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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Studies on the Postnatal Development of the Rat Liver Plasma Membrane Following Maternal Ethanol Ingestion

Rovinski, Benjamin 03 1900 (has links)
No description available.
2

Estudi estructural i cinètic de l'especificitat de coenzim d'una alcohol deshidrogenasa d'amfibi dependent de Nadp(h)

Rosell Febres, Albert 09 June 2004 (has links)
Els teixits gàstrics de l'amfibi Rana perezi expressen una alcohol deshidrogenasa (ADH8) dependent de NADP(H), una especificitat de coenzim única dintre de les ADHs de vertebrat. Aquest enzim és actiu amb etanol i retinoides. Han estat resoltes i refinades les estructures tridimensionals de l'ADH8 i del complex binari ADH8-NADP+ a 2,2 i 1,8 Å, respectivament. ADH8 presenta una identitat de seqüència amb ADHs dependents de NAD(H) de vertebrat inferior al 60%. La seva especificitat de coenzim i de substrat suggereixen un paper d'aquest enzim en la reducció d'aldehids més que en l'oxidació d'alcohols. El gran volum del seti d'unió al substrat pot explicar les altes eficiències catalítiques d'ADH8 amb retinoides. La preferència per el NADP(H) sembla ser deguda a la presència en ADH8 de la triada Gly223-Thr224-His225, i al manteniment dels residus conservats Lys228 i Leu200, que defineixen un seti d'unió per el grup fosfat terminal del cofactor. El NADP(H) s'uneix a l'ADH8 amb una conformació extesa. Aquest coenzim es pot superposar amb el NAD(H) unit en complexos d'ADHs dependents de NAD(H). Les desviacions conformacionals més grans corresponen al grup adenina-ribosa de la molècula. No s'observaren diferències adicionals en el seti d'unió al dinucleòtid, fet que explicaria que el NAD(H) pugui també ser utilitzat com a cofactor per ADH8.En el complex crystal.logràfic ADH8-NADP+, el seti d'unió del grup fosfat extra del coenzim està format per Gly223-Thr224-His225, residus específics de l'ADH8, i per les posicions conservades Leu200 i Lys228. Amb l'objectiu d'investigar els mínims determinants estructurals responsables de l'especificitat de coenzim, diferents mutants d'ADH8 en les posicions 223-225 foren dissenyats i cinèticament caracteritzats. El mutant G223D, tot i presentar una càrrega negativa en el seti d'unió al fosfat, encara preferia el NADP(H) com a coenzim, de la mateixa manera que els mutants T224I i H225N. Les eficiències catalítiques amb NADP(H) disminuiren dramàticament en els dobles mutants, G223D/T224I i T224I/H225N, i en el triple mutant G223D/T224I/H225N (kcat/KmNADPH = 760 mM-1min-1), respecte l'enzim silvestre (kcat/KmNADPH = 133330 mM-1min-1). Aquest resultat fou associat amb una més baixa afinitat per el NADP+ i amb un canvi en l'etapa limitant de la reacció. Contràriament, en el triple mutant, l'eficiència catalítica amb NAD(H) incrementà, assolint valors (kcat/KmNADH = 155000 mM-1min-1) similars als de l'enzim silvestre amb NADP(H). La reversió completa de l'especificitat de coenzim de l'ADH8 doncs, fou aconseguida amb únicament la substitució de tres residus consecutius en el seti d'unió del fosfat extra, un resultat sense precedents dintre de la família ADH. / Gastric tissues from amphibian Rana perezi express an alcohol dehydrogenase (ADH8) with a distinct specificity for NADP(H) and active with ethanol and retinoids.The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP+ have been determined and refined to resolutions of 2.2 and 1.8 Å, respectively. ADH8 exhibits less than 60% sequence identity with vertebrate NAD(H)-dependent ADHs. Its coenzyme and substrate specificity support a role in aldehyde reduction rather than in alcohol oxidation. The large volume of the substrate-binding pocket can explain the high catalytic efficiency of ADH8 with retinoids. Preference for NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225, and the recruitment of conserved Lys228 and Leu200, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds ADH8 in an extended conformation and can be superimposed with NAD(H) in NAD(H)-dependent ADH complexes, the largest conformational deviations being for the adenine-ribose moiety of molecule. No additional reshaping of the dinucleotide binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8.In the crystallographic ADH8-NADP+ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly223-Thr224-His225, and the highly conserved Leu200 and Lys228. In order to investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (kcat/KmNADPH = 760 mM-1min-1), as compared to the wild-type enzyme (kcat/KmNADPH = 133330 mM-1min-1). This was associated with a lower affinity for NADP+ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (kcat/KmNADH = 155000 mM-1min-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.
3

