Tesis: "Inborn errors of"

1

Ristoff, Ellinor. "Inborn errors in the metabolism of glutathione /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-392-9/.

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2

Pastore, Nunzia. "Gene therapy for inborn errors of metabolism." Thesis, Open University, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590807.

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Inborn errors of liver metabolism are frequent causes of morbidity and mortality especially in children. For several of these diseases, treatment approaches depend on ,manipulation of the affected metabolic pathway by diet, drugs, vitamin cofactors, enzyme induction, end-product replacement, and alternative pathway activation. Unfortunately, these approaches often remain unsatisfactory especially in the face of illness or catabolism. Ideally, transfer of the normal genes in the liver cells that are defective might restore the metabolic function. The goal of my PhD thesis was to develop gene-based therapeutic strategies to correct a life-threatening inborn error of liver metabolism, Crigler-Najjar syndrome type I (CNI). CNI is a severe inborn error of bilirubin metabolism due to mutations of the uridine diphospho-glucuronosyl transferase lAl (UGTIA1) gene, Affected patients have elevations of serum bilirubin, and they have to spend extended hours under bilirubin lights throughout childhood and adolescence. Despite this therapy, they remain at risk of brain damage when intercurrent infections may increase production of bilirubin above that which can be controlled by the bilirubin light therapy. Thus, patients with CNI often are advised to consider liver transplantation. Therefore, alternative therapies are highly needed to overcome the mortality and morbidity associated with transplantation procedure, and risks of life-long immunosuppression. Gene therapy has the potential to provide a definitive cure for patient with CNI My studies have focused on the development of gene therapy strategies for this disease. First, I investigated in Gunn rats, the animal model for CNI, the efficacy of adeno-associated viral (AA V) vector-mediated muscledirected gene therapy and I found that serotype I AA V vector expressing UOTIAI resulted in expression of UGTIAl protein and functionally active enzyme in injected muscles, and aj 50% reduction in serum bilirubin levels for at least 1 year post-injection. Taken together, these data show that clinically relevant and sustained reduction of serum bilirubin levels can be achieved by simple and safe intramuscular injections. Following initial problems with intravenous injections of AA V2 vector, a major success has been achieved with AA V2/8 vectors for liver-directed gene therapy of hemophilia. Encouraged by these results and by the possibility of achieving full correction of the hyperbilirubineotia with systemic delivery, next I focused on the design and optimization of an AA V2I8 vector for liver-directed gene therapy of CNI. I generated multiple expression cassettes expressing the UGTIAl gene inserted into the AA V2/8 vectors for in vivo testing. The results of these studies showed that AAV2/8 vector with codon optimized UGTlAI gene tender the control of the hepatocyte-specific LP} promoter resulted in improved and sustained correction of hyperbilirubinemia in Gunn rats. Taken together, these data demonstrate the development of an optimal expression cassette for liver-directed gene therapy of CNI and form the preclinical basis for the development of a gene therapy trial for this severe disorder.

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3

Kocic, Vesna Garovic. "Methionine auxotrophy in inborn errors of cobalamin metabolism." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=56756.

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Several of the inborn errors of vitamin B$ sb{12}$ (cobalamin, Cbl) metabolism (cblC, cblD, cblE, cblF, cblG) are associated with homocystinuria and hypomethioninemia due to a functional deficiency of the cytoplasmic enzyme methionine synthase which requires methylcobalamin (MeCbl) as a cofactor. We compared the growth of cultured fibroblasts from controls with those from patients with a selective deficiency of MeCbl (cblE and cblG) and with those from patients with a defect in both MeCbl and adenosylcobalamin (AdoCbl) (cblC, cblD and cblF). Cells were grown in methionine and folic acid free media and in fully supplemented medium. Control cells were able to grow in the deficient medium supplied with homocysteine, cobalamin and folate, while mutant cells were not, due to their inability to synthesize methionine from its immediate metabolic precursor, homocysteine. This differential growth is useful for screening for genetic defects of methionine biosynthesis. Moreover, by correcting methionine auxotrophy in these cells, it may be possible to isolate genes which code for the products that are deficient in these disorders.

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4

Byck, Susan. "Cross-correctional studies in inborn errors of vitamin B12 metabolism." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59259.

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Human skin fibroblasts derived from patients with all 7 known inborn errors of vitamin B$ sb{12}$ metabolism have been studied for functional integrity of methylmalonyl CoA mutase and methionine synthase. Cocultivation of cblC and cblF fibroblasts in the absence of polyethylene glycol resulted in a twofold increase over the expected in both ($ sp{14}$C) propionate and ($ sp{14}$C) methyltetrahydrofolate incorporation into acid-precipitable material, suggesting that metabolic cooperation between cells occurs. CblD fibroblasts, which are biochemically similar to cblC cells (Goodman et al, 1970; Willard et al, 1977), do not cooperate metabolically when mixed with cblF cells. Partial correction in phenotype was seen in mixtures of cblD and cblG cells, but not cblC and cblG cells. These observations lend further support for the division of cblC and cblD disease into two discrete complementation classes. Cocultivation of cblF fibroblasts with both cblE and cblG cells also resulted in partial correction in phenotype. ($ sp{14}$C) Propionate incorporation in both cblC and cblF cells exposed to conditioned medium from control cells was increased more than twofold. ($ sp{14}$C) methyltetrahydrofolate incorporation in cblC cells exposed to conditioned medium from cblF cells was increased twofold. This suggests the presence of a diffusible factor correcting the defect in the mutant cell lines.

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5

Black, Duncan Arthur. "Aspects of purine and pyrimidine metabolism." Doctoral thesis, University of Cape Town, 1989. http://hdl.handle.net/11427/26590.

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In Chapter 1 a review of the literature concerning aspects of erythrocyte membrane transport and metabolism, and purine and pyrimidine metabolism is presented. The effects of pH, pO₂ and inorganic phosphate (Pi) on the uptake and metabolism of hypoxanthine by erythrocytes has been studied in Chapter 2. Uptake of hypoxanthine and accumulation of inosine 5'-monophosphate (IMP) were markedly increased at acid pH, high external phosphate concentrations, and low pO₂. Release of accumulated IMP as hypoxanthine occurred at alkaline pH values and low external phosphate concentrations. Conditions favouring IMP accumulation gave rise, in the absence of hypoxanthine, to a corresponding increase in 5'-phosphoribosyl-1-pyrophosphate (PRPP). Intracellular phosphate concentrations were markedly pH dependent and a model is presented whereby hypoxanthine uptake and release are controlled by intracellular concentrations of inorganic phosphate and 2,3- bisphosphoglycerate (2,3-DPG). These allosteric effectors influence, in opposing ways, two enzymes governing IMP accumulation, namely PRPP synthetase and 5'-nucleotidase. These metabolic properties suggest that the erythrocyte could play a role in the removal of hypoxanthine from anoxic tissue. In Chapter 3 the kinetics and mechanism of transport of orotate across the human erythrocyte membrane and the effect of pH and inorganic phosphate on its metabolism (in the erythrocyte) have been studied. It has been shown that orotate enters erythrocytes with non-saturable kinetics and with a capacity of 190 μmoles/1 packed cells/min at a concentration of 4-6 mmolar. The presence of competition for transport by a number of anions and the lack of competition by uridine is indicative of transport by a general anion transporter, with the ability for concentrative uptake in the absence of other external anions being compatible with transport via a ping-pong mechanism. Inhibition of transport by the specific band 3 inhibitors DIDS and CHCA confirm that transport is via the band 3 anion transporter. This explains the lack of significant uptake of orotate by most differentiated tissues which lack the intact band 3 protein. However, the demonstration of band 3 in rat hepatocytes (Cheng and Levy, 1980) provides a mechanism for the orotate transport which has been observed in liver (Handschumacher and Coleridge, 1979). Changes in pH and inorganic phosphate (Pi) concentrations have been shown to have marked effects on the relative quantities of metabolic products produced by the erythrocyte from orotate. There was an increase in orotate metabolised with increasing Pi, an effect augmented by lowering the pH, and most easily explained by the allosteric activation of PRPP synthetase by Pi. The increase in UTP levels with decreasing pH may be the consequence of both increased PRPP availability for the formation of uridine nucleotide from orotate, and decreased conversion of UMP to uridine by pyrimidine 5'-nucleotidase, which is known to be inhibited by phosphate. The accumulation of UDP sugars is optimal at a phosphate concentration of 10 mmolar, which is unexplained but would be compatible with an inhibitory effect of Pi on CTP synthetase. A PRPP wasting cycle at alkaline pH values is proposed to explain the apparent paradox where no PRPP was observed to accumulate in erythrocytes (Chapter 2) at pH values of 7.6 and above in the presence of 10 mmolar phosphate and no added hypoxanthine, yet the metabolism of orotate, which is a PRPP utilising reaction, at alkaline pH values was readily demonstrable here. This (apparent paradox) can be resolved if one assumes that even in the absence of added hypoxanthine and demonstrable intracellular IMP there are sufficient quantities of hypoxanthine and/or IMP to maintain a PRPP wasting cycle at alkaline pH values. The cycle is interrupted at acidic pH values as phosphate levels rise and inhibit 5'-nucleotidase, an effect augmented by the decreasing levels of 2,3-DPG which accompany decreasing pH. This wasting cycle has recently been confirmed by P. Berman (unpublished). The kinetics of orotate uptake by erythrocytes and its eventual release as uridine provides a role for the erythrocyte in the transport and distribution of pyrimidines to peripheral tissues. A model is proposed and involves the de novo production of orotate in the liver. In the next step erythrocytes take up the orotate secreted by the liver into the circulation, convert it into an intermediate buffer store of uridine nucleotides, whose distribution is a function of pH and phosphate concentration, and eventually release it as uridine, which is a readily available form of pyrimidine for utilisation by peripheral nucleated cells. The enhancement of uptake of labelled orotate into nucleic acids of cultured cells is demonstrated here. The degradative half of the cycle proposes that uracil and palanine are the predominant degradative forms of pyrimidines produced by peripheral cells, and their ultimate metabolic fate is complete catabolism in the liver to CO₂ and water. In the final chapter the possible role of the human erythrocyte in the prevention of reperfusion injury has been investigated. The development of a model of renal ischaemia in the rat is described. The ability of human erythrocytes, "primed" by preincubating in acid medium of high Pi concentration and low pO₂, to take up hypoxanthine in a concentrative manner when perfused through ischaemic rat kidney is demonstrated. Attempts to demonstrate improved survival and renal function in rats with "primed" human erythrocytes prior to reperfusion were, however, unsuccessful. It is further demonstrated that "unprimed" human erythrocytes, resident in ischaemic rat kidney for 3 hours, take up hypoxanthine and convert it to IMP. that erythrocytes could play a physiological prevention of reperfusion injury.

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6

Moras, Emily. "Mitochondrial cobalamin binding proteins in patients with inborn errors of cobalamin metabolism." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=97972.

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Vitamin B12 (cobalamin, Cbl) is required as a cofactor for two human enzymes: methylmalonyl-CoA mutase (MCM) and methionine synthase (MS). Fibroblasts from patients with inborn errors of cobalamin metabolism have been classified into nine distinct complementation classes ( cblA-cblH and mut). Previous studies have shown that cobalamin binds MCM in mitochondria and MS in the cytosol. Cobalamin binding patterns were analyzed in crude mitochondrial fractions obtained from normal and mutant fibroblasts. Crude mitochondrial fractions from wildtype fibroblasts confirmed that the majority of [57Co]Cbl eluted with MCM. However, in six of the nine disorders, at least one previously unidentified mitochondrial cobalamin binding protein was observed to bind [57Co]Cbl. The proportion of [57Co]Cbl that binds, is increased when a deficiency in either adenosylcobalamin synthesis or utilization prevents binding to MCM. Furthermore, unique cobalamin binding profiles emerged, demonstrating how known mutations in these patients affect cobalamin binding to accessory proteins.

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7

MIGNANI, LUCA. "Study of inborn errors of Coenzyme A biosynthesis using Danio rerio animal models." Doctoral thesis, Università degli studi di Brescia, 2022. http://hdl.handle.net/11379/558461.

