No description available.

No description available.

No description available.

This study set out to express, purify and characterize twelve ammonia lyase enzymes for potential application as a supplement to a treatment of an inborn error of metabolism disease. The DNA sequence for two wild-type ammonia lyases, three modified ammonia lyases and seven resurrected ancestral ammonia lyases had been synthesized and cloned in vectors. These were transformed into Escherichia coli, expressed, purified using immobilized metal affinity chromatography and size exclusion chromatography and characterized. Ten of the enzymes were successfully expressed and purified. All enzymes had a higher turnover number with substrate 1 than with substrate 2. The wild-types showed the highest catalytic turnover and one of them displayed substrate cooperativity. The modified enzymes were inactive. Some ancestral enzymes were active and had decreasing kcat with age. A promising ancestral enzymes was found that showed a kcat of 2,85 s-1 with substrate 1 and 1,82 s-1 with substrate 2. The ancestral enzymes had a lower Km with substrate 2 compared to substrate 1, while one of the wild-types had a higher Km with substrate 2 than with substrate 1, indicating that the substrate affinity has switched. The ancestral enzymes had increased thermostability compared to the wild-types which increased with age. Ranging from a +7C increase in melting temperature with the youngest ancestral enzyme to +10,7C with the oldest tested enzyme, comparing with one of the wild-types. The promising ancestral enzyme displayed a higher stability than the wild-types during long term incubation in 37_C and 25_C, since it did not become prone to aggregation,it did not show visible degradation on SDS-PAGE and it retained the highest activity following incubation. It was also demonstrated that neither wild-types nor the promising ancestral enzyme were stable in a simulated gut environment. The promising ancestral enzyme and one of the wild-types degraded substrate 1 and 2 in serum. Using the resurrection of ancestral sequences a promising enzyme has been produced and characterized, displaying properties that are desired in therapeutic enzymes. The enzyme did not aggregate or become prone to aggregation over time, it was thermostable, it was active in serum and had acceptable catalytic properties. For therapeutic application of the ancestral enzyme, immunogenicty should be analyzed in silico and in vitro followed by further investigation in vivo. / Målet med denna studie var att uttrycka, rena och karaktärisera tolv ammonia lyase enzymer, för potentiell användning som komplement till en behandling utav en sjukdom, som tillhör sjukdomsgruppen medfödda ämnesomsättningsrubbningar. DNA sekvensen för två vild-typammonia lyaser, tre modifierade ammonia lyaser och sju återuppväckta ammonia lyaser hade blivit syntetiserade och klonade i vektorer. E.coli celler blev transformerade med vektorerna, vilka uttryckte enzymerna, som renades med hjälp av immobilized metal affinity chromatography och gelfiltrering och karaktäriserades. Tio utav enzymerna kunde uttryckas och renas. Alla enzymer hade högre katalytisk omsättning av substrat 1 än substrat 2. Vildtyperna hade högst kcat med båda substrat och en utav dem uppvisade substratsammarbete. De modifierade enzymerna var inaktiva. Några av de återuppväckta ammonia lyaserna var aktiva och kcat minskade med ålder. Ett av de återuppväckta enzymerna var lovande och hade ett kcat värde av 2,85 s-1 med substrat 1 och 1,82 s-1 med substrat 2. De återuppväckta enzymerna hade ett lägre Km värde för substrat 2 än substrat 1, jämfört med en utav vildtyperna som hade ett högre Km värde för substrat 2 än substrat 1, vilket indikerar ett skifte i substrataffinitet. De återuppväckta enzymerna var mer termostabilia än vild-typerna och termostabiliteten ökar med ålder. Ökningen i smälttemperatur låg i spannet av +7C för de yngsta återuppväckta enzymerna till + 10,7C för det äldsta testade återuppväckta enzymet, vid jämförelse med en utav vild-typerna. Det lovande återuppväckta enzymet demonstrerade även en högre stabilitet än vild-typerna under långtidsinkubering, eftersom den inte blev benägen att aggregera, den uppvisade ingen nedbrytning på SDS-PAGE och den behöll högst aktivitet efter inkubering. Det bevisades även att varken vild-typerna eller det lovande återuppväckta enzymet var stabila i en simulerad magsäcksmiljö. Både det lovande återuppväckta enzymet och en av vild-typerna bröt ner substrat 1 och 2 i serum. Genom att återuppväcka sekvenser kunde ett lovande enzym produceras och karaktäriseras, vilket uppvisade egenskaper som är eftertraktade i terapeutiska enzymer. Enzymet aggregerade ej, det blev inte benäget att aggregera över tid, det var termostabilt, det var aktivt i serum och hade acceptabla katalytiska egenskaper. För terapeutisk applikation av det återuppväckta enzymet, borde analys av dess immunogenicitet utföras in silico och in vitro följt av vidare undersökning in vivo.