Psychological and educational outcome of Very Low Birthweight children at 12yrs

Botting, Nicola Fay January 1997 (has links)
No description available.
4

Urinary antidiuretic hormone excretion during mechanical ventilation and weaning in man

Viquerat, Christian E. January 1979 (has links)
Thesis (doctoral)--University of Geneva, 1979. / "Thèse No. 3753." Includes bibliographical references (p. 17-19).
5

Análise da constituição genética de linhagens industriais da levedura Dekkera bruxellensis

LIBERAL, Anna Theresa de Souza 31 January 2010 (has links)
Made available in DSpace on 2014-06-12T18:02:04Z (GMT). No. of bitstreams: 2 arquivo767_1.pdf: 2982127 bytes, checksum: ec4ba15e127a34669d0fe0a656f86bc3 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / A levedura Dekkera bruxellensis vem se mostrando um importante microrganismo industrial, não apenas por causar eventos de contaminação em diversos processos fermentativos como também pela alta adaptação a esses processos. Este trabalho teve como objetivo principal identificar características genéticas básicas como composição genômica e cariotípica de linhagens industriais da levedura Dekkera bruxellensis a partir do uso de técnicas moleculares. Nesse trabalho, conseguimos visualizar o número e tamanho dos cromossomos das linhagens industriais de álcool combustível através da técnica de PFGE. Identificamos e analisamos estruturalmente os genes envolvidos na via do metabolismo central fermentativo através de busca no genoma seqüenciado, depositado no Banco de Dados do Projeto Dekkera bruxellensis. Analisamos a constituição genética e o status filogenético da D. bruxellensis em relação ao grupo ascomiceto através da análise dos genes piruvato descarboxilase (PDC) e álcool desidrogenase (ADH). Além disso, realizamos experimentos de expressão gênica por PCR em Tempo Real com os genes dbARO10 e dbADH em diversos meios com diferentes fontes de Carbono e Nitrogênio, verificando a resposta dessa levedura e analisando sua atividade enzimática para a fenilpiruvato descarboxilase e a álcool desidrogenase. Este foi um trabalho prospectivo, que fornece um painel inicial sobre a constituição genética desta levedura. Entretanto, algumas perguntas foram respondidas a partir dos resultados obtidos. Nos nossos trabalhos anteriores foi verificada a presença de dois padrões distintos de fingerprinting dos isolados de fermentação alcoólica. Esta variação de perfis refletiu-se nas variações cromossômicas, tanto em número quanto em tamanho. Pelas análises filogenéticas foi possível também, posicionar a D. bruxellensis dentro do grupo dos ascomicetos para os genes estudados
6

Development of a reporter system for the study of gene expression for solvent production in Clostridium beijerinckii NRRL B592 and Clostridium acetobutylicum ATCC 824

Li, Guang-Shan 11 December 1998 (has links)
To study the regulation of gene expression, a good reporter system is very useful. The lack of a good reporter system for the solvent-producing clostridia hindered the progress of research in this area. The objective of this study was to develop a reporter system to facilitate the elucidation of the control mechanism for the expression of solvent-producing genes. A potential reporter gene was found in Clostridium beijerinckii NRRL B593, which contains an adh gene encoding a primary-secondary alcohol dehydrogenase and this adh gene is not present in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NRRL B592. The adh gene was cloned into the E. coli -Clostridium shuttle vectors to generate plasmids. An electro-transformation procedure was developed for C. beijerinckii NRRL B592. Shuttle plasmids were transformed into C. beijerinckii NRRL B592 or C. acetobutylicum ATCC 824. The copy number of the plasmids in C. beijerinckii was 4. Isopropanol production suggested that the adh gene was expressed in transformants of C. acetobutylicum ATCC 824 and C. beijerinckii NRRL B592. Northern analysis indicated that the expression of the adh gene was regulated at the transcriptional level in the transformants of C. beijerinckii. The transcriptional start site for the adh gene was identified by the primer extension method. A promoter-probing vector was constructed and tested with the promoter from the ferredoxin(fer) gene. The expression of the adh gene under the control of the fer promoter was at a low and similar level during acidogenesis and solventogenesis. The expression pattern of the adh gene under the control of the promoter of the adh gene differed from that under the control of the promoter of the fer gene. / Ph. D.
7