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Animal models are widely used to investigate the pathogenesis and molecular processes involved in the development and progression of human diseases. The Danio rerio, assumed a predominant role in the last decade due to some characteristics such as ease of use, the high number of embryos obtainable, the possibility to follow embryonic development and a completely sequenced genome. Neurodegeneration with Brain Iron Accumulation (NBIA) is a family of diseases characterized by a peculiar accumulation of iron in the brain. All known forms of NBIA are induced by mutations in single genes that code for proteins involved in different pathways. PKAN and CoPAN are two neuropathologies of this group, respectively caused by mutations in PANK2 and COASY genes, which encode for proteins directly involved in the synthesis of Coenzyme A. The transient downregulation of the two zebrafish orthologues of PANK2 and CoASY, respectively pank2 and coasy, induces the onset of specific phenotypes comparable to human pathologies, suggesting the possibility of using Danio rerio as a model for the study of XXX and a tool to discover potential therapeutic strategies. The study was followed by the generation of Knock-Out models for pank2 and coasy and their subsequent characterization. CRISPR/Cas9 technology was used, which allowed us to obtain mutant lines for pank2 and coasy. Analysis performed on pank2 Knock-Out, revealed no significant changes in gene expression that could compensate the lack of pank2 (pank1a and pank1b), or in genes that could compensate the absence of the protein (coasy). However, Western Blot analysis demonstrated a significant reduction in pank2 levels in mutant embryos. Based on previously obtained data which indicated aberrant neuronal development, evaluation of the nervous system development of mutant embryos and the brains of adult individuals was performed using several techniques. The results obtained do not show significant defects in the development of the main neuronal regions both in embryos and in mutant adults. The analysis of the vascular system instead highlighted a morphological alteration at the level of the caudal venous plexus. This results partially confirms what was found in Knock-Down models, suggesting the importance of pank2 for the correct development of the vessels. Specific stains for primordial germ cells showed a decrease in this population in mutant embryos at early stages of development, defects that are probably implicated in the testicular atrophy observed in adults of 1 year of age. This result is similar to what was observed in the mouse model for PKAN which is mainly characterized by azoospermia. A preliminary analysis of the behaviour of the mutant larvae did not reveal any particular difference, while the study of the stress-induced response in adults showed a decrease in the behaviour associated with stress in the mutants. The results indicate that pank2 is involved in various pathways in zebrafish, specifically the formation of vessels, the correct development of germ cells and in the neural circuits involved in the stress response. Furthermore, we successfully generated a CoPAN model in zebrafish inducing mutations in coasy. To date, the phenotypical analysis of this model demonstrate that individuals carrying homozygotes mutation cannot reach adulthood and die before day 15 of development. We also noted the absence of swimming bladder in mutant individuals at 5 days of development. Further analyses will be carried out to better understand the mechanisms that link the onset of this phenotype and the lack of coasy. In conclusion, we generated two models one for for inborn errors of CoA biosynthesis in Danio rerio, their characterization highlights different phenotypes related to the absence of pank2 and coasy that could be useful to better understand the mechanisms underlying these pathologies and can be used to screen molecules with therapeutic potential. I modelli animali vengono utilizzati per investigare la patogenesi e i processi molecolari coinvolti nell’insorgenza di malattie umane. Danio rerio, ha assunto nell’ultimo decennio un ruolo predominante per via di alcune caratteristiche come la facilità d’utilizzo, l’elevato numero di embrioni ottenibili, la possibilità di seguire lo sviluppo embrionale e un genoma completamente sequenziato. Con il termine Neurodegenerazione con accumulo di ferro cerebrale (NBIA) si indica una famiglia di malattie, caratterizzate da un accumulo di ferro a livello cerebrale. Tutte le forme di NBIA conosciute sono indotte da mutazioni in singoli geni che codificano per proteine coinvolte in diversi processi cellulari. PKAN e CoPAN sono due di queste patologie causate rispettivamente da mutazioni dei geni PANK2 e COASY, che codificano per proteine coinvolte nella sintesi del Coenzima A. La downregolazione dei due ortologhi di zebrafish di PANK2 e CoASY, rispettivamente pank2 e coasy, ha dimostrato l’insorgenza di fenotipi paragonabili alle patologie umane suggerendo la possibilità di usare Danio rerio come modello per lo studio di PKAN e CoPAN. Lo studio è proseguito con la generazione di modelli Knock-Out per pank2 e coasy e dalla loro caratterizzazione. È stata utilizzata la tecnologia CRISPR/Cas9, che ha permesso di ottenere linee mutanti per pank2 e coasy. L’analisi di espressione genica sui KO di pank2 non ha rilevato cambiamenti significativi in geni che potrebbero sopperire alla funzione di pank2 (pank1a e pank1b), o in geni coinvolti nella sintesi del CoA (coasy). Analisi dei livelli proteici hanno dimostrato una significativa riduzione dei livelli di pank2 negli embrioni mutanti. Sulla base dei dati ottenuti in precedenza che indicavano uno sviluppo neuronale aberrante, è stata eseguita una valutazione dello sviluppo del sistema nervoso degli embrioni e del cervello di individui adulti utilizzando diverse tecniche. I risultati ottenuti dimostrano che sia negli embrioni che negli adulti mutanti non sono presenti rilevanti difetti nello sviluppo delle principali aree neuronali. L’analisi del sistema vascolare ha evidenziato un’alterazione morfologica del plesso venoso causale, risultato che conferma in parte quello visto in modelli KO per pank2 suggerendo ulteriormente l’importanza di pank2 nello sviluppo dei vasi. È stata osservata una diminuzione del numero di cellule primordiali germinali negli embrioni, difetti che probabilmente è implicato nell’atrofia testicolare osservata negli individui adulti. Questo risultato è in linea con quanto documentato nel modello murino per PKAN che è caratterizzato soprattutto per azoospermia. Un’analisi comportamentale delle larve non ha rilevato alcuna differenza, mentre lo studio della risposta indotta da stress nell’adulto ha dimostrato una diminuzione del comportamento associato a stress nei mutanti. I risultati ottenuti ci hanno permesso di dimostrare che pank2 è implicato in diversi pathways in zebrafish, nello specifico la formazione di vasi, nel corretto sviluppo di cellule germinali e nei circuiti neurali implicati nella risposta allo stress. Il secondo obiettivo della nostra analisi è stata la generazione di una linea mutante per coasy, creando un modello di CoPAN in zebrafish. La caratterizzazione di questo modello a livello fenotipo ha dimostrato che individui omozigoti per la mutazione non riesco a raggiungere lo stadio adulto ma muoiono entro il 15 giorno di sviluppo, inoltre è stata documentata l’assenza della vescia natatoria in individui mutanti a 5 giorni. Ulteriori analisi andranno effettuate per meglio comprendere i meccanismi che legano questo fenotipo alla mancanza di coasy. I risultati ottenuti evidenziano differenze rispetto ai controlli che potrebbero essere utili per comprendere i meccanismi alla base di queste patologie e/o per lo screening di potenziali target terapeutici.

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8

MIGNANI, LUCA. "Study of inborn errors of Coenzyme A biosynthesis using Danio rerio animal models." Doctoral thesis, Università degli studi di Brescia, 2022. http://hdl.handle.net/11379/558476.

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Animal models are widely used to investigate the pathogenesis and molecular processes involved in the development and progression of human diseases. The Danio rerio, assumed a predominant role in the last decade due to some characteristics such as ease of use, the high number of embryos obtainable, the possibility to follow embryonic development and a completely sequenced genome. Neurodegeneration with Brain Iron Accumulation (NBIA) is a family of diseases characterized by a peculiar accumulation of iron in the brain. All known forms of NBIA are induced by mutations in single genes that code for proteins involved in different pathways. PKAN and CoPAN are two neuropathologies of this group, respectively caused by mutations in PANK2 and COASY genes, which encode for proteins directly involved in the synthesis of Coenzyme A. The transient downregulation of the two zebrafish orthologues of PANK2 and CoASY, respectively pank2 and coasy, induces the onset of specific phenotypes comparable to human pathologies, suggesting the possibility of using Danio rerio as a model for the study of XXX and a tool to discover potential therapeutic strategies. The study was followed by the generation of Knock-Out models for pank2 and coasy and their subsequent characterization. CRISPR/Cas9 technology was used, which allowed us to obtain mutant lines for pank2 and coasy. Analysis performed on pank2 Knock-Out, revealed no significant changes in gene expression that could compensate the lack of pank2 (pank1a and pank1b), or in genes that could compensate the absence of the protein (coasy). However, Western Blot analysis demonstrated a significant reduction in pank2 levels in mutant embryos. Based on previously obtained data which indicated aberrant neuronal development, evaluation of the nervous system development of mutant embryos and the brains of adult individuals was performed using several techniques. The results obtained do not show significant defects in the development of the main neuronal regions both in embryos and in mutant adults. The analysis of the vascular system instead highlighted a morphological alteration at the level of the caudal venous plexus. This results partially confirms what was found in Knock-Down models, suggesting the importance of pank2 for the correct development of the vessels. Specific stains for primordial germ cells showed a decrease in this population in mutant embryos at early stages of development, defects that are probably implicated in the testicular atrophy observed in adults of 1 year of age. This result is similar to what was observed in the mouse model for PKAN which is mainly characterized by azoospermia. A preliminary analysis of the behaviour of the mutant larvae did not reveal any particular difference, while the study of the stress-induced response in adults showed a decrease in the behaviour associated with stress in the mutants. The results indicate that pank2 is involved in various pathways in zebrafish, specifically the formation of vessels, the correct development of germ cells and in the neural circuits involved in the stress response. Furthermore, we successfully generated a CoPAN model in zebrafish inducing mutations in coasy. To date, the phenotypical analysis of this model demonstrate that individuals carrying homozygotes mutation cannot reach adulthood and die before day 15 of development. We also noted the absence of swimming bladder in mutant individuals at 5 days of development. Further analyses will be carried out to better understand the mechanisms that link the onset of this phenotype and the lack of coasy. In conclusion, we generated two models one for for inborn errors of CoA biosynthesis in Danio rerio, their characterization highlights different phenotypes related to the absence of pank2 and coasy that could be useful to better understand the mechanisms underlying these pathologies and can be used to screen molecules with therapeutic potential. I modelli animali vengono utilizzati per investigare la patogenesi e i processi molecolari coinvolti nell’insorgenza di malattie umane. Danio rerio, ha assunto nell’ultimo decennio un ruolo predominante per via di alcune caratteristiche come la facilità d’utilizzo, l’elevato numero di embrioni ottenibili, la possibilità di seguire lo sviluppo embrionale e un genoma completamente sequenziato. Con il termine Neurodegenerazione con accumulo di ferro cerebrale (NBIA) si indica una famiglia di malattie, caratterizzate da un accumulo di ferro a livello cerebrale. Tutte le forme di NBIA conosciute sono indotte da mutazioni in singoli geni che codificano per proteine coinvolte in diversi processi cellulari. PKAN e CoPAN sono due di queste patologie causate rispettivamente da mutazioni dei geni PANK2 e COASY, che codificano per proteine coinvolte nella sintesi del Coenzima A. La downregolazione dei due ortologhi di zebrafish di PANK2 e CoASY, rispettivamente pank2 e coasy, ha dimostrato l’insorgenza di fenotipi paragonabili alle patologie umane suggerendo la possibilità di usare Danio rerio come modello per lo studio di PKAN e CoPAN. Lo studio è proseguito con la generazione di modelli Knock-Out per pank2 e coasy e dalla loro caratterizzazione. È stata utilizzata la tecnologia CRISPR/Cas9, che ha permesso di ottenere linee mutanti per pank2 e coasy. L’analisi di espressione genica sui KO di pank2 non ha rilevato cambiamenti significativi in geni che potrebbero sopperire alla funzione di pank2 (pank1a e pank1b), o in geni coinvolti nella sintesi del CoA (coasy). Analisi dei livelli proteici hanno dimostrato una significativa riduzione dei livelli di pank2 negli embrioni mutanti. Sulla base dei dati ottenuti in precedenza che indicavano uno sviluppo neuronale aberrante, è stata eseguita una valutazione dello sviluppo del sistema nervoso degli embrioni e del cervello di individui adulti utilizzando diverse tecniche. I risultati ottenuti dimostrano che sia negli embrioni che negli adulti mutanti non sono presenti rilevanti difetti nello sviluppo delle principali aree neuronali. L’analisi del sistema vascolare ha evidenziato un’alterazione morfologica del plesso venoso causale, risultato che conferma in parte quello visto in modelli KO per pank2 suggerendo ulteriormente l’importanza di pank2 nello sviluppo dei vasi. È stata osservata una diminuzione del numero di cellule primordiali germinali negli embrioni, difetti che probabilmente è implicato nell’atrofia testicolare osservata negli individui adulti. Questo risultato è in linea con quanto documentato nel modello murino per PKAN che è caratterizzato soprattutto per azoospermia. Un’analisi comportamentale delle larve non ha rilevato alcuna differenza, mentre lo studio della risposta indotta da stress nell’adulto ha dimostrato una diminuzione del comportamento associato a stress nei mutanti. I risultati ottenuti ci hanno permesso di dimostrare che pank2 è implicato in diversi pathways in zebrafish, nello specifico la formazione di vasi, nel corretto sviluppo di cellule germinali e nei circuiti neurali implicati nella risposta allo stress. Il secondo obiettivo della nostra analisi è stata la generazione di una linea mutante per coasy, creando un modello di CoPAN in zebrafish. La caratterizzazione di questo modello a livello fenotipo ha dimostrato che individui omozigoti per la mutazione non riesco a raggiungere lo stadio adulto ma muoiono entro il 15 giorno di sviluppo, inoltre è stata documentata l’assenza della vescia natatoria in individui mutanti a 5 giorni. Ulteriori analisi andranno effettuate per meglio comprendere i meccanismi che legano questo fenotipo alla mancanza di coasy. I risultati ottenuti evidenziano differenze rispetto ai controlli che potrebbero essere utili per comprendere i meccanismi alla base di queste patologie e/o per lo screening di potenziali target terapeutici.

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9

MIGNANI, LUCA. "Study of inborn errors of Coenzyme A biosynthesis using Danio rerio animal models." Doctoral thesis, Università degli studi di Brescia, 2022. http://hdl.handle.net/11379/558458.