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No description available.

No description available.

Introdução: A enzima 3-hidroxiacil CoA desidrogenase de cadeia longa (LCHAD) é uma das enzimas envolvidas na beta-oxidação mitocondrial de ácidos graxos e faz parte do complexo enzimático chamado proteína trifuncional. Sua deficiência segue um modelo de herança autossômica recessiva com uma mortalidade maior que 70%, porém um tratamento dietoterápico adequado reduz substancialmente a morbimortalidade. Estudos recentes descreveram que gestantes de fetos homozigóticos para defeitos de LCHAD apresentam grandes chances de desenvolver síndrome HELLP e doença hepática aguda da gestação, com risco de morte materna, fetal e do recém-nascido. A síndrome HELLP é caracterizada por plaquetopenia, enzimas hepáticas elevadas e hemólise, podendo se apresentar na forma completa ou parcial. Estudos mostram que investigar a relação entre síndrome HELLP e defeitos de LCHAD, possibilita a prevenção de futuras gestações de risco por meio de aconselhamento genético e o diagnóstico precoce deste erro inato do metabolismo. Objetivos: Verificar a associação entre gestantes com síndrome HELLP e lactentes com defeitos LCHAD e identificar problemas de saúde gerados pela doença nesses conceptos. Metodologia: Análise de prontuários, necropsias e desfecho atual dos conceptos de 42 gestantes com síndrome HELLP e 84 controles. Resultados: Entre as gestantes que compunham os casos, a maioria apresentou proteinúria; além de sintomas como vômitos e epigastralgia. O parto cesárea foi realizado em 93% das gestantes. Quase metade das puérperas apresentou algum tipo de complicação materna. Quanto ao desfecho perinatal, 90% dos conceptos apresentaram baixo peso ao nascer e 23,8% evoluíram para óbito. Entre esses 10 óbitos, resgatamos 7 imagens histopatológicas com esteatose hepática. Inferimos doença metabólica nesses casos, que levou a uma associação de 11% com a síndrome HELLP. Entre o grupo controle, 46,2% das mulheres já haviam sofrido pelo menos um aborto. Na atual gestação 6,4% desenvolveram pré-eclampsia; entre outras complicações. Encontramos gravidezes subsequentes das gestantes do grupo Caso com recorrência de HELLP e óbito. Conclusão: Os resultados reforçam a importância do diagnóstico precoce de síndrome HELLP, além da investigação da associação do defeito de LCHAD e HELLP mesmo post morten afim de evitar futuras gestações de risco e diminuir a morbimortalidade materna e neonatal. / Long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) is one of the enzymes involved in the mitochondrial fatty acids beta-oxidation and part of the enzymatic complex called trifunctional protein. Its deficiency follows an autosomal recessive model with a higher mortality, but an adequate dietary treatment reduces its morbimortality. Recent studies reported that mothers of fetuses homozygous for LCHAD deficiency have higher chances of developing HELLP and acute fatty liver of pregnancy with risk of maternal, infant and fetal death. Thrombocytopenia, elevated liver enzymes and hemolysis characterize HELLP syndrome, which can be diagnosed as complete or partial. Studies demonstrate that investigate the association between HELLP syndrome and LCHAD defects can prevent future risk pregnancies through genetic counseling and early diagnosis of this inborn error of metabolism. Objectives: To investigate the association between pregnant women and concepts with LCHAD deficiency and identify health problems caused by the disease in these fetuses. Methodology: Analysis of medical records, autopsy reports and current outcome of fetuses of 42 pregnant women with HELLP syndrome and 84 controls. Results: In case group, most patients presented proteinuria; as well as symptoms as vomiting and epigastric pain. The cesarean delivery was performed in 93% of pregnant women. Almost half of women presented maternal complications. In perinatal outcome, 90% of fetuses has low weight at birth and 23.8% died. Among these 10 deaths, we rescued 7 hystopathological images with hepatic steatosys. We could infer metabolic disease in these cases, which led to an association of 11% to the HELLP syndrome. Among the control group, 46.2% of women had at least one abortion before this pregnancy. During the pregnancy 6.4% developed pre-eclampsia among other complications. In control group, we find HELLP syndrome recurrence and death in subsequent pregnancy. Conclusion: The results reinforce the importance of early diagnosis of HELLP syndrome, as well as research LCHAD and HELLP association even post mortem to avoid future risk pregnancies and reduce maternal and neonatal morbidity and mortality.

