enzymes. Cette stratégie a été développée récemment pour l'inhibition et l'activation d'CA, certaines études ayant démontré des améliorations dans la puissance d'inhibition et la sélectivité. Dans ce projet, différentes plateformes (peptides, nanoparticules de silice) multifonctionnels ont été revêtus de sulfonamides comme inhibiteurs de l'CA par bioconjugaison. L'effet d'inhibition et la spécificité de la multivalence ont été étudiés sur les isoformes CA. / Carbonic anhydrases (CAs, EC. 4.2.1.1) are ubiquitous zinc metalloenzymes which catalyze the reversible hydration of CO2 with formation of bicarbonate and release of a proton. On the 13 active isoforms present in human, some of them are involved in pathological processes. CAs are known for more than 50 years as a therapeutic targets, and some inhibitors are currently in clinic or in (pre)clinical studies for the treatment of glaucoma, epilepsy and cancer. Nevertheless the lack of selectivity against the different isoforms responsible of side-effects requires the development of new strategies. The aim of this work is to develop a new way for CA inhibition by taking advantage of multivalent interaction to selectively and efficiently inhibit CA isoforms. Indeed, multivalent clusters represent an emerging class of compounds for enzymes inhibition. This strategy has been recently developed for CA inhibition and activation, some studies reporting improvements in inhibitory potency and selectivity. In this project, different platforms (peptides, polymers, silica nanoparticles) multifunctional were coated with sulfonamides as inhibitors of CA by bioconjugation. The inhibitory effect and specificity of the multivalency were studied isoforms CA.
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Characterisation of surface traits of Helicobacter pylori and their role in the infectious process /Petersson, Christoffer January 2003 (has links) (PDF)
Diss. Linköping : Univ., 2003.
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X-ray crystallographic analysis of three proteins : the novel structures of the corn Hageman factor inhibitor, the G-protein coupled receptor rhodopsin, and the ultra-high resolution structure of carbonic anhydrase /Behnke, Craig A. January 2000 (has links)
Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 73-77).
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Kinetic and structural studies on the activation of the proton transfer in catalysis by carbonic anhydraseElder, Ileana, January 2004 (has links)
Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 116 pages. Includes Vita. Includes bibliographical references.
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Caracterização de anidrases carbônicas do fungo Paracoccidioides brasiliensis / Characterization of carbonic anhydrases of Paracoccidioides brasiliensisTomazett, Mariana Vieira 29 August 2011 (has links)
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Previous issue date: 2011-08-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Paracoccidioidomycosis (PCM) is the most important endemic deep mycosis in Latin
America. Understanding of the complex interactions between the fungus and host must
include the identification of gene expression patterns during infection. Carbonic anhydrase
(CA) belongs to the family of zinc metalloenzymes that catalyzes the reversible hydratation of
carbon dioxide to bicarbonate. Transcriptional studies have shown that carbonic anhydrase of
P. brasiliensis is expressed in yeast cells recovered from liver of infected mice. In the present
work, we characterized the cDNAs encoding for four carbonic anhydrases of P.brasiliensis
(PbCA1, PbCA2, PbCA3, PbCA4). Recombinant PbCA1, 3 and 4 were obtained in
heterologous systems with 33 kDa, 28 kDa and 32 kDa respectively. Mass spectrometry
analysis confirmed the sequences of the produced proteins. The expression of PbCAs
transcripts was evaluated by using real-time RT-PCR in mycelium, yeast cells and mycelium
to yeast transition, in yeast cells exposed to CO2 and yeast cells recovery directly from liver
and spleen. In the presence of CO2, PbCA1, PbCA2 and PbCA4 gene expression was reduced
in the course of time. PbCA1 transcript expression was induced during the mycelium to yeast
transition. PbCA2 and PbCA4 gene expression was higher in yeast cells, when compared to
mycelium and mycelium to yeast transition. Pbca1 was induced yeast cells recovery directly
from liver and spleen while the transcripts for Pbca2 and Pbca4 were repressed in all
condictions. The gene expression data suggest differential roles of the CAs in the fungal
physiology. / Paracoccidioidomicose (PCM) é a mais importante micose endêmica na América Latina. A
compreensão das interações complexas entre o fungo e o hospedeiro deve incluir a
identificação de padrões de expressão gênica durante a infecção. Anidrase carbônica (CA)
pertence à família de metaloenzimas de zinco que catalisa a hidratação reversível do dióxido