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Resumen

Animal models are widely used to investigate the pathogenesis and molecular processes involved in the development and progression of human diseases. The Danio rerio, assumed a predominant role in the last decade due to some characteristics such as ease of use, the high number of embryos obtainable, the possibility to follow embryonic development and a completely sequenced genome. Neurodegeneration with Brain Iron Accumulation (NBIA) is a family of diseases characterized by a peculiar accumulation of iron in the brain. All known forms of NBIA are induced by mutations in single genes that code for proteins involved in different pathways. PKAN and CoPAN are two neuropathologies of this group, respectively caused by mutations in PANK2 and COASY genes, which encode for proteins directly involved in the synthesis of Coenzyme A. The transient downregulation of the two zebrafish orthologues of PANK2 and CoASY, respectively pank2 and coasy, induces the onset of specific phenotypes comparable to human pathologies, suggesting the possibility of using Danio rerio as a model for the study of XXX and a tool to discover potential therapeutic strategies. The study was followed by the generation of Knock-Out models for pank2 and coasy and their subsequent characterization. CRISPR/Cas9 technology was used, which allowed us to obtain mutant lines for pank2 and coasy. Analysis performed on pank2 Knock-Out, revealed no significant changes in gene expression that could compensate the lack of pank2 (pank1a and pank1b), or in genes that could compensate the absence of the protein (coasy). However, Western Blot analysis demonstrated a significant reduction in pank2 levels in mutant embryos. Based on previously obtained data which indicated aberrant neuronal development, evaluation of the nervous system development of mutant embryos and the brains of adult individuals was performed using several techniques. The results obtained do not show significant defects in the development of the main neuronal regions both in embryos and in mutant adults. The analysis of the vascular system instead highlighted a morphological alteration at the level of the caudal venous plexus. This results partially confirms what was found in Knock-Down models, suggesting the importance of pank2 for the correct development of the vessels. Specific stains for primordial germ cells showed a decrease in this population in mutant embryos at early stages of development, defects that are probably implicated in the testicular atrophy observed in adults of 1 year of age. This result is similar to what was observed in the mouse model for PKAN which is mainly characterized by azoospermia. A preliminary analysis of the behaviour of the mutant larvae did not reveal any particular difference, while the study of the stress-induced response in adults showed a decrease in the behaviour associated with stress in the mutants. The results indicate that pank2 is involved in various pathways in zebrafish, specifically the formation of vessels, the correct development of germ cells and in the neural circuits involved in the stress response. Furthermore, we successfully generated a CoPAN model in zebrafish inducing mutations in coasy. To date, the phenotypical analysis of this model demonstrate that individuals carrying homozygotes mutation cannot reach adulthood and die before day 15 of development. We also noted the absence of swimming bladder in mutant individuals at 5 days of development. Further analyses will be carried out to better understand the mechanisms that link the onset of this phenotype and the lack of coasy. In conclusion, we generated two models one for for inborn errors of CoA biosynthesis in Danio rerio, their characterization highlights different phenotypes related to the absence of pank2 and coasy that could be useful to better understand the mechanisms underlying these pathologies and can be used to screen molecules with therapeutic potential. I modelli animali vengono utilizzati per investigare la patogenesi e i processi molecolari coinvolti nell’insorgenza di malattie umane. Danio rerio, ha assunto nell’ultimo decennio un ruolo predominante per via di alcune caratteristiche come la facilità d’utilizzo, l’elevato numero di embrioni ottenibili, la possibilità di seguire lo sviluppo embrionale e un genoma completamente sequenziato. Con il termine Neurodegenerazione con accumulo di ferro cerebrale (NBIA) si indica una famiglia di malattie, caratterizzate da un accumulo di ferro a livello cerebrale. Tutte le forme di NBIA conosciute sono indotte da mutazioni in singoli geni che codificano per proteine coinvolte in diversi processi cellulari. PKAN e CoPAN sono due di queste patologie causate rispettivamente da mutazioni dei geni PANK2 e COASY, che codificano per proteine coinvolte nella sintesi del Coenzima A. La downregolazione dei due ortologhi di zebrafish di PANK2 e CoASY, rispettivamente pank2 e coasy, ha dimostrato l’insorgenza di fenotipi paragonabili alle patologie umane suggerendo la possibilità di usare Danio rerio come modello per lo studio di PKAN e CoPAN. Lo studio è proseguito con la generazione di modelli Knock-Out per pank2 e coasy e dalla loro caratterizzazione. È stata utilizzata la tecnologia CRISPR/Cas9, che ha permesso di ottenere linee mutanti per pank2 e coasy. L’analisi di espressione genica sui KO di pank2 non ha rilevato cambiamenti significativi in geni che potrebbero sopperire alla funzione di pank2 (pank1a e pank1b), o in geni coinvolti nella sintesi del CoA (coasy). Analisi dei livelli proteici hanno dimostrato una significativa riduzione dei livelli di pank2 negli embrioni mutanti. Sulla base dei dati ottenuti in precedenza che indicavano uno sviluppo neuronale aberrante, è stata eseguita una valutazione dello sviluppo del sistema nervoso degli embrioni e del cervello di individui adulti utilizzando diverse tecniche. I risultati ottenuti dimostrano che sia negli embrioni che negli adulti mutanti non sono presenti rilevanti difetti nello sviluppo delle principali aree neuronali. L’analisi del sistema vascolare ha evidenziato un’alterazione morfologica del plesso venoso causale, risultato che conferma in parte quello visto in modelli KO per pank2 suggerendo ulteriormente l’importanza di pank2 nello sviluppo dei vasi. È stata osservata una diminuzione del numero di cellule primordiali germinali negli embrioni, difetti che probabilmente è implicato nell’atrofia testicolare osservata negli individui adulti. Questo risultato è in linea con quanto documentato nel modello murino per PKAN che è caratterizzato soprattutto per azoospermia. Un’analisi comportamentale delle larve non ha rilevato alcuna differenza, mentre lo studio della risposta indotta da stress nell’adulto ha dimostrato una diminuzione del comportamento associato a stress nei mutanti. I risultati ottenuti ci hanno permesso di dimostrare che pank2 è implicato in diversi pathways in zebrafish, nello specifico la formazione di vasi, nel corretto sviluppo di cellule germinali e nei circuiti neurali implicati nella risposta allo stress. Il secondo obiettivo della nostra analisi è stata la generazione di una linea mutante per coasy, creando un modello di CoPAN in zebrafish. La caratterizzazione di questo modello a livello fenotipo ha dimostrato che individui omozigoti per la mutazione non riesco a raggiungere lo stadio adulto ma muoiono entro il 15 giorno di sviluppo, inoltre è stata documentata l’assenza della vescia natatoria in individui mutanti a 5 giorni. Ulteriori analisi andranno effettuate per meglio comprendere i meccanismi che legano questo fenotipo alla mancanza di coasy. I risultati ottenuti evidenziano differenze rispetto ai controlli che potrebbero essere utili per comprendere i meccanismi alla base di queste patologie e/o per lo screening di potenziali target terapeutici.

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10

Yamani, Lama. "Studies on transcobalamin in cultured fibroblasts from patients with inborn errors of cobalamin metabolism." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112320.

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Cobalamin must be metabolized intracellularly in order to bind two enzymes: methionine synthase in cytoplasm and methylmalonyl-CoA mutase in mitochondria. Defects in this process cause different inborn errors of cobalamin metabolism (cblA-cblG and mut). A previous study described a cobalamin-binding protein, in addition to methylmalonyl-CoA mutase, in crude mitochondrial fractions. The amount of [57Co]cobalamin bound to this protein was increased in cblB, mut and cblD variant2 cell lines, compared to control cell lines. In the present study, this protein was identified as transcobalamin (TC). Mitochondrial fractions from a cblB cell line were incubated with anti-TC antibodies, which precipitated the cobalamin-bound protein. Analysis of mitochondrial and cytoplasmic fractions isolated from a chloroquine-incubated cblF cell line showed that isolated mitochondrial fractions contain lysosomal material, suggesting that the identified TC is lysosomal. Quantification of cobalamin-bound TC levels in whole cell extracts showed significant increases in cblB and mut groups compared to control cell lines.

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11

Lerner-Ellis, Jordan. "The molecular characterization of inborn errors of vitamin B₁₂ metabolism : cblA, cblB and cblC." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111863.

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This work investigates the molecular basis of three genetic diseases of vitamin B12 metabolism: cblA, cblB and cblC. Two genes responsible for isolated forms of methylmalonic aciduria types cblA and cblB, called MMAA and MMAB respectively, were recently identified. We sequenced the coding sequence and flanking regions of the MMAA and MMAB genes from the gDNA of 37 cblA and 35 cblB patient cell lines and identified 31 novel mutations in total. The biochemical properties of these cell lines were examined in cell culture. Haplotype analysis was used to investigate the history of mutations. The occurrence of both rare and common mutations were identified. Attempts were made to make genotype-phenotype correlations and to understand the effects of mutations on protein function. In the MMAA gene 18 novel mutations were identified, eight of which were common to two or more individuals. One mutation, c.433C>T represented 43% of pathogenic alleles and a common haplotype was identified. Diagnostic tests were designed for every mutation identified. In the MMAB gene, 13 novel mutations were identified. Most mutations were clustered in exon 7. One mutation, c.556C>T accounted for 33% of pathogenic alleles, associated with disease presentation in the first year of life, was observed on a common haplotype and seen almost exclusively in European individuals. We used a combination of linkage, sibling pair, homozygosity mapping and haplotype analyses to refine the disease locus and identify the gene responsible for cblC disease on chromosome 1p called MMACHC. We examined the gDNA of 244 cblC patient cell lines and identified 42 different mutations. The large number of patient samples allowed for the identification of specific genotype phenotype correlations. Of the mutations with elevated frequency in the patients examined, the c.271dupA and c.331C>T mutations were associated with early onset disease whereas c.394C>T was associated with late onset disease. Other missense mutations were also associated with onset of disease later in life. Seven mutations showed clustering by ethnicity. Eight SNPs were identified spanning the gDNA of the MMACHC gene and allowing for the identification of specific haplotypes and the determination of recurrent vs common mutations. Infection of the wild-type MMACHC gene into cblC patient fibroblast cell lines showed correction of the cellular phenotype. Examination of EST databases and northern blot analysis demonstrated MMACHC is ubiquitously expressed in humans with higher levels in fetal liver. Multiple sequence alignment of genomic DNA in eukaryotes and of the polypeptide sequence demonstrated that MMACHC is well conserved in eukaryotes. Two functional domains were identified in the MMACHC gene product by comparison with bacterial genes involved in vitamin B12 related functions: a putative vitamin B 12 binding domain and a TonB-like domain. Molecular modeling demonstrated that the C-terminal region of the gene product folds similarly to TonB from E. coli and suggesting that the C-terminal region of MMACHC may function in a similar manner.

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12

Dolenga, Michael Peter. "Metabolic studies of prolidase deficiency in cultured human fibroblasts." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61190.

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Prolidase deficiency (McKusick 26413) is a rare autosomal recessive disorder characterized by iminodipeptiduria, skin lesions and mental retardation. The enzyme prolidase hydrolyzes dipeptides containing C-terminal proline or hydroxyproline. The results presented here indicate that prolidase plays a major role in the recycling of dipeptide bound proline. Control fibroblasts were able to use iminodipeptides in lieu of proline to sustain normal growth and protein synthesis whereas prolidase deficient cells were not. Iminodipeptides added to the media of control and mutant cells showed no adverse effects on protein synthesis or cell growth. These results are consistent with a mechanism of biochemical pathology in which proline deprivation caused by the enzyme deficit is the cause of damage to skin cells. Prolidase regulation by product and substrate was studied. A two fold decrease of prolidase activity was observed in fibroblasts grown in excess proline. However, cells grown in medium in which iminodipeptides replaced proline showed no significant difference in prolidase activity.

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13

Mandla, Suzan (Suzan G. ). "Studies on mammalian 25-hydroxyvitamin D3-24-hydroxylase." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39363.

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This thesis describes three studies on mammalian 25-hydroxyvitamin D$ sb3$-24-hyroxylase (24-hydroxylase), the first enzyme in the C24-oxidation pathway, a major catabolic pathway for vitamin D metabolites in kidney and other target tissues for vitamin D hormone. The first study examines the involvement of protein kinase C in the regulation of 24-hydroxylase activity in mouse kidney. Evidence is presented supporting a stimulatory role for protein kinase C in the regulation of constitutive, but not inducible, renal 24-hydroxylase. The kinase is also implicated in the aberrant expression of renal vitamin D metabolism in the mutant X-linked hypophosphatemic (Hyp) mouse. The second study investigates the mechanism(s) by which forskolin, a classic activator of adenylate cyclase, inhibits 24-hydroxylase activity in mouse kidney. Both the traditional cAMP-dependent mechanism and a novel cAMP-independent mode of action are observed. A direct interaction between forskolin and the substrate binding site of 24-hydroxylase is suggested for the latter based on kinetic analyses and structural similarities between the diterpene and the steroid substrate for the hydroxylase. The third study addresses the structural relationship between renal 1-hydroxylase and renal and target cell 24-hydroxylase(s) by assessing 24-hydroxylase activity in patients with vitamin D dependency rickets type I (VDDR-I), a Mendelian disorder of 1-hydroxylase function. Both constitutive renal 24-hydroxylase, indirectly ascertained through measurement of circulating levels of relevant vitamin D metabolites, and inducible target cell 24-hydroxylase, directly measured in cultured skin fibroblasts, are shown to be intact in VDDR-I patients undergoing calcitriol therapy. These findings suggest that the 1- and 24-hydroxylase activities likely represent or contain distinct gene products.

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14

Buist, Neil R. M. "Fifty years in inborn errors of metabolism : from urine ferric chloride to mass spectrometry and gene analysis." Thesis, University of St Andrews, 2014. http://hdl.handle.net/10023/12724.