Introdução: A enzima 3-hidroxiacil CoA desidrogenase de cadeia longa (LCHAD) é uma das enzimas envolvidas na beta-oxidação mitocondrial de ácidos graxos e faz parte do complexo enzimático chamado proteína trifuncional. Sua deficiência segue um modelo de herança autossômica recessiva com uma mortalidade maior que 70%, porém um tratamento dietoterápico adequado reduz substancialmente a morbimortalidade. Estudos recentes descreveram que gestantes de fetos homozigóticos para defeitos de LCHAD apresentam grandes chances de desenvolver síndrome HELLP e doença hepática aguda da gestação, com risco de morte materna, fetal e do recém-nascido. A síndrome HELLP é caracterizada por plaquetopenia, enzimas hepáticas elevadas e hemólise, podendo se apresentar na forma completa ou parcial. Estudos mostram que investigar a relação entre síndrome HELLP e defeitos de LCHAD, possibilita a prevenção de futuras gestações de risco por meio de aconselhamento genético e o diagnóstico precoce deste erro inato do metabolismo. Objetivos: Verificar a associação entre gestantes com síndrome HELLP e lactentes com defeitos LCHAD e identificar problemas de saúde gerados pela doença nesses conceptos. Metodologia: Análise de prontuários, necropsias e desfecho atual dos conceptos de 42 gestantes com síndrome HELLP e 84 controles. Resultados: Entre as gestantes que compunham os casos, a maioria apresentou proteinúria; além de sintomas como vômitos e epigastralgia. O parto cesárea foi realizado em 93% das gestantes. Quase metade das puérperas apresentou algum tipo de complicação materna. Quanto ao desfecho perinatal, 90% dos conceptos apresentaram baixo peso ao nascer e 23,8% evoluíram para óbito. Entre esses 10 óbitos, resgatamos 7 imagens histopatológicas com esteatose hepática. Inferimos doença metabólica nesses casos, que levou a uma associação de 11% com a síndrome HELLP. Entre o grupo controle, 46,2% das mulheres já haviam sofrido pelo menos um aborto. Na atual gestação 6,4% desenvolveram pré-eclampsia; entre outras complicações. Encontramos gravidezes subsequentes das gestantes do grupo Caso com recorrência de HELLP e óbito. Conclusão: Os resultados reforçam a importância do diagnóstico precoce de síndrome HELLP, além da investigação da associação do defeito de LCHAD e HELLP mesmo post morten afim de evitar futuras gestações de risco e diminuir a morbimortalidade materna e neonatal. / Long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) is one of the enzymes involved in the mitochondrial fatty acids beta-oxidation and part of the enzymatic complex called trifunctional protein. Its deficiency follows an autosomal recessive model with a higher mortality, but an adequate dietary treatment reduces its morbimortality. Recent studies reported that mothers of fetuses homozygous for LCHAD deficiency have higher chances of developing HELLP and acute fatty liver of pregnancy with risk of maternal, infant and fetal death. Thrombocytopenia, elevated liver enzymes and hemolysis characterize HELLP syndrome, which can be diagnosed as complete or partial. Studies demonstrate that investigate the association between HELLP syndrome and LCHAD defects can prevent future risk pregnancies through genetic counseling and early diagnosis of this inborn error of metabolism. Objectives: To investigate the association between pregnant women and concepts with LCHAD deficiency and identify health problems caused by the disease in these fetuses. Methodology: Analysis of medical records, autopsy reports and current outcome of fetuses of 42 pregnant women with HELLP syndrome and 84 controls. Results: In case group, most patients presented proteinuria; as well as symptoms as vomiting and epigastric pain. The cesarean delivery was performed in 93% of pregnant women. Almost half of women presented maternal complications. In perinatal outcome, 90% of fetuses has low weight at birth and 23.8% died. Among these 10 deaths, we rescued 7 hystopathological images with hepatic steatosys. We could infer metabolic disease in these cases, which led to an association of 11% to the HELLP syndrome. Among the control group, 46.2% of women had at least one abortion before this pregnancy. During the pregnancy 6.4% developed pre-eclampsia among other complications. In control group, we find HELLP syndrome recurrence and death in subsequent pregnancy. Conclusion: The results reinforce the importance of early diagnosis of HELLP syndrome, as well as research LCHAD and HELLP association even post mortem to avoid future risk pregnancies and reduce maternal and neonatal morbidity and mortality.

Copies of author's previously published articles inserted / Includes bibliographic references / 2 v. (various foliations) : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Summary: Studies the enzymology of some of the nine lysosomal exo-enzyme activities which act together to degrade the more highly sulphated regions of the glycosaminoglycans heparin and heparan sulphate. A deficiency of any one of these enzyme activities can result in one of the lysosomal storage disorders collectively known as the Mucopolysaccharidoses (MPS) / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1991