de carbono para bicarbonato. Estudos transcricionais têm mostrado que uma anidrase
carbônica de P. brasiliensis é expressa em células de levedura recuperadas de fígado de
camundongos infectados. No presente trabalho, nós caracterizamos os cDNAs que codificam
para quatro anidrases carbônicas de P.brasiliensis (PbCA1, PbCA2, PbCA3, PbCA4). As
recombinante de PbCA1, PbCA3 e PbCA4 foram obtidas em sistemas heterólogos com 33
kDa, 28 kDa e 32 kDa, respectivamente. Análise por espectrometria de massas confirmou as
seqüências das proteínas produzidas. A expressão dos transcritos de Pbcas foi avaliada usando
real-time RT-PCR em micélio, levedura e transição de micélio para levedura; em células de
levedura expostas a CO2 e células de leveduras recuperadas diretamente do fígado e baço. Na
presença de CO2, a expressão dos genes de Pbca1, Pbca2 e Pbca4 foi reduzida no decorrer do
tempo. A expressão do transcrito de Pbca1 foi induzida durante a transição de micélio para
levedura. A expressão de Pbca2 e Pbca4 foi maior em células de levedura, quando comparada
com micélio e transição de micélio para levedura.O transcrito para Pbca1 foi induzido em
células de leveduras recuperadas diretamente do fígado e baço, enquanto que os transcritos
para Pbca2 e Pbca4 foram reprimidos em todas as conduções. Os dados de expressão gênica
sugerem diferentes papéis das CAs na fisiologia dos fungos.
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Approches physiologique et moléculaire de la calcification chez le corail rouge de méditerranée Corallium rubrum / Molecular and physiological approaches to study calcification in the mediterranean red coral Corallium rubrumLe Goff, Carine 14 December 2016 (has links)
Le processus de calcification chez Corallium rubrum conduit à la formation de deux structures squelettiques composées de CaCO3, l’axe squelettique et les sclérites, de taille et de forme différentes. Comme chez de nombreuses espèces calcifiantes, la calcification se fait sous contrôle biologique impliquant notamment des enzymes et des transporteurs ioniques. Une question centrale est d’identifier les mécanismes communs ou propres à chaque espèce qui sous-tendent leur convergence fonctionnelle envers ce processus. Deux approches ont été utilisées pour caractériser ces mécanismes chez C. rubrum: 1) Une approche physiologique avec le développement d’une technique de culture de microcolonies sur lamelles permettant d’observer différents stades de calcification, et de mesurer le pH aux sites de calcification par imagerie confocale ; 2) Une approche moléculaire afin de caractériser une famille d’enzymes, les anhydrases carboniques (ACs), qui jouent un rôle clef dans la calcification.Nous avons réalisé une cartographie du pH en effectuant des mesures dans différents compartiments intra- et extracellulaires. Nos résultats montrent notamment que le pH aux sites de calcification est supérieur à celui du milieu circulant dans les canaux gastrodermiques et non à celui l’eau de mer. Les mesures d’expression différentielle des ACs dans différents tissus mettent en évidence une isozyme préférentiellement exprimée dans les cellules calcifiantes.Ces résultats intégrés dans un contexte de calcification comparée pointent sur la convergence fonctionnelle des ACs et de la régulation du pH par les cellules calcifiantes, tout en soulignant des divergences évolutives. / The calcification process in Corallium rubrum leads to the formation of two skeletal structures made of calcium carbonate, the skeletal axis and sclerites, of different size and shape. As in many calcifying species, calcification occurs under a biological control that involves enzymes and ion transporters. A central issue is to determine the common and the species-specific mechanisms of calcification in order to identify functional convergences in this process. Two approaches were used to characterize these mechanisms in C. rubrum: 1) A physiological approach involving the development of a microcolony culture technique on glass coverslips, allowing the observation of the different stages of calcification, and the measurement of pH at the sites of calcification by the use of confocal microscopy; 2) A molecular approach to characterize an enzyme family, the carbonic anhydrases, which play a key role in calcification.We performed pH mapping by making measurements in different intra- and extracellular compartments. Our results show higher pH values at the sites of calcification compared with the fluid circulating in the gastrodermal canals, but not with the seawater surrounding the microcolony. Measurements of differential expression of carbonic anhydrases in different tissue fractions highlight an isozyme preferentially expressed in the calcifying cells.Within comparative calcification perspectives, these results point towards the functional convergence of carbonic anhydrases and pH regulation by the calcifying cells, while highlighting evolutionary divergences.
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