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Prefatory material introducing a collection of articles spanning fifty years of research into inborn errors of metabolism. Table of Contents: 1. Introduction -- 2. Background information about inborn errors of metabolism -- 3. Lessons from phenylketonuria [PKU] -- 4. My role in developing new medical foods -- 5. My role in solving an epidemic of benzyl alcohol poisoning in premature infants -- 6. My role in galactosaemia research -- 7. My start in the metabolic world - screening tests in urine -- 8. My experiences in disaster relief -- 9. My first appearance in the medical literature -- 10. A selection of rare and unusual diseases -- 11. Tyrosinaemia type II; tyrosine aminotransferase deficiency -- 12. Iminodipeptiduria due to prolidase deficiency -- 13. Citrullinaemia -- 14. Rippling muscle disease -- 15. A fatal X-linked disorder of diarrhoea, diabetes mellitus and immune dysregulation -- 16. Infantile Refsum disease -- 17. Hereditary hypocalcuric hypercalcaemia -- 18. Carbohydrate deficient glycoprotein disease type IAPKU -- 19. Thiamine-responsive diabetes and deafness -- 20. Folinic acid-responsive seizures: a false alarm -- 21. S-adenosylmethionine hydrolase deficiency -- 22. Deficiency of complex III of the respiratory chain -- 23. Current research: quantitation of infant sucking behaviour -- 24. Discussion and summary.

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15

Mak, Miu, and 麥苗. "Chemical pathology analysis of inborn errors of metabolism for expanded newborn screening in Hong Kong." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48540924.

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Inborn errors of metabolism (IEM) are under international spotlight because of the recent tremendous development in expanded newborn screening (NBS) and molecular genetics. IEM is a difficult subject involving more than 1,000 different disorders with protean clinical presentations and complicated diagnostic pathways. Cumulative incidence of IEM was reported up to 1 in 800. Patients can be affected in any ages. High clinical suspicion alone is not sufficient to reduce morbidities and mortalities. Notably, some IEM are amenable to treatment with promising outcome. Local data regarding the disease spectrum and incidences is largely lacking. Public awareness and readiness for expanded NBS is unknown. This renders difficulties in the consideration of expanded NBS in Hong Kong. In this study, laboratory data of classical IEM from 2005 to 2009 were retrospectively analyzed (Chapter 2). Local incidence was 1 in 4,122 and that of hyperphenylalaninemias was 1 in 29,542, similar to worldwide figures. Majority (69%) was amino acid disorders, 12% was organic acidemias and 19% was fatty acid oxidation defects. Most of these diseases are effectively amenable to treatment. Local cases including hyperphenylalaninemia, tyrosinemia type I, arginase deficiency, ornithine transcarbamylase deficiency, very long-chain acyl-CoA dehydrogenase deficiency, tyrosine hydroxylase deficiency, thermolabile carnitine palmitoyltransferase II variants and adults IEM with X-linked adrenoleukodystrophy, cerebrotendinous xanthomatosis, familial transthyretin amyloidosis, Wilson disease and PANK2-associated young-onset Parkinsonism were described (Chapters 1.1.3 and 3). Electronic chemical pathology consultation service and dried blood spot metabolic screening were implemented (Chapter 4). There were 279 consultations and 158 screening in a 12-month period. Major referral reasons were developmental delay, neurological defects and unexplained biochemical abnormalities. The incidence in high risk screening was 1 in 158. A non-derivatized tandem mass spectrometry assay for amino acids and acylcarnitines was evaluated for its precision, accuracy and reference intervals (Chapter 5). The concordance rate was 100% in inter-laboratory comparison and external quality assurance programs. The method was proven to be accurate, rapid and affordable. It is suitable for large volume testing and emergency diagnostic needs. A feasibility study of a hospital-based expanded NBS service model was conducted on 360 newborns (Chapter 6). More than 90% of babies were older than 48 hours before discharge and were fit for blood collection. The service model consisted of parent education, consent, postnatal sample collection, technical analysis, clinical interpretation, reporting and follow-up actions. Questionnaire on the knowledge and attitude towards IEM and expanded NBS was surveyed on 172 parents to investigate the psychological, social and ethical aspects (Chapter 7). Here, 99.4% demanded more education on expanded NBS; 97.6% supported to implement the program; 97.7% supported population screening even though some diseases are incurable. Availability of treatment is not the most important pre-requisite for NBS; 93.9% accepted the possibility of false positive and false negative results. Acceptance towards expanded NBS among parents was high. Our data indicate that IEM is not uncommon in Hong Kong and it is indisputable for the introduction of a local expanded NBS program. Our data serve as groundwork for policy decision and further discussion on expanded NBS. published_or_final_version Pathology Master Doctor of Medicine

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16

Farah, Rita S. "Intragenic complementation in methylmalonyl CoA mutase." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=55444.

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Methylmalonic aciduria (MMA) is an autosomal recessive metabolic disorder with an incidence of 1 in 48,000, which may be due to a defect in the mitochondrial homodimeric enzyme methylmalonyl CoA mutase (mut MMA). mut MMA is subdivided into $mut sp circ$ and $mut sp-$ subclasses on the basis of complementation analysis; $mut sp circ$ cell lines have very low incorporation of ($ sp{14}$C) from propionate into acid precipitable material while incorporation in $mut sp-$ cells is increased when cells are incubated in cobalamin. Intragenic complementation was first observed with WG 1130, a $mut sp circ$ fibroblast line with a homozygous R93H mutation, that is capable of complementing MCM activity when fused with some $mut sp circ$ and some $mut sp-$ cells (1). Extensive intragenic complementation in mut MMA was subsequently observed. Fibroblasts cultured from thirteen unrelated patients (6 $mut sp-$, 7 $mut sp circ$) were fused in all possible pairwise combination and MCM activity was assayed in the heterokaryons by measuring the incorporation of ($ sp{14}$C) from propionate into acid precipitable material. Intragenic complementation, indicated by stimulation of ($ sp{14}$C) -propionate incorporation following cell fusion with polyethylene glycol, was observed in fusions involving twelve of the thirteen strains. Of these thirteen strains, mutations have been identified in six; four have a homozygous mutation (WG 1130 (R93H), WG 1511 (H678R), WG 1610 (G717V), WG 1609 (G630E)), and two cell lines are compound heterozygous (WG 1681 (G623R and G703R), WG 1607 (W105R and A377E)); the remainders are yet to be determined. These intragenic complementations will provide information for grouping the mutations in defined domains in order to correlate structure and function of MCM.

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17

Qureshi, Amber A. (Amber Ateef). "The molecular characterization of mutations at the methylmalonyl CoA mutase locus involved in interallelic complementation /." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69686.

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Methylmalonic aciduria is an autosomal recessive metabolic disorder, which may be due to a defect in the methylmalonyl CoA mutase (MCM) apoenzyme. The mut$ sp circ$ mutation is characterized by undetectable enzyme activity in cell extracts, and by the low incorporation of ($ sp{14}$C) propionate in the presence of hydroxocobalamin in culture. A mut$ sp circ$ fibroblast cell line, WG 1681, from an African-American male infant was shown to complement another mut$ sp circ$ cell line, WG 1130. Subsequent cloning and sequencing of cDNA from WG 1681 identified two previously described homozygous polymorphisms: H532R and V671I(1). In addition, compound heterozygosity was observed for two novel changes at highly conserved sites: G623R and G703R. Hybridization of allele specific oligonucleotides to PCR amplified MCM exons from WG 1681 and family members identified a clinically normal mother, sister and half-brother as carriers of the G703R change in cis with both polymorphisms. The putative father was not identified as a carrier of the G623R change. transfection of each change, singly and in cis with both polymorphisms, into GM1673 cells demonstrated a lack of stimulation of ($ sp{14}$C) propionate uptake in the absence and presence of OH-Cbl, in comparison to controls. Co-transfection of each separate mutation with the previously identified R93H mutation of WG 1130 (2) stimulated propionate uptake. These results indicate that G623R and G703R are novel mutations responsible for deficient MCM activity and the mut$ sp circ$ phenotype in WG 1681, and both mutations are independently capable of complementing the R93H mutation of WG 1130.

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18

Boright, Andrew Pepler. "Prolidase deficiency : studies in human dermal fibroblasts." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75956.

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Prolidase deficiency (MIM 26413), an autosomal recessive phenotype, is caused by rare alleles at a locus on chromosome 19cent.-q13.2. The clinical phenotype is pleiotropic (affecting skin, brain, etc.) and of variable expressivity (benign to early death). I established skin fibroblast cultures from 6 homozygous probands and 6 obligate heterozygotes, purified prolidase (E.C. 3.4.13.9, a homodimer) from normal human fibroblasts, raised a monospecific rabbit antiserum to the subunit, and studied its biosynthesis. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized in the cytosol as a 58 KDa. polypeptide and not processed further. Homozygous prolidase-deficient cell strains expressed 3 classes of mutant alleles which by complementation analysis mapped to one locus. The alleles were designated CRM$-$ (nul), CRM+ activity/size variant, and CRM+ activity variant. Heterozygotes carrying CRM$-$ alleles have heat stable prolidase (50$ sp circ$C, 1hr); heterozygotes carrying CRM+ variant alleles have heat labile enzyme. The finding implies that variant CRM+ allele(s) can confer negative allelic complementation on the dimeric enzyme (dominant relative phenotype). CRM$-$ homozygous cells contain varying amounts of an alternative imidodipeptidase-like activity. The variant prolidase allele (major gene) and amount of alternative "prolidase" activity (modifier gene) are apparently both determinants of the associated clinical phenotype in prolidase deficiency. I obtained and sequenced a tryptic peptide from human kidney prolidase for synthesis of oligonucleotide probes in the future.

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19

Vieira, Tatiane Alves. "Fatores associados a adesão ao tratamento dos pacientes com fenilcetonúria acompanhados pelo Serviço de Genética Médica do Hospital de Clinícas de Porto Alegre." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2010. http://hdl.handle.net/10183/26896.

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Introdução: Os Erros Inatos do Metabolismo ocorrem devido a um defeito enzimático que determina um bloqueio em uma via metabólica, sendo a maioria herdada de forma autossômica recessiva. A fenilcetonúria (PKU) é um erro inato do metabolismo associado à atividade deficiente da enzima hepática fenilalanina hidroxilase, a qual converte a fenilalanina em tirosina. Em conseqüência, os pacientes apresentam níveis séricos elevados de fenilalanina. Quando não diagnosticada e tratada precocemente a PKU causa danos neurológicos irreversíveis. O tratamento consiste em uma dieta restrita em fenilalanina, complementada com uma fórmula metabólica. Semelhante a outras doenças crônicas, o sucesso do tratamento depende dos pacientes e suas famílias. O Ambulatório de Tratamento de Distúrbios Metabólicos do Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre iniciou suas atividades em 1991 e atualmente tem 65 pacientes em acompanhamento, sendo um serviço de referência no tratamento da fenilcetonúria. Estima-se que a adesão dos pacientes seja insatisfatória. Objetivo: O presente trabalho tem por objetivo identificar os fatores associados à adesão ao tratamento dos pacientes acompanhados no Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre. Métodos: Estudo transversal de base ambulatorial que incluiu 56 pacientes com diagnóstico de fenilcetonúria clássica ou atípica. Os pacientes foram classificados em aderentes e não aderentes de acordo com a mediana de phe dos últimos 12 meses de tratamento. Os dados foram coletados a partir de revisão de prontuário e entrevista com pacientes e familiares. Três questionários foram aplicados aos pacientes ou familiares e continham questões sobre condições socioeconômicas, escolaridade, variabilidade intra-familial, conhecimento sobre a doença, percepção da dieta e principais problemas associados ao tratamento da fenilcetonúria. Resultados: A mediana de idade dos pacientes foi de 12 anos. Dezoito pacientes (32,1%) pacientes foram classificados como aderentes, sendo que 11 deles apresentavam idade superior a 13 anos. Fatores como convívio com os familiares e nível de escolaridade da mãe influenciaram na adesão dos pacientes ao tratamento. Outros fatores analisados como classe socioeconômica e conhecimento sobre a doença não foram associados à adesão ao tratamento. Conclusão: Os pacientes possuem uma adesão insatisfatória ao tratamento. Embora diversos fatores possam estar associados à mesma, outros parecem não ter influência direta sobre a adesão. A adesão ao tratamento é um assunto complexo, e que necessita do entendimento e conhecimento por parte dos pacientes e de seus familiares em relação ao tratamento e a doença, e principalmente suporte e confiança na equipe médica. As dificuldades associadas ao tratamento da PKU devem ser trabalhadas em conjunto a fim de serem encontradas para cada caso, as intervenções mais efetiva. Background: The Inborn Errors of Metabolism occurs, mostly inherited as an autosomal recessive trait. Phenylketonuria (PKU) is an inborn error of metabolism associated with deficient activity of the hepatic enzyme phenylalanine hydroxylase, which converts phenylalanine to tyrosine. As a result, patients have high serum levels of phenylalanine . When not diagnosed and treated promptly cause irreversible neurological damage. The treatment involves a diet restricted in phenylalanine, complemented with a metabolic formula . Similar to other chronic treatment success depends on patients and their families. The Ambulatory Treatment of Metabolic Disorders, Medical Genetics Service, Hospital de Clinicas de Porto Alegre starting its activities in 1991 and currently has 65 patients monitored, and a referral service for the treatment of PKU. It is estimated that compliance of patients is unsatisfactory. Objective: This study aims to identify factors associated with treatment adherence of patients followed in the Department of Medical Genetics, Hospital de Clinicas de Porto Alegre. Methods: Cross-sectional study that included 56 ambulatory patients with classical or atypical phenylketonuria. Patients were divided into compliant and noncompliant according to the median of phenylalanine in the last 12 months of treatment. Data were collected from chart review and interviews with patients and families. Three questionnaires were administered to patients or relatives and contained questions on socioeconomic status, educational level, family status, housing conditions and intra-familial variability, knowledge about the disease, perception of diet and major problems related to treatment of phenylketonuria. Results: The median age of the 56 patients was 12 years. Eighteen patients (32.1%) patients were classified as compliant, with 11 of them were older than 13 years. Factors such as contact with family members and mother's education level influenced the patients' adherence to treatment. Other factors such as socioeconomic status and knowledge about the disease were not associated with adherence to treatment. Conclusions: Patients have a poor adherence to treatment. Although several factors may be associated with it, others seem to have no direct influence on adherence. Treatment adherence is a complex issue and requires the understanding and knowledge of patients and their families regarding treatment and disease, and mainly support and confidence in the medical team. The difficulties associated with the treatment of PKU should be worked together in order to be found for each case, the most effective interventions.