Estudo sobre o polimorfismo dos genes álcool desidrogenase e aldeído desidrogenase (gene adh e aldh) e a dependência ao álcool em uma população da cidade de Goiânia-GO, Brasil / Polymorphism study of alcohol dehydrogenase and aldehyde dehydrogenase (adh and aldh) genes and the alcohol dependence in a population of Goiania, GO, Brazil

Teixeira, Thallita Monteiro 28 March 2016 (has links)
Submitted by Jaqueline Silva (jtas29@gmail.com) on 2017-01-04T16:37:13Z No. of bitstreams: 2 Dissertação - Thallita Monteiro Teixeira - 2016.pdf: 2002902 bytes, checksum: 6b37cff3f40bb0248879b27757987f0d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2017-01-04T16:37:24Z (GMT) No. of bitstreams: 2 Dissertação - Thallita Monteiro Teixeira - 2016.pdf: 2002902 bytes, checksum: 6b37cff3f40bb0248879b27757987f0d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-04T16:37:24Z (GMT). No. of bitstreams: 2 Dissertação - Thallita Monteiro Teixeira - 2016.pdf: 2002902 bytes, checksum: 6b37cff3f40bb0248879b27757987f0d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Alcoholism is a disease defined as a physical and / or mental condition, which characterized the need of the individual to ingest the substance constantly in order to experience its psychic effects or stop the withdrawal symptoms of the same. Heavy alcohol consumption brings many consequences for the health and quality of life by increasing the frequency of morbidity and mortality, causing several functional limitations. Worldwide, enormous resources are being invested in campaigns against alcohol abuse. WHO, for example, establishing a standard dose contains about 10 to 12 g of pure alcohol equivalent to a can of beer (350 mL) and draft (330 mL), a glass of wine (100ml), or distillate dose (30 mL). Some individuals metabolise alcohol better than others. The main ethanol elimination pathway occurs through oxidation of acetaldehyde to the same and then acid and water. These reactions are catalyzed by the enzyme alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). In this context, the scientific endeavor of this study to characterize the presence of polymorphisms of ADH and ALDH genes in alcohol users region of the state of Goiás, it is necessary to establish a possible correlation with alcoholism. This study was conducted in Goiás Alcoholics Recovery Center (GO CEREA) and Psychosocial Care Center Alcohol and Drugs (CAPS AD) both in Goiania. For the experiments, a sample of blood from each patient for DNA extraction was collected. Assays were performed by RT-PCR TaqMan SNP Genotyping Assays system. Statistical analyzes were made through GENEPOP 4.5 and Haploview software. Significant differences were found for SNPs studied in the analysis “Case vs. Control” and “Female vs. Male”. Furthermore, binding was observed moderate linkage disequilibrium between the ADH1B*2 and ADH1C*2 SNPs and the presence of haplotype by segregation dependent thereof. These results therefore suggest that these genetic variants may be associated with protection or addiction to alcoholism in the population studied in the city of Goiania, GO, Brazil. / O alcoolismo é uma doença definida como um estado físico e/ou psíquico onde se caracteriza a necessidade do indivíduo de ingerir a substância constantemente, no intuito de sentir seus efeitos psíquicos ou fazer cessar os sintomas da abstinência da mesma. O consumo abusivo de álcool traz inúmeras consequências para a saúde e qualidade de vida, aumentando a frequência de morbimortalidade, causando diversas limitações funcionais.Em todo o mundo, enormes recursos estão sendo investidos em campanhas contra o abuso do álcool.A OMS, por exemplo, estabelece que uma dose padrão contenha aproximadamente de 10 a 12 g de álcool puro, o equivalente a uma lata de cerveja (350 mL), ou chope (330 mL), uma taça de vinho (100 mL), ou uma dose de destilado (30 mL). Alguns indivíduos metabolizam o álcool melhor que outros. A principal via de eliminação do etanol ocorre por meio de oxidação do mesmo à acetaldeído e posteriormente em ácido e água. Estas reações são catalisadas pelas enzimas Álcool desidrogenase (ADH) e Aldeído desidrogenase (ALDH). Neste contexto, o empenho científico deste estudo para caracterizar a presença de polimorfismos nos genes ADH e ALDH nos usuários de álcool da região do Estado de Goiás, faz-se necessário para se estabelecer uma possível correlação com o alcoolismo. Este estudo foi realizado no Centro de Recuperação de Alcoólatras de Goiás – regional Goiânia (CEREA GO) e no Centro de Atenção Psicossocial Álcool e Drogas (CAPS AD). Para a realização dos experimentos, foi coletado uma amostra de sangue de cada paciente para extração de DNA. Os ensaios foram realizados através da PCR em Tempo Real pelo sistema TaqMAn® SNP Genotyping Assays. As análises estatísticas foram feitas através dossoftware GENEPOP 4.5 e Haploview.Foram encontradas diferenças significativas para os SNPs estudados nas análises Caso versus Controle e Feminino versus Masculino. Além disso, foi observada ligação moderada de Linkage disequilibrium entre os SNPs ADH1B*2 e ADH1C*2 e a presença de haplótipos através da segregação dependente destes. Estes resultados sugerem, portanto, que estas variantes genéticas possam estar associadas à proteção ou dependência ao alcoolismo na população estudada na cidade de Goiânia, GO, Brasil.
8

Estudo da bioeletrocatálise de oxidação de etanol utilizando extrato bruto de sementes de Helianthus annuus como fonte de álcool desidrogenase / Booeletrocatalysis study for ethanol oxidation on crude extract of seeds of Helianthus annuus as a source of alcohol dehydrogenase

Silva, Manoel Lucas da 11 November 2011 (has links)
Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-04T20:15:19Z No. of bitstreams: 2 Dissertação - Manoel Lucas da Silva - 2011.pdf: 1006184 bytes, checksum: 822278eea13e5a9617ab8622362a7f8e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-05T10:50:05Z (GMT) No. of bitstreams: 2 Dissertação - Manoel Lucas da Silva - 2011.pdf: 1006184 bytes, checksum: 822278eea13e5a9617ab8622362a7f8e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-05T10:50:05Z (GMT). No. of bitstreams: 2 Dissertação - Manoel Lucas da Silva - 2011.pdf: 1006184 bytes, checksum: 822278eea13e5a9617ab8622362a7f8e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-11-11 / Outros / In this work, the enzyme alcohol dehydrogenase (ADH), extracted from sunflower seeds, was used in the preparation of base electrodes of carbon paste. Thus studies have been conducted concerning the electrochemical properties and the catalytic ability of this enzyme for the conversion of ethanol into acetaldehyde, which leads to oxidation of the cofactor β-nicotidamina adenine dinucleotide (NADH). We constructed three working electrodes (A, B and C) for use in the detection of ethanol in phosphate buffer 0.1 mol L-1 (supporting electrolyte) at pH 7.4 and 8.0. Other variables in this study was the mode of extraction of the enzyme using deionized water or 0.1 mol L-1 phosphate buffer (pH 7.4) and form of the enzyme in carbon powder, were used for this mineral oil Nujol ® and Teflon ® solutions and glutaraldehyde. The cyclic voltammetry and chronoamperometry were the two methods used to verify the action of the enzyme in solution. The results showed that the electrodes A and B have inferior performance compared with the electrode C. Finally, electrochemical experiments showed that the enzyme alcohol dehydrogenase derived from crude extract of sunflower seeds has catalytic activity for oxidation of ethanol, but the enzyme does not maintain this constant activity, probably due to the absence of the cofactor which can be in the crude extract in a very low concentration, acting as a limiting reagent in the reaction. / Neste trabalho, a enzima álcool desidrogenase (ADH), extraída das sementes de girassol, foi utilizada para a elaboração de eletrodos a base de pasta de carbono. Assim, foram realizados estudos a respeito das propriedades eletroquímicas e da capacidade catalítica desta enzima para a conversão do etanol em acetaldeído, que tem como consequência a oxidação do cofator β-nicotidamina adenina dinucleotídeo (NADH). Foram construídos três eletrodos de trabalho (A, B e C) para serem utilizados na detecção de etanol em solução tampão fosfato 0,1 mol L-1 (eletrólito de suporte) em pH 7,4 e 8,0. Outras variáveis deste estudo foram o modo de extração da enzima utilizando água deionizada ou tampão fosfato 0,1 mol L-1 (pH 7,4) e a forma de aglutinação da enzima no pó de carbono, para isso foram utilizados o óleo mineral Nujol® e soluções de Teflon® e glutaraldeído. As técnicas de voltametria cíclica e cronoamperometria foram os dois métodos utilizados para verificar a ação da enzima em solução. Os resultados mostraram que os eletrodos A e B tiveram o desempenho inferior em comparação com o eletrodo C. Por fim, os experimentos eletroquímicos mostraram que a enzima álcool desidrogenase derivada do extrato bruto das sementes de girassol tem atividade bioeletrocatalítica para a oxidação do etanol, mas a enzima não mantém essa atividade constante, provavelmente devido à ausência do cofator que no extrato bruto possa estar em uma concentração muito baixa, agindo como reagente limitante da reação.
9

Prevention of Cardiometabolic Disease in Familial Hypercholesterolemia

Awan, Zuhier 11 1900 (has links)
L’hypercholestérolémie familiale (FH) est un désordre lipidique associé aux maladies cardiovasculaires les plus fréquentes. La FH est causée par des mutations dans les gènes LDLR, APOB et PCSK9. Toutefois, chez 20% des patients souffrant de FH, aucune mutation dans ces gènes n'a été détectée et ceci suggère que d’autres gènes seraient à l’origine de la FH. Actuellement, le seul traitement de la FH est une thérapie aux statines. En général les statines sont bien tolérées, cependant, une monothérapie ne permet pas d’atteindre des niveaux thérapeutiques acceptables et dans bien des cas, une thérapie combinée devient nécessaire. De plus, l’intolérance aux statines est présente dans environ 12% des patients. Dans les trois dernières décennies, la survie des patients avec la FH a augmentée de façon notoire mais on observe aussi l’apparition d’une calcification vasculaire sévère chez certains d’entre eux. Il est donc primordial de développer des nouvelles approches thérapeutiques afin de prévenir ces complications tardives. Dans cette thèse doctorat, nous présentons l’étude d’une famille avec un phénotype de FH sévère non causé par des mutations dans les gènes LDLR, APOB et PCSK9. Par des études biochimiques et par séquençage d’ADN utilisant les technologies de nouvelle génération (NextGenSeq), nous avons découvert une mutation dans le gène de l’APOE (Leu167del). Ceci nous permet de proposer le gène codant pour l’APOE comme le 4e locus responsable de la FH (FH4). Par la suite, nous avons effectué deux études de cohortes chez les patients atteints de FH. Premièrement, dans l’étude JUPITER, nous avons démontré que la rosuvastatin augmente les niveaux sanguins de la protéine PCSK9 et ceci limiterait l’efficacité du traitement aux statines. Nous avons aussi étudié l’influence du mutant naturel R46L (perte de fonction de la PCSK9) dans la réponse aux statines. Deuxièmement, nous avons examiné les effets de la perte de fonction de la PCSK9 sur le profil cardiométabolique au sein d’une population pédiatrique. Nous avons déterminé que le génotype de l’APOE est déterminant dans ce profil cardiométabolique. Enfin, nous avons étudié la calcification vasculaire chez les patients atteints de FH. Cette calcification vasculaire progresse de façon indépendante des niveaux de cholestérol sérique et n’est pas associée aux anomalies de l’homéostasie du calcium. En utilisant des modèles murins, nous avons démontré que les souris Ldlr-/- et Tg(Pcsk9) développent des calcifications vasculaires semblables à celles observées chez l’homme. De plus, nous avons confirmé l’implication de la voie de signalisation LRP5/Wnt dans la pathophysiologie de la calcification artérielle. Avec une étude interventionnelle, nous avons trouvé que l’inhibition de l’interleukine 1β (IL-1β) diminue fortement l’apparition de calcifications vasculaire dans notre modèle murin. En conclusion, nos études ont permis l’identification d’un nouveau gène impliqué dans la FH, ont démontré aussi que les statines augmentent les niveaux sériques de PCSK9 et que la perte de fonction de la PCSK9 altère le profil cardiométabolique. Enfin, nous avons établi que la calcification vasculaire représente une complication tardive chez les patients atteints de FH et que, dans notre modèle murin, la calcification vasculaire peut être retardée par l’inhibition d’IL-1β. Ces découvertes peuvent avoir d’importantes répercussions cliniques chez l’humain. / Familial Hypercholesterolemia (FH) is the most common lipoprotein disorder associated with premature cardiovascular disease. Mutations in the LDLR, APOB and PCSK9 genes cause the FH phenotype, but in 20% of FH patients, no mutations in these genes are identified, suggesting that mutations in other genes cause FH. Treatment with statins has been the cornerstone of therapy. While statins are generally well tolerated, statin intolerance is found in approximately 12% of patients. Furthermore, statin use may not allow reaching LDL-C goals and combination therapy is often required. Nevertheless, survival of FH patients over the past 3 decades has improved significantly. As FH patients live longer, severe vascular calcifications have been described as a late complication in these patients. Given the increased survival rate and late complications, novel approaches and therapies are needed. In the present thesis we examined a kindred with a severe FH phenotype, where sequencing of candidate genes failed to identify a causal mutation. Through biochemical analysis and next-generation exome sequencing we report a mutation (Leu167del) within the APOE gene that identifies the 4th locus causing FH (FH4). Next, we performed two cohort-based studies. Firstly, in the JUPITER trial we report that 20mg rosuvastatin treatment increases PCSK9 levels by 30%, thereby possibly limiting the efficacy of statin therapy. Then we show the effect of a loss-of-function (LOF) mutation of PCSK9, p.R46L, on the response to rosuvastatin. Secondly, we report that two PCSK9 gene variants, p.R46L and insLEU, were more frequent in French Canadian individuals. We also report that the APOE genotype determine the metabolic risk profile in these mutations. Finally, we studied vascular calcifications in FH individuals. These calcifications appear to progress independently of cholesterol levels and are not associated with disturbances in calcium homeostasis. Using mouse models, we show that Ldlr-/- and Tg(Pcsk9) mice develop aortic calcifications similar to that observed in humans. Furthermore, the involvement of the LRP5/Wnt pathway in the pathogenesis of calcification is illustrated. In a proof-of-concept experiment, inhibiting the upstream pro-inflammatory cytokine IL-1β attenuates calcification in mice. In conclusion, we have contributed to the identification of a novel locus responsible for FH, reported the increase in PCSK9 levels with a statins treatment and the associated altered cardiometabolic profile in PCSK9 LOF. Finally, we demonstrated that vascular calcifications represent a severe complication of FH that can be prevented by inhibiting IL-1β in a mouse model. The latter novel approach may have an important translational application in human.
10

Prevention of Cardiometabolic Disease in Familial Hypercholesterolemia

Awan, Zuhier 11 1900 (has links)
L’hypercholestérolémie familiale (FH) est un désordre lipidique associé aux maladies cardiovasculaires les plus fréquentes. La FH est causée par des mutations dans les gènes LDLR, APOB et PCSK9. Toutefois, chez 20% des patients souffrant de FH, aucune mutation dans ces gènes n'a été détectée et ceci suggère que d’autres gènes seraient à l’origine de la FH. Actuellement, le seul traitement de la FH est une thérapie aux statines. En général les statines sont bien tolérées, cependant, une monothérapie ne permet pas d’atteindre des niveaux thérapeutiques acceptables et dans bien des cas, une thérapie combinée devient nécessaire. De plus, l’intolérance aux statines est présente dans environ 12% des patients. Dans les trois dernières décennies, la survie des patients avec la FH a augmentée de façon notoire mais on observe aussi l’apparition d’une calcification vasculaire sévère chez certains d’entre eux. Il est donc primordial de développer des nouvelles approches thérapeutiques afin de prévenir ces complications tardives. Dans cette thèse doctorat, nous présentons l’étude d’une famille avec un phénotype de FH sévère non causé par des mutations dans les gènes LDLR, APOB et PCSK9. Par des études biochimiques et par séquençage d’ADN utilisant les technologies de nouvelle génération (NextGenSeq), nous avons découvert une mutation dans le gène de l’APOE (Leu167del). Ceci nous permet de proposer le gène codant pour l’APOE comme le 4e locus responsable de la FH (FH4). Par la suite, nous avons effectué deux études de cohortes chez les patients atteints de FH. Premièrement, dans l’étude JUPITER, nous avons démontré que la rosuvastatin augmente les niveaux sanguins de la protéine PCSK9 et ceci limiterait l’efficacité du traitement aux statines. Nous avons aussi étudié l’influence du mutant naturel R46L (perte de fonction de la PCSK9) dans la réponse aux statines. Deuxièmement, nous avons examiné les effets de la perte de fonction de la PCSK9 sur le profil cardiométabolique au sein d’une population pédiatrique. Nous avons déterminé que le génotype de l’APOE est déterminant dans ce profil cardiométabolique. Enfin, nous avons étudié la calcification vasculaire chez les patients atteints de FH. Cette calcification vasculaire progresse de façon indépendante des niveaux de cholestérol sérique et n’est pas associée aux anomalies de l’homéostasie du calcium. En utilisant des modèles murins, nous avons démontré que les souris Ldlr-/- et Tg(Pcsk9) développent des calcifications vasculaires semblables à celles observées chez l’homme. De plus, nous avons confirmé l’implication de la voie de signalisation LRP5/Wnt dans la pathophysiologie de la calcification artérielle. Avec une étude interventionnelle, nous avons trouvé que l’inhibition de l’interleukine 1β (IL-1β) diminue fortement l’apparition de calcifications vasculaire dans notre modèle murin. En conclusion, nos études ont permis l’identification d’un nouveau gène impliqué dans la FH, ont démontré aussi que les statines augmentent les niveaux sériques de PCSK9 et que la perte de fonction de la PCSK9 altère le profil cardiométabolique. Enfin, nous avons établi que la calcification vasculaire représente une complication tardive chez les patients atteints de FH et que, dans notre modèle murin, la calcification vasculaire peut être retardée par l’inhibition d’IL-1β. Ces découvertes peuvent avoir d’importantes répercussions cliniques chez l’humain. / Familial Hypercholesterolemia (FH) is the most common lipoprotein disorder associated with premature cardiovascular disease. Mutations in the LDLR, APOB and PCSK9 genes cause the FH phenotype, but in 20% of FH patients, no mutations in these genes are identified, suggesting that mutations in other genes cause FH. Treatment with statins has been the cornerstone of therapy. While statins are generally well tolerated, statin intolerance is found in approximately 12% of patients. Furthermore, statin use may not allow reaching LDL-C goals and combination therapy is often required. Nevertheless, survival of FH patients over the past 3 decades has improved significantly. As FH patients live longer, severe vascular calcifications have been described as a late complication in these patients. Given the increased survival rate and late complications, novel approaches and therapies are needed. In the present thesis we examined a kindred with a severe FH phenotype, where sequencing of candidate genes failed to identify a causal mutation. Through biochemical analysis and next-generation exome sequencing we report a mutation (Leu167del) within the APOE gene that identifies the 4th locus causing FH (FH4). Next, we performed two cohort-based studies. Firstly, in the JUPITER trial we report that 20mg rosuvastatin treatment increases PCSK9 levels by 30%, thereby possibly limiting the efficacy of statin therapy. Then we show the effect of a loss-of-function (LOF) mutation of PCSK9, p.R46L, on the response to rosuvastatin. Secondly, we report that two PCSK9 gene variants, p.R46L and insLEU, were more frequent in French Canadian individuals. We also report that the APOE genotype determine the metabolic risk profile in these mutations. Finally, we studied vascular calcifications in FH individuals. These calcifications appear to progress independently of cholesterol levels and are not associated with disturbances in calcium homeostasis. Using mouse models, we show that Ldlr-/- and Tg(Pcsk9) mice develop aortic calcifications similar to that observed in humans. Furthermore, the involvement of the LRP5/Wnt pathway in the pathogenesis of calcification is illustrated. In a proof-of-concept experiment, inhibiting the upstream pro-inflammatory cytokine IL-1β attenuates calcification in mice. In conclusion, we have contributed to the identification of a novel locus responsible for FH, reported the increase in PCSK9 levels with a statins treatment and the associated altered cardiometabolic profile in PCSK9 LOF. Finally, we demonstrated that vascular calcifications represent a severe complication of FH that can be prevented by inhibiting IL-1β in a mouse model. The latter novel approach may have an important translational application in human.

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