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20

Brewer, Judy. "Metabolic Modeling of Inborn Errors of Metabolism: Carnitine Palmitoyltransferase II Deficiency and Respiratory Chain Complex I Deficiency." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:24078365.

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The research goal was to assess the current capabilities of a metabolic modeling environment to support exploration of inborn errors of metabolism (IEMs); and to assess whether, drawing on evidence from published studies of EMs, the current capabilities of this modeling environment correlate with clinical measures of energy production, fatty acid oxidation, accumulation of toxic by-products of defective metabolism, and mitigation via therapeutic agents. IEMs comprise several hundred disorders of energy production, often with significant impact on morbidity and mortality. Despite advances in genomic medicine, currently the majority of therapeutic options for IEMs are supportive only, and most only weakly evidenced. Metabolic modeling could potentially offer an in silico alternative for exploring therapeutic possibilities. This research established models of two inborn errors of metabolism (IEMs), carnitine palmitoyltransferase (CPT) II deficiency and respiratory chain complex I deficiency, allowing exploration of combinations of IEMs at different degrees of enzyme deficiency. It utilized a modified version of the human metabolic network reconstruction, Recon 2, which includes known metabolic reactions and metabolites in human cells, and which allows constraint-based modeling within a computational and mathematical representation of human metabolism. It utilized the Matlab-based COBRA (Constraint-based Reconstruction and Analysis) Toolbox 2.0, and a customized suite of functions, to model ATP production, long-chain fatty acid oxidation (LCFA), and acylcarnitine accumulation in response to varying defect levels, inputs and a simulated candidate therapy. Following significant curation of the metabolic network reconstruction and customization of COBRA/Matlab functions, this study demonstrated that ATP production and LCFA oxidation were within expected ranges, and correlated with clinical data for enzyme deficiencies, while acylcarnitine accumulation inversely correlated with the degree of enzyme deficiency; and that it was possible to simulate upregulation of enzyme activity with a therapeutic agent. Results of the curation effort contributed to development of an updated version of the metabolic reconstruction Recon 2. Customization of modeling approaches resulted in a suite of re-usable Matlab functions and scripts usable with COBRA Toolbox methods available for further exploration of IEMs. While this research points to potentially greater suitability of kinetic modeling for some aspects of metabolic modeling of IEMs, it helps to demonstrate potential viability of constraint-based steady state modeling as a means to explore some clinically relevant measures of metabolic function for single and combined inborn errors of metabolism.

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21

Wilson, Aaron. "Molecular genetics and characterisation of functional methionine synthase deficiency : mutation analysis and gene cloning." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21665.

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Methionine synthase (MS) is a vitamin B12(cobalamin;cbl) dependent enzyme that catalyses the methylation of homocysteine to methionine. It uses methyl-cbl as coenzyme and in ethyl tetrahydrofolate as the methyl donor. Methionine sythase reductase (MSR) maintains MS in it active state using S-adenosyl methionine as the methyl donor. Functional MS deficiency may occur as a result of a defect in either enzyme. Patients with this disorder have been classified into two complemetation groups according to which protein is defective: cblG patients are deficient in MS and cblE patients in MSR. A subset of cblG, known as cblG variant, is unique in showing barely detectable MS activity and failure of cbl incorporation into MS in patient fibroblasts. I report the mutations responsible for three cblG variant patients, two of them siblings, and connect their phenotype to lack of protein expression. I also report the cloning of the MSR cDNA, aided by confirming the identity of the cDNA through the discovery of two deleterious mutations in three cblE patients. These findings contribute to the overall understanding of functional MS deficiency.

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22

Nowacki, Piotr Marek. "Design, development, and deployment of a locus specific mutation database : the PAHdb example." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0004/MQ44234.pdf.

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23

Roy, Stéphane. "Regulation of 1,25 dihydroxyvitamin D3-24-hydroxylase gene expression." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=34441.

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The first three studies in this thesis address the mechanism for the aberrant fall in serum 1,25-dihydroxyvitamin D$ sb3$ (1,25-(OH)$ sb2$D$ sb3 rbrack$ and increase in renal 1,25-(OH)$ sb2$D$ sb3$-24-hydroxylase(24-hydroxylase) activity in X-linked hypophosphatemic mice (Hyp). The 24-hydroxylase is the first enzyme in the C-24 oxidation pathway that degrades the vitamin D hormone to its final inactivation product, calcitroic acid. We demonstrated that: (i) the aberrant increase in 24-hydroxylase activity in Pi-deprived Hyp mice is specific to the kidney and is the result of an increase in enzyme Vmax, immunoreactive protein and mRNA abundance; (ii) the increase in 24-hydroxylase mRNA in both Pi-deprived Hyp mice and 1,25-(0H)$ sb2$D$ sb3$-treated normal littermates can be ascribed to an increase in the transcriptional activity of the 24-hydroxylase gene; (iii) 24-hydroxylase transcripts in normal mice, Pi-deprived Hyp and normal mice and 1,25-(OH)$ sb2$D$ sb3$-treated normal mice are localized to the proximal tubule by in situ hybridization; and (iv) recombinant human growth hormone administration normalizes the aberrant increase in 24-hydroxylase but that this response is not sufficient to correct serum 1,25-(OH)$ sb2$D$ sb3$ levels in Pi-deprived Hyp mice. The fourth study addresses the mechanism whereby EB 1089, an analogue of 1,25-(OH)$ sb2$D$ sb3,$ is less calcemic than the vitamin D hormone, while being more potent in its antiproliferative action. We demonstrate that: (i) EB 1089 has a 50-fold lower affinity than 1,25-(OH)$ sb2$D$ sb3$ for the vitamin D catabolic enzyme, 24-hydroxylase; and (ii) EB 1089 and 1,25-(OH)$ sb2$D$ sb3$ exhibit tissue-specific differences in vitamin D receptor-mediated responses in vivo that may be ascribed, at least in part, to differences in binding affinities for the vitamin D receptor.

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24

Lewis, Martin David. "Human lysosomal sulphate transport." Title page, contents and abstract only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phl6752.pdf.

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Addendum inserted at back Includes bibliographical references (leaves 266-287). 1. Introduction -- 2. Materials and general methods -- 3. Characterisation and partial purification of the lysosomal sulphate transporter -- 4. Identification of proteins involved in lysosomal sulphate transport -- 5. The relationship between a sulphate anion transporter family and the lysosomal sulphate transporter -- 6. Investigation of sulphate transport in human skin fibroblasts -- 7. Concluding remarks

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25

Cochran, Brittany Paige. "Nutrition Support and Newborn Screening in the NICU Population: Is There a Link?" Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/76756.

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Background: Recent research is revealing the high rate of false-positive screening results for IEMs in the NICU population. No study published to date has specifically studied the possible relationship between nutrition and newborn screening in this population. Objective: It is suspected that NICU infants who receive PN are more likely to have abnormal newborn screening results than infants who receive EN. An understanding of the role of nutrition will assist in developing protocols for screening in the NICU and decrease false-positives. Design: Infants admitted to the NICU between January 1-June 30, 2009 were included in this retrospective chart review study (n=339). The type of nutrition and timing of its initiation was recorded and compared to newborn screening results to identify correlations with false-positives. Statistical analysis included means, percentages, Fisher's exact test, Chi-square test, and the Cochran-Mantel-Haenszel test. Results: Nutrition type was significantly associated with newborn screening (p<0.001); those who received parenteral nutrition were more likely to have a false-positive. For infants who also received PN, EN of breast milk exclusively increased risk of an abnormal screen more than formula exclusively or breast milk plus formula. The timing of parenteral nutrition had no effect on screening. Premature infants who received PN exclusively had a higher percentage of false-positives than those who received EN Conclusions: Although the hypothesis could not be statistically supported, PN appears to contribute to false-positive newborn screens. More research is needed to ascertain the role of EN and GA in newborn screening and to develop standardized protocols. Master of Science

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26

Tappin, David Michael. "Prevalence of HIV infection in childbearing women in Scotland : a project testing inborn errors screening cards for HIV-1 antibodies." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338468.

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27

Marx, Laura. "Parents’ Reflections of their Child’s Initial Visit to Metabolic Clinic: A Qualitative Study." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1553513682882592.

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28

Spiegel, Erin Kathleen. "Psychomotor deficits in mice transgenic for a mutant adenylosuccinate lyase associated with autism in humans /." Connect to full text via ProQuest. IP filtered, 2006.

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Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006. Typescript. Includes bibliographical references (leaves 127-143). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

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29

Lalovic, Aleksandra. "The relationship between lipid metabolism and suicidal behaviour : clinical and molecular studies." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103207.

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Suicide continues to claim hundreds of thousands of lives worldwide each year, in spite of the significant progress of research efforts aimed at understanding the complexity of this tragic behaviour. Data accumulated over the last decades suggest a certain biological predisposition to suicidal behaviour. Among the possible biological risk factors, cholesterol has frequently been cited. Several lines of evidence support the relationship between altered lipid metabolism, particularly low levels of serum cholesterol, and suicidal behaviour, yet the possible mechanisms governing the relationship remain to be elucidated. Three separate strategies were employed in order to explore the link between lipid metabolism and suicidal behaviour, each one from a novel perspective on this issue. The first approach aimed to substantiate the existing evidence of an association between low serum cholesterol and suicidality by examining psychiatric data, suicidality and related behavioural characteristics in a sample of Smith-Lemli-Opitz syndrome heterozygotes---a clinically normal population with altered cholesterol metabolism due to an inherited partial deficiency in the 7-dehydrocholesterol reductase enzyme---compared with controls. The second approach consisted in measuring the lipid profile in brain tissue from suicide completers, in order to address whether there are alterations in cholesterol and/or fatty acids in the brain. The final approach involved the use of exploratory gene expression studies to identify novel candidate genes and proteins that may be involved in mediating the link between lipid metabolism and suicidality. The results of these studies will be presented and discussed.

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30

Nalin, Tatiéle. "Hiperfenilalaninemia por deficiência de fenilalanina hidroxilase : avaliação da responsividade ao BH4 em pacientes acompanhados no Serviço de Genética Médica do HCPA e que apresentam controle metabólico adequado." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2011. http://hdl.handle.net/10183/31887.

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Introdução: Estudos recentes, utilizando vários protocolos, têm demonstrado que pacientes com Hiperfenilalaninemia por deficiência de fenilalanina hidroxilase (HPA-PAH) podem apresentar redução das concentrações plasmáticas de fenilalanina (Phe) mediante a administração de tetrahidrobiopterina (BH4). Objetivo: Determinar, em uma amostra de pacientes brasileiros com HPA-PAH, a responsividade à administração de dose única de BH4 por meio de um protocolo incluindo o teste de sobrecarga simples de Phe e o teste combinado de sobrecarga de Phe+BH4. Métodos: Foram incluídos no estudo pacientes com idade ≥ 4 anos, em tratamento dietético e que possuíam mediana de Phe plasmática inferior a 10mg/dL no ano anterior à inclusão. Foram realizados teste de sobrecarga simples de Phe, utilizando 100mg/kg de L-Phe (Teste 1) e teste combinado de Phe+BH4, utilizando 100mg/kg de L-Phe e 20mg/kg de BH4 (Teste 2), com intervalo de uma semana entre ambos. A ingestão do BH4 ocorreu após três horas da ingestão da Phe. Foram realizadas coletas de sangue no ponto basal e após três, 11 e 27h da ingestão de Phe (T0, T1, T2 e T3 dos testes 1 e 2, respectivamente). Para ser considerado responsivo, o paciente deveria apresentar evidência de redução dos níveis de Phe associada à administração do BH4 de acordo com pelo menos um dos seguintes critérios: critério A – análise das diferenças, em percentual, dos valores de Phe nos pontos T1 e T2 dos Testes 1 e 2; critério B – análise das diferenças, em percentual, dos valores de Phe nos pontos T1 e T3 dos Testes 1 e 2 e critério C – análise da diferença, em percentual, das áreas abaixo da curva de Phe entre os Testes 1 e 2. A classificação de responsividade foi também comparada com quatro critérios adicionais: critério D – análise da diferença, em percentual, dos valores de Phe nos pontos T1 e T2 do Teste 2; critério E – análise da diferença, em percentual, dos valores de Phe nos pontos T1 e T3 do Teste 2; cinco pacientes participaram de estudo anterior do grupo e foram também classificados através do critério F – análise da diferença, em percentual, dos valores de Phe após 8h da sobrecarga simples com BH4 e do critério G – análise da diferença, em percentual, dos valores de Phe após 24h da sobrecarga simples com BH4. Para todos os critérios foi utilizado como ponte de corte redução ≥ 30%. Resultados: Dezoito pacientes, com mediana de idade de 12 anos, participaram do estudo. Dez pacientes apresentavam a forma Leve de HPA-PAH e oito a forma Clássica. Seis pacientes foram considerados responsivos de acordo com os critérios adotados (Clássica: 2; Leve: 4). Houve concordância de responsividade entre os critérios A e B em relação ao C (Índice Kappa=0,557; p=0,017). Dos pacientes com genótipo disponível (n=16), seis possuíam dados de responsividade ao BH4 descritos na literatura, que foram concordantes com os encontrados no presente estudo. Conclusão: Dados relativos à responsividade, tipo de HPA-PAH e genotipagem estão de acordo com descrito na literatura. Haja vista a diferença de responsividade dos pacientes ao BH4 conforme o critério de classificação utilizado, salienta-se a importância de uma definição cautelosa de responsividade e que não seja baseada em um único critério. A comparação entre a sobrecarga simples de Phe e combinada de Phe+BH4 parece ser um critério adequado para avaliar responsividade ao BH4 em pacientes com HPA-PAH que apresentam bom controle metabólico quando em tratamento dietético. Introduction: Recent studies using different protocols showed that patients with hyperphenylalaninemia due to phenylalanine hydroxylase deficiency (HPA-PAH) may have a reduction in phenilalanine (Phe) plasma concentrations after tetrahydrobiopterin (BH4) administration. Objective: To determine responsiveness to the administration of a single dose of BH4, in a sample of Brazilian patients with HPA-PAH using a protocol that includes the simple Phe loading test and the combined Phe+BH4 loading test. Methods: Patients included in the study were ≥ 4 years of age; their median Phe plasma concentration was ≤ 10mg/dL, and all underwent dietary treatment in the 12 months before inclusion in the study. Participants received a simple Phe loading test using 100mg/kg L-Phe (Test 1) and a combined Phe+BH4 loading test using 100mg/kg L-Phe and 20mg/kg /BH4 (Test 2) at a one-week interval. BH4 was ingested three hours after Phe ingestion. Blood samples were collected at baseline and three, 11 and 27h after Phe ingestion (time points T0, T1, T2 and T3 in Tests 1 and 2). To be classified as responsive, there should be evidence that the patient had a reduction in Phe levels associated with BH4 administration according to at least one of the criteria used: criterion A – analysis of percentage differences of the Phe values at time points T1 and T2 for Tests 1 and 2; criterion B – analysis of percentage differences of Phe values at time points T1 and T3 for Tests 1 and 2; and criterion C – analysis of percentage differences of the areas under the Phe curve for Tests 1 and 2. Responsiveness classifications were also compared according to four additional criteria: criterion D – analysis of percentage differences of the Phe values at time points T1 and T2 for Test 2; criterion E – analysis of percentage differences of Phe values at time points T1 and T3 for Test 2; criterion F – analysis of percentage difference of Phe values 8h after simple BH4 loading used for five patients that participated in a previous study conducted by the same authors; and criterion G – analysis of percentage difference of Phe values 24 h after simple BH4 loading, also used for the patients in the same previous study. The cut-off point for all criteria was a reduction of ≥ 30%. Results: Eighteen patients (median age = 12 years) were included in the study. Ten patients had mild HPA-PAH and eight, classical HPA-PAH. Six patients were responsive according to the criteria used (Classical: 2; Mild: 4). Responsiveness was concordant for criteria A and B when compared with criterion C (kappa=0.557; p=0.017). Of the patients whose genotype was available (n=16), six had data about BH4 responsiveness described in the literature, and these data were in agreement with our findings. Conclusion: Data about responsiveness, HPA-PAH type and genotype were in agreement with those described in the literature. The difference in BH4 responsiveness of patients according to classification criterion emphasizes the importance of a careful definition of responsiveness not based on a single criterion. The comparison of simple Phe loading and combined Phe+BH4 loading seems to be an adequate criterion to evaluate responsiveness to BH4 in patients with HPA-PAH and good metabolic control when following a dietary treatment.

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31

Grecco, Mariana Setanni. "Síndrome HELLP e defeitos de beta oxidação de ácidos graxos de cadeia longa hidroxi-acil: um estudo de caso-controle." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17144/tde-29082016-114203/.

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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.

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32

Freeman, Craig. "The lysosomal degradation of heparan sulphate : a comparative study of the physical and catalytic properties of the heparan sulphate degradative enzymes /." Title page, contents and abstract only, 1991. http://web4.library.adelaide.edu.au/theses/09PH/09phf855.pdf.

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Moreno, Carolina Araujo 1981. "Estudo da etiopatogenia da hidropisia fetal não-imune a partir de uma série de casos utilizando um protocolo de investigação ampliado." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/308790.

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Orientador: Denise Pontes Cavalcanti Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Made available in DSpace on 2018-08-22T09:53:23Z (GMT). No. of bitstreams: 1 Moreno_CarolinaAraujo_M.pdf: 4072523 bytes, checksum: 09921eee06d7c1098048c98a3f891b33 (MD5) Previous issue date: 2013 Resumo: A hidropisia fetal não-imune (HFNI) é causada por um grupo heterogêneo de condições e atualmente corresponde à maior parte dos casos de hidropisia fetal. Em função da ampla diversidade etiopatogênica, a investigação dos casos de HFNI constitui um desafio diagnóstico. Esse estudo teve como objetivo a avaliação prospectiva e sistemática de uma série de casos de HFNI a partir de um protocolo de investigação ampliado, que incluiu a pesquisa de doenças metabólicas. O presente estudo também incluiu a revisão dos casos de HFNI registrados previamente pelo Programa de Genética Perinatal na maternidade da Unicamp. Durante aproximadamente dois anos (2010-2012), foram identificados 53 casos de HFNI. Nesse período, ocorreram 6.129 nascimentos na maternidade local, com registro de HFNI em 37 recém-nascidos, conferindo uma prevalência de 60 por 10.000 nascimentos, valor maior do que o observado no período anterior ao estudo (1987 a 2009). Para o restante da análise, quatro casos foram excluídos devido à impossibilidade de estudá-los adequadamente. A maioria dos hidrópicos nasceu pré-termo (43 - 73,5%). Houve registro de 23 nativivos (47%), 10 óbitos no período neonatal e 26 óbitos durante a gestação (53%), resultado em uma mortalidade geral (pré-natal e neonatal) de 73,4%. A hidropisia foi identificada no pré-natal na maioria dos casos (44 - 89,8%) e, apesar da condição ser comumente associada a mau prognóstico, em três pacientes (6,1%) houve resolução completa e espontânea da hidropisia durante a gestação. Os principais grupos diagnósticos encontrados foram: anomalias cromossômicas (17 casos - 34,7%), quadros sindrômicos (16,4% - oito casos), cardiopatias e infecções congênitas (8,2% - quatro casos cada). Os erros inatos do metabolismo (EIM) corresponderam a 6,1% da amostra (três casos de doenças de depósito lisossômico). Três casos (6,1%) foram classificados como idiopáticos. Comparando os diagnósticos da casuística prospectiva com aqueles observados no período 1987-2009, observou-se que os grupos diagnósticos mais frequentes, ou seja, anomalias cromossômicas, quadros sindrômicos e cardiopatias, foram os mesmos. No entanto, algumas diferenças foram observadas na casuística prospectiva, como a frequência maior de anomalias cromossômicas e de EIM, que podem ser explicadas respectivamente pela inclusão de abortos e pesquisa sistemática de doenças metabólicas. Por outro lado, a menor frequência do grupo de idiopáticos foi decorrente da ampliação do protocolo de investigação e da eliminação de casos inadequadamente estudados. Ressalta-se também que a frequência de EIM registrada no trabalho prospectivo (6%) foi superior à usualmente descrita na literatura e provavelmente aproxima-se da frequência real das doenças metabólicas em HFNI, justificando desse modo a pesquisa das mesmas na investigação de hidrópicos de causa não-imune após exclusão das condições mais comuns, como as anomalias cromossômicas, cardiopatias isoladas, infecções congênitas e síndromes conhecidas. Sendo assim, observou-se que o protocolo de investigação proposto permitiu o diagnóstico clínico-etiológico ou patogênico de mais de 90% dos casos, evidenciando que uma avaliação ampla e sistemática é capaz de identificar a maior parte dos fatores etiopatogênicos envolvidos na HFNI Abstract: Non-immune hydrops fetalis (NIHF) is caused by a hetereogenous group of conditions, currently accounting for the most cases of hydrops fetalis. Because of the wide etiopathogenic diversity, the investigation of NIHF cases constitutes a real diagnostic challenge. This study aimed to evaluate prospectively and systematically a series of NIHF cases from an expanded research protocol including the investigation of metabolic diseases. The present study also aimed to revise the NIHF cases previously recorded by Perinatal Genetics Program (PGP) in the maternity hospital of Unicamp. During approximately two years (2010-2012), 53 cases were identified. In this period, among 6,129 births that occurred in our hospital, NIHF was identified in 37 newborns, given a birth prevalence of 60 per 10,000, higher than that was observed in the previous period - 23:10,000 (1987-2009). For purpose of all other analysis, four of the 53 cases evaluated had to be excluded due to inability to assess them correctly. Most hydropic individuals were born preterm (43 - 73.5%). Twenty-three patients (47%) were live births, 10 of them died before hospital discharge; and 26 (53%) died in the prenatal period, given an overall mortality of 73.4%. The hydrops were identified in prenatal period in most cases (44 - 89.8%), and despite being commonly associated with poor prognosis, three cases (6.1%) had complete and spontaneous resolution of hydrops during pregnancy. The main diagnostic groups were chromosomal abnormalities (17 - 34.7%), syndromic (8 - 16.4%), isolated heart defects (4 - 8.2%), and congenital infections (4 - 8.2%). Inborn errors of metabolism (IEM) occurred in three cases (6.1%), all represented by lysosomal storage diseases. Three cases (6.1%) were classified as idiopathic. The comparison of these diagnostic groups with those found during the retrospective period (1987-2009) showed that the most frequent groups, i.e. chromosomal abnormalities, syndromic and isolated cardiopathy, were the same. However, some differences were observed in the prospective series. For instance, the higher frequency of chromosomal abnormalities and IEM can be respectively explained because of the inclusion of abortions and due to systematic investigation of metabolic diseases. The lower frequency of idiopathic group, by the other hand, can be regarded as close related, first, to the expanded investigation and, second, to the exclusion of cases poorly studied. It is noteworthy that the recorded IEM frequency in the present prospective series (6%) was higher than the usually reported in the literature and seems to be more realistic, thus, justifying the inclusion of a systematic approach of these conditions in NIHF. This investigation, however, should be initiated after the exclusion of the more common causes, i.e., chromosomal abnormalities, isolated cardiopathy, congenital infections and known syndromes. In conclusion, the proposed investigation protocol allowed the clinical-etiological or pathogenic diagnosis in more than 90% of the cases, suggesting that the most etiopathogenic factors related to NIHF can be identified if a wide and systematic evaluation is performed Mestrado Genetica Medica Mestra em Ciências Médicas

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Mezzomo, Nathana Jamille. "AVALIAÇÃO DE LIPOSSOMAS COM CREATINA SOBRE O METABOLISMO CEREBRAL DE RATOS WISTAR COM HIPERFENILALANINEMIA." Centro Universitário Franciscano, 2015. http://www.tede.universidadefranciscana.edu.br:8080/handle/UFN-BDTD/535.

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Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-16T19:41:42Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_NathanaJamilleMezzomo.pdf: 2403536 bytes, checksum: a811beb07b8f995e286c3f3f76fad6bd (MD5) Made available in DSpace on 2018-08-16T19:41:42Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_NathanaJamilleMezzomo.pdf: 2403536 bytes, checksum: a811beb07b8f995e286c3f3f76fad6bd (MD5) Previous issue date: 2015-03-27 Phenylketonuria (PKU) is an inborn error of phenylalanine (Phe) metabolism caused by a deficiency of hepatic phenylalanine hydroxylase enzyme (EC 1.14.16.1 - PAH), which converts Phe tyrosine (Tyr), resulting in hyperphenylalaninemia. Excess Phe compromises the developing brain of affected individuals. Considering the creatine energy and neuroprotective substance, the aim of use it in liposomal form was to increase its concentration in the brain and evaluate parameters of energy metabolism, oxidative stress and behavioral usually changed by excess Phe in PKU patients or hyperphenylalaninemia (HPA) animals. Also, check whether the administrations of nanoparticles could affect non-PAH rats parameters, thus evaluating a neurotoxic action. The ethanol injection method for production of liposomes containing creatine allowed the formation of nanoscale particles with pH around the saline, with an average size of 236.44 nm, a polydispersity index below 0.3, mean zeta potential of -23.9 mV, encapsulation efficiency of 33.65% and creatine content of 93%. The HPA alter the activity of cytosolic and mitochondrial fractions of creatine kinase (CK) in the cerebral cortex and the hippocampus in the mitochondrial fraction, however, did not affect the activity of pyruvate kinase (PK) and adenilato kinase (AK) the analyzed brain structures. The administration of liposomes containing creatine (LCr) possibly prevented the alterations of cytosolic and mitochondrial CK activity in the cerebral cortex of the HPA group. Induction HPA did not change the oxidative stress parameters evaluated in the brain cortex and hippocampus. However, administration of LCr in animals non-HPA, increased levels of GSH antioxidant and superoxide dismutase activity (SOD) and in contrast, stimulated an increased of dihydrodichlorofluorescein oxidation (DCFH) in cerebral cortex. The blank liposomes (LBr) also increased the reduced glutathione (GSH) content in the cerebral cortex. Associated with these results, one can also check the cerebral impairment of the HPA animals through the reduced brain mass, open field test, tail suspension and elevated zero maze. The administration of LCr the HPA animals can partially prevent changes in enzyme activity, as well as the number of rearings improved open field test and the number of head-dipping of the elevated zero maze test. In short, we believe that the LCr may have crossed the blood-brain barrier, since its effects were differentiated administration of free creatine (Cr), thus preventing changes caused by Phe administration on behavioral tests and enzyme activity energy metabolism in the cortex and hippocampus. A fenilcetonúria (PKU) é um erro inato do metabolismo do aminoácido fenilalanina (Phe) provocado pela deficiência da enzima hepática fenilalanina hidroxilase (EC 1.14.16.1 - PAH), que converte Phe em tirosina (Tyr), resultando em hiperfenilalaninemia. O excesso de Phe compromete o desenvolvimento cerebral dos indivíduos afetados. Considerando a creatina uma substância energética e neuroprotetora, o objetivo de utilizá-la na forma lipossomal foi o de aumentar sua concentração no cérebro e avaliar parâmetros do metabolismo energético, do estresse oxidativo e comportamentais, normalmente alterados pelo excesso de Phe em pacientes PKU ou em animais com hiperfenilalaninemia (HPA). Além de verificar se as administrações das nanopartículas poderiam afetar os parâmetros de ratos não-HPA, avaliando dessa forma uma ação neurotóxica. O método de injeção de etanol utilizado para a produção dos lipossomas contendo creatina permitiu a formação de partículas nanométricas com pH próximo ao da salina, com tamanho médio de 236,44 nm, índice de polidispersão abaixo de 0,3, potencial zeta médio de -23,9 mV, eficiência de encapsulação de 33,65 % e teor de 93 %. A HPA alterou a atividade das frações citosólica e mitocondrial da creatinacinase (CK) no córtex cerebral e na fração mitocondrial no hipocampo, porém, não afetou a atividade da piruvatoquinase (PK) e adenilatoquinase (AK) nas estruturas cerebrais estudadas. A administração de lipossomas contendo creatina (LCr), possivelmente preveniu as alterações da atividade da CK citosólica e mitocondrial no córtex cerebral dos grupo HPA. A indução a HPA não alterou os parâmetros de estresse oxidativo avaliados no córtex cerebral e hipocampo. No entanto, a administração de LCr nos animais não-HPA, aumentou os níveis do antioxidante glutationa reduzida (GSH) e a atividade da superóxido dismutase (SOD) e em contrapartida, estimularam uma maior oxidação de diclodihidrofluoresceína (DCFH) em córtex cerebral. Os lipossomas brancos (LBr) também elevaram a concentração de GSH no córtex cerebral. Associado a esses resultados, pode-se também verificar o comprometimento cerebral dos animais HPA por meio da redução da massa cerebral, testes de campo aberto, suspensão da cauda e labirinto zero elevado. A administração de LCr nos animais HPA, pode prevenir parcialmente alterações das atividades enzimáticas, assim como melhorou o número de levantamentos do teste de campo aberto e o número de espiadas do teste labirinto zero elevado. Em suma, acreditamos que os LCr possam ter atravessado a barreira sangue-cérebro, uma vez que seus efeitos foram diferenciados da administração da creatina livre (Cr), conseguindo prevenir alterações causadas pela administração de Phe sobre o testes comportamentais e a atividade de enzimas do metabolismo energético do córtex e hipocampo.

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35

Carter, Kevin C. (Kevin Craig). "Population genetic variation at the human phenylalanine hydroxylase locus." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=23991.

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Denaturing gradient gel electrophoresis (DGGE) and sequencing of the PAH locus has found 38 different mutations on 141 chromosomes in the PKU patients resident in Quebec; mutation analysis is now 92.5% complete. Two novel disease producing alleles (K421, R157N) and one silent allele (IVS6 nt-55) were discovered in this project; these mutations remain unique to the Quebec population. Three novel mutation-(haplotype) combinations were also found (S67P (H1), G218V (H2), V245A (H7)); they are not at hypermutable sites and are therefore compatible with a single homologous recombination event between two different haplotypes. Whereas mutation types (missense 64%, nonsense 6%, splice 9%, frameshifts 6%, silent 15%), resemble those in world populations, the Quebec allele profile differs from that of any European population, reflecting range expansion, founder effects, genetic drift and assimilation. Furthermore, when analyzed by geographic region a stratification of PAH alleles is apparent, reflecting the different demographic histories of Western and Eastern Quebec and Montreal.

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36

Sim, Keow Giak. "Quantitative Fibroblast Acylcarnitine Profiling In The Diagnostic and Prognostic Assessment of Mitochondrial fatty acid [beta]-oxidation disorders." Thesis, The University of Sydney, 2002. http://hdl.handle.net/2123/801.

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Mitochondrial fatty acid ß-oxidation disorders are a group of clinically and biochemically heterogeneous defects mainly associated with intolerance to catabolic stress. The diseases are potentially fatal, but treatable and the prognosis for most diagnosed disorders is generally favourable. Early diagnosis is thus important to prevent morbidity and mortality. This project describes an improved and validated quantitative fibroblast acylcarnitine profile assay for the investigation of suspected fatty acid ß-oxidation disorders. Intact cells were incubated with deuterium-labelled hexadecanoate and L-carnitine, and the accumulated acylcarnitines in the medium analysed using electrospray tandem mass spectrometry. This modified procedure is less demanding technically, requires fewer cells and better reflects the in vivo acylcarnitine status than previously published methods. Mitochondrial fatty acid ß-oxidation is coupled to the respiratory chain. Functional defects of one pathway may lead to secondary alterations in flux through the other. The diagnostic specificity and the prognostic potential of the in vitro acylcarnitine profile assay were investigated in fibroblasts from 14 normal controls, 38 patients with eight enzyme deficiencies of fatty acid ß-oxidation presenting with various phenotypes, and 16 patients with primary respiratory chain defects including both isolated and multiple enzyme complex defects. All fatty acid ß-oxidation deficient cell lines revealed disease-specific acylcarnitine profiles related to the sites of defects irrespective of the severity of symptoms or of different mutation. Preliminary studies suggested a correlation between severity of symptoms and higher concentrations of long-chain acylcarnitine species. However, the fibroblast acylcarnitine profiles from some patients with respiratory chain defects were similar to those of controls, whereas others had abnormal profiles resembling those found in fatty acid ß-oxidation disorders. In vitro acylcarnitine profiling is useful for the detection of fatty acid ß-oxidation deficiencies, and perhaps the prediction of disease severity and prognostic evaluation facilitating decisions of therapeutic intervention and genetic counselling. However, abnormal profiles do not exclusively indicate these disorders, and primary defects of the respiratory chain remain a possibility. Awareness of this diagnostic pitfall will aid in the selection of subsequent confirmatory tests and therapeutic options.

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37

Harper, Peter Andrew Windsor. "Studies on neurological disorders of neonatal calves associated with spongy changes in the central nervous system : neuroaxial oedema and the inborn errors of amino acid metabolism." Thesis, The University of Sydney, 1987. https://hdl.handle.net/2123/25996.

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Investigations of neurological disease in neonatal calves were conducted over a four and a half year period. The studies commenced with so called Hereditary Neuraxial Oedema of Poll Hereford calves. It was determined that two distinct disease entities in this breed had led to confusion regarding the diagnosis of this disorder.

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38

Rahman, Alvi. "Estimating Screening Results Following the Introduction of Next-generation Sequencing Into Newborn Screening." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36988.

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Objective: The objective of this thesis was to estimate the impact on newborn screening (NBS) results of changing screening technology from tandem mass spectrometry (MS/MS) to an approach using targeted next-generation sequencing (T-NGS) and MS/MS in parallel. Methods: We integrated results of an analysis of MS/MS screening data for phenylketonuria (PKU) and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency; and a query of genetic compendia for variants of genes associated with the two disorders. Results: The introduction of T-NGS into NBS may reduce nearly 80% of false positives that are generated using the current screening approach. Based on estimated NBS results, T-NGS may be applied using a second-tier approach, which may improve specificity while maintaining sensitivity at its current level. Discussion: T-NGS may enhance the performance of NBS for PKU by improving specificity when used as a second tier test, but may be limited by feasibility and cost under current circumstances. Future studies should consider the cost-effectiveness of T-NGS for all infants undergoing NBS.

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Gradinger, Abigail. "Atypical methylmalonic aciduria : frequency of mutations in the methylmalonyl-CoA epimerase (MCEE) gene." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101848.

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Methylmalonic aciduria results from defects in the enzyme methylmalonyl-CoA mutase and from defects in the synthesis of the enzyme's cofactor adenosylcobalamin. Two patients who excrete methylmalonic acid have been shown to have a homozygous nonsense mutation in the methylmalonyl-CoA epimerase gene (MCEE). To further understand the causes of methylmalonic acid excretion, the MCEE gene was sequenced in 229 patients who excreted methylmalonic acid for which no cause was known. Mutations were detected in five patients. Fusion of fibroblast lines from two patients with a homozygous nonsense mutation in MCEE did not result in correction of [14C]propionate incorporation toward control values while the defect in these fibroblasts was complemented by mut, cblA, and cblB fibroblasts. Transfection with wild-type MCEE cDNA resulted in correction of the biochemical phenotype in cells from both patients. These experiments support the hypothesis that a defective epimerase enzyme can be a cause of elevated methylmalonic acid excretion.

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40

Mirandola, Sandra Regina. "Disfunção mitocondrial induzida por metilmalonato e 3-nitropropionato." [s.n.], 2004. http://repositorio.unicamp.br/jspui/handle/REPOSIP/311828.

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Orientador: Roger Frigerio Castilho Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Made available in DSpace on 2018-08-11T10:41:34Z (GMT). No. of bitstreams: 1 Mirandola_SandraRegina_D.pdf: 3705031 bytes, checksum: e1e34dd5e1b3949d9c308b3c20a19787 (MD5) Previous issue date: 2008 Resumo: A acidemia metilmalônica (MMAemia) é uma desordem metabólica hereditária do metabolismo de aminoácidos com cadeia ramificada e de ácidos graxos com cadeia ímpar, envolvendo um defeito na conversão de metilmalonil-CoA a succinil-CoA. Manifestações sistêmicas e neurológicas nesta doença são relacionadas com o acúmulo de metilmalonato (MMA) em tecidos e fluidos biológicos e com o comprometimento do metabolismo energético. Neste trabalho, verificou-se que o MMA inibiu com grande intensidade a conversão de lactato a piruvato catalisada pela enzima lactato desidrogenase (LDH) em homogenatos de fígado e cérebro de rato. A conversão de piruvato a lactato, catalisada pela LDH, foi menos sensível à inibição por MMA. Estudos de cinética enzimática sobre a inibição da LDH de cérebro, utilizando-se lactato como substrato, indicaram que o MMA inibe esta enzima competitivamente (Ki = 3,02 ± 0,59 mM). Propôs-se que a inibição da conversão lactato/piruvato por MMA contribui para a fisiopatologia da MMAemia, resultando, dentre outras alterações, em acúmulo de lactato e acidemia metabólica. Mostrou-se que, em mitocôndrias isoladas de cérebro e músculo de rato, concentrações milimolares de MMA inibiram o consumo de O2 mantido por succinato, enquanto nenhum efeito inibitório foi observado quando substratos para os complexos I ou IV foram utilizados. Notadamente, o efeito inibitório de MMA, mas não de malonato, no consumo mitocondrial de O2 mantido por succinato foi minimizado quando uma permeabilização não-seletiva das mitocôndrias foi induzida por alameticina. Em adição, o MMA apresentou apenas um pequeno efeito inibitório no consumo de O2 por partículas submitocondriais invertidas na presença de succinato. Não se obteve evidência de produção de malonato nas mitocôndrias tratadas com MMA. Conclui-se que o MMA inibe o consumo mitocondrial de O2 na presença de succinato por interferir na captação deste substrato pela mitocôndria. A inibição do transporte mitocondrial de substratos, induzida pelo MMA, através do carreador de dicarboxilatos, pode ter importantes implicações fisiopatológicas na MMAemia. Comparou-se a suscetibilidade de mitocôndrias isoladas de fígado, rim e coração de rato, assim como de diferentes subregiões cerebrais quanto à transição de permeabilidade mitocondrial (MPT) induzida por 3-nitropropionato (3-NP) e Ca2+. A MPT foi estimada pela queda do potencial elétrico transmembrana e inchamento mitocondrial sensíveis à ciclosporina A. Mitocôndrias de cérebro e coração foram mais suscetíveis à MPT induzida por 3-NP e Ca2+ que organelas isoladas de fígado e rim. A comparação de mitocôndrias de diferentes regiões cerebrais indicou que uma inibição parcial da respiração por 3-NP resultou em MPT mais rapidamente em organelas estriatais que corticais ou cerebelares. Em ratos tratados sistemicamente com 3-NP, verificou-se uma inibição de mesma magnitude da succinato desidrogenase em todos os tecidos estudados. Notadamente, mitocôndrias isoladas de cérebro de ratos tratados sistemicamente com 3-NP apresentaram uma maior suscetibilidade à MPT induzida por Ca2+ quanto comparadas a controles. Propôs-se que a maior suscetibilidade do estriado à neurodegeneração induzida por 3-NP pode ser, pelo menos em parte, explicada por uma maior vulnerabilidade desta região cerebral à MPT, juntamente com a vulnerabilidade desta região ao influxo de Ca2+ citosólico mediado pelo estímulo de receptores de glutamato Abstract: Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism. In the present work it was observed that MMA potently inhibited lactate dehydrogenase (LDH)-catalyzed conversion of lactate to pyruvate in liver and brain homogenates. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (Ki = 3.02 ± ?0.59 mM). We proposed that inhibition of the lactate/pyruvate conversion by MMA contributes to the MMAemia physiophatology, leading to lactate accumulation and metabolic acidemia. While millimolar concentrations of MMA inhibit succinate-supported O2 consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial O2 consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported O2 consumption by inside-out submitochondrial particles. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial O2 consumption by interfering with the uptake of this substrate. MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier may have important physiopatological implications. The susceptibility of isolated mitochondria from liver, kidney and heart and different rat brain regions (striatum, cortex and cerebellum) was compared regarding to mitochondrial permeability transition (MPT) evoked by 3-nitropropionate (3-NP) and Ca2+ ions. In general, isolated brain mitochondria from different regions were more sensitive to 3-NP and Ca2+ toxicity than mitochondria from liver and kidney as estimated by decrease in the transmembrane electrical potential and mitochondrial swelling. The comparision of different brain regions revealed that the inhibition of 50% of the mitochondrial succinate-supported respiration elicited by 3-NP resulted in a Ca2+-induced MPT pore opening, inhibited by cyclosporin A, faster in striatal than in cortical and cerebellar mitochondria. It was verified an inhibition of succinate dehydrogenase activity from the same magnitude in all tissues studied after a 3-NP systemic treatment. Interestingly, isolated forebrain mitochondria obtained from rats systemically treated with 3-NP showed a more pronounced susceptibility to Ca2+-induced MPT pore opening when compared to control rats. We proposed that the increased susceptibility of rat striatum to 3-NP-induced neurodegeneration could be in part explain by a region-specific susceptibility to MPT together with increase vulnerability of this brain region to glutamate receptors-mediated cytosolic Ca2+ influx Doutorado Biologia Estrutural, Celular, Molecular e do Desenvolvimento Doutor em Fisiopatologia Medica

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Herber, Silvani. "Doença da urina do xarope do bordo no Brasil : um panorama das duas últimas décadas." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2012. http://hdl.handle.net/10183/48974.

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Introdução: A Doença da Urina do Xarope do Bordo (DXB) é um Erro Inato do Metabolsimo (EIM), causada pela deficiência da atividade do complexo enzimático desidrogenase dos L-cetoácidos de cadeia ramificada (CACR). O programa público brasileiro de triagem neonatal não inclui o diagnóstico e o tratamento da DXB. Objetivo: Caracterizar aspectos relacionados ao diagnóstico e ao tratamento de pacientes brasileiros com DXB. Metodologia: Estudo retrospectivo. Os pacientes foram identificados a partir dos registros de laboratório considerado referência nacional para o diagnóstico de DXB, e de contato com demais serviços nacionais de referência em genética médica. O diagnóstico foi realizado entre 1992-2011. Os dados analisados foram obtidos a partir da revisão de prontuários. Resultados: Oitenta e três pacientes, oriundos de 75 famílias, foram incluídos no estudo (mediana de idade= 3 anos; IQ25- 75= 0,57-7). A mediana da idade de início dos sintomas foi de 10 dias (IQ= 5-30), enquanto que a mediana da idade ao diagnóstico foi de 60 dias (IQ= 29-240, p= 0,001). Apenas três (3,6%) pacientes foram diagnosticados antes de desenvolverem manifestações clínicas. A comparação entre os pacientes com (n=12) e sem (n=71) diagnóstico precoce mostrou que o mesmo associa-se com a presença de história familiar positiva e diminuição da prevalência de manifestações clínicas ao diagnóstico, mas não com melhor resultado; além disso, a maioria desses diagnósticos foi realizada entre 2002-2011 (n= 10/12). Considerando a amostra total, 98,8% dos pacientes apresentam atraso de desenvolvimento neuropsicomotor. Conclusão: Os pacientes com DXB são diagnosticados tardiamente no Brasil, de forma que as complicações associadas a esta condição não são prevenidas. Entretanto, existem indícios de que está havendo um melhora gradual desse panorama desde a última década. Os nossos dados indicam que o diagnóstico precoce dessa condição, mesmo que em fase sintomática, associa-se a um melhor prognóstico do paciente. Sugere-se a elaboração de políticas públicas específicas para doenças raras no país. Introduction: Maple syrup urine disease (MSUD) is an Inborn Errors of Metabolism, is caused by a deficiency in activity of the branched-chain L-keto acid dehydrogenase complex. The Brazilian neonatal screening program does not include the diagnosis and treatment of MSUD. Objective: To draw a profile of aspects related to the diagnosis and treatment of Brazilian patients who received. Methods: In this retrospective study, patients were identified through a search of records from a national reference laboratory for the diagnosis of MSUD and through contact with other medical genetics services across Brazil. A diagnosis of MSUD between 1992 and 2011. Data were collected by means of a chart review. Results: Eighty-three patients from 75 families were enrolled in the study (median age 3 years; interquartile range, 0.57–7). Median age at onset of symptoms was 10 days (IQR 5–30), whereas median age at diagnosis was 60 days (IQR 29–240, p=0.001). Only three (3.6%) patients were diagnosed before the onset of clinical manifestations. A comparison between patients with (n=12) and without (n=71) an early diagnosis show that early diagnosis is associated with the presence of positive familial history and decreased prevalence of clinical manifestations at the time of diagnosis, but not with a better outcome. Overall, 98.8% of patients have some psychomotor or neurodevelopmental delay. Conclusion: In Brazil, patients with MSUD receive a late diagnosis and show neurological compromise and poor survival even with an early diagnosis. We suggest that specific public policies for rare diseases should be developed and implemented in the country.

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Holmberg, Larsson Albin. "Biochemical characterization of resurrected ancestral ammonia lyases." Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-261355.

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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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Alves, Sofia Alexandra Micaelo. "Plano de comunicação integrada de marketing para um Laboratório de Análises Clínicas." Master's thesis, Instituto Superior de Economia e Gestão, 2013. http://hdl.handle.net/10400.5/11086.

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Mestrado em Marketing O principal objetivo da Comunicação Integrada de Marketing é juntar todas as ferramentas de marketing e comunicar de forma clara e consistente para que a empresa possa alcançar o público pretendido. Este Plano de Comunicação Integrada de Marketing destina-se a apresentar à população todos os serviços, conhecimento e tecnologia de que dispõe o Laboratório de Análises Clínicas da Faculdade Farmácia da Universidade de Lisboa. Pretende-se aumentar a notoriedade do laboratório bem como a preferência pelo mesmo. Para alcançar este objetivo recorre-se à comunicação interna e junto da comunidade local. Adicionalmente também se pretende comunicar ao nível dos Media, melhorar a comunicação na Internet, aumentar o número de parcerias, organizar eventos e comunicar ao nível da publicidade. Para mensurar os resultados das ações consideram-se indicadores como a quota de mercado, o aumento de utentes no laboratório e um questionário para se compreender de que forma os utentes tomaram conhecimento do laboratório e se ficaram satisfeitos com o serviço que lhes foi prestado. The main goal of Integrated Marketing Communication is put together all of the marketing tools and communicate the clearest and consistent way that the company can for achieve the people that they want. This Integrated Marketing Communication Plan is meant to present to the population all of the services, knowledge and technology that is at the disposal of the Clinical Analysis Laboratory of the Faculdade de Farmácia - Universidade de Lisboa. It is intended to increase the awareness of the laboratory as well as the preference for the aforementioned one. To reach this goal internal communication tactics and communication with the local community are suggested. It is also intended to communicate with the Media, improving the internet communication, increasing the number of partnerships, organizing events and communicating through publicity. To measure the actions it is planned to take some indicators in consideration as the market share, the increase of users of the laboratory and the level of satisfaction with the service that was provided to them.

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44

García, Villoria Judit. "Deficiencia de 2-metil-3-hidroxibutiril-CoA deshidrogenasa (MHBD o HSD10) e implicaciones en la enfermedad de Alzheimer." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/398985.

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Esta tesis se basa en diagnóstico de pacientes con deficiencia de 2 metil 3 hidroxibutiril CoA deshidrogenasa (MHBD o HSD10) y en el estudio de la fisiopatología de esta enfermedad y de su implicación en la Enfermedad de Alzheimer (EA). El desarrollo de un método cuantitativo de metabolitos clave en orina y un método de análisis enzimático de la proteína HSD10 en fibroblastos cultivados, así como el análisis molecular, de los niveles de HSD10 y de los estudios patogenicidad de las mutaciones encontradas, nos ha permitido el diagnóstico de 6 pacientes y 8 portadoras, que representan el 37% de los pacientes descritos en la literatura. La herencia de esta enfermedad se halla ligada al cromosoma X. Se había descrito que el gen HSD17B10, que codifica para la proteína HSD10, se escapaba de la inactivación del cromosoma X. Sin embargo, el fenotipo bioquímico y clínico de nuestros pacientes no apoyaban este hallazgo. Mediante estudios de expresión de HSD17B10 por PCR cuantitativa se demostró que HSD17B10 no escapaba a la inactivación del cromosoma X. HSD10 es una proteína mitocondrial multifuncional que además de su papel en el metabolismo de la isoleucina y de los ácidos grasos, desempeña otras funciones en el metabolismo de hormonas esteroideas sexuales, esteroides neuroactivos, en la detoxificación de aldehídos citotóxicos y en la metabolización de cardiolipina peroxidada. La clínica neurológica de los pacientes con deficiencia de HSD10 difiere de otras deficiencias de la vía metabólica de la isoleucina. Por ello, realizamos estudios de microarrays de expresión. Obtuvimos una expresión significativamente diferente en 31 genes. Los resultados se confirmaron mediante PCR cuantitativa. La diferencia de expresión de estos genes podrían explicar los síntomas clínicos de los pacientes con deficiencia de HSD10. La proteína HSD10 parece estar implicada en la EA, debido a su interacción con el péptido β amiloide (pßA), habiéndose sugerido que dicha interacción es una de las causas de la disfunción mitocondrial en la EA. Para determinar la implicación de HSD10 en la EA se realizaron una serie de estudios en homogenados de cerebro de pacientes con EA y controles. Encontramos una disminución de la actividad de HSD10 en homogenados de cerebro de pacientes con EA y esta disminución no se debía a una disminución de los niveles de proteína, ya que el wester blot no reveló diferencias entre pacientes y controles. Se observó también que la actividad HSD10 disminuye con la progresión de la enfermedad. Por otro lado, comprobamos que la incubación de homogenados de cerebro control con pßA provocaba una inhibición de la actividad HSD10, que se rescataba al incubar con elevadas concentraciones de NAD +. Esta observación también fue corroborada en la EA. Este hecho se podía explicar por una interacción competitiva entre NAD+ y pßA, ya que ambos interaccionan con HSD10 en la misma posición aminoacídica. Por ello, al incubar con elevadas concentraciones NAD+ se inhibe la interacción pßA HSD10. Por tanto, la medición de la actividad HSD10 podría ser utilizada como diana terapéutica para la EA. Con esta finalidad, obtuvimos un sistema “in vitro” con la valoración de la proteína recombinante HDSD10 isoforma 1, que es la que expresa mayoritariamente en cerebro humano, antes y después de la incubación con pßA, con y sin adición de NAD+. Los resultados obtenidos fueron los mismos que al utilizar homogenados de cerebro humano. Por lo tanto, quedaba validado un sistema para cribar moléculas terapéuticas que pudieran inhibir la interacción pßA HSD10 y restablecer la actividad HSD10. Se probaron 117 compuestos de una librería peptídica, Coenzima Q10 y otros antioxidantes, pero no se consiguió rescatar la actividad HSD10. La actividad enzimática sólo se consiguió rescatar con NAD+ . Así, proponemos que los precursores de NAD+ podrían ser considerados una terapia coadyuvante en la EA. Estudios recientes apoyan nuestros resultados, ya que la administración de precursores de NAD+ a ratones modelo para la EA, mejoran la disfunción mitocondrial así como la cognición. This thesis is based on diagnosis of patients with 2 methyl 3 hydroxybutyryl CoA dehydrogenase (MHBD or HSD10) deficiency and the study of its pathophysiology and its involvement in Alzheimer's Disease (AD). The development of different methods for quantification of key metabolites in urine and for enzymatic analysis in cultured fibroblasts, as well as the molecular studies performed, has allowed us the diagnosis of 6 patients and 8 carriers, which represents 37% of the patients described in the literature. Expression studies of HSD17B10 demonstrated that this gen not escapes inactivation of the X chromosome, contrary to that published by other authors. The neurological symptoms of patients with deficiency HSD10 differs from other deficiencies in the isoleucine's metabolic pathway, maybe due to the alteration of other functions involved in both, brain and mitochondrial homeostasis. The expression "microarray" studies performed revealed significantly different expression in 31 genes, which could explain the clinical symptoms of the patients. The HSD10 protein appears to be involved in AD, due to their interaction with β amyloid peptide (Aβ). Studies in brain homogenates of AD patients showed decreased HSD10 activity from the early stages of the disease, which could explain the early mitochondrial dysfunction observed in AD. Furthermore, the Aβ incubation caused inhibition of HSD10 activity in control brain homogenates, which was rescued by incubating with high NAD+ concentrations. This observation was also corroborated in AD. This fact could be explained by a competitive interaction that occurs between NAD+ and Aβ. Therefore, the measurement of HSD10 activity could be used as a therapeutic target for AD. To this end, we obtained "in vitro" system to the evaluation of the HDSD10 isoform 1 recombinant protein, which is the mainly expressed in human brain, before and after incubation with Aβ, with and without addition of NAD+. The results were the same as when using human brain homogenates. Therefore, the system was validated for screening therapeutic molecules that could inhibit the Aβ HSD10 interaction and restore HSD10 activity. Among 117 compounds tested, only NAD+ was able to rescue HSD10 activity. Thus, we propose that the precursors of NAD+ could be considered an adjunctive therapy in AD. Recent studies support our results, since administration of NAD precursors mice model for AD, improve mitochondrial dysfunction and cognition.

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45

Hinttala, R. (Reetta). "Genetic causes of mitochondrial complex I deficiency in children." Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514282884.

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Abstract The mitochondrial oxidative phosphorylation system is composed of five multisubunit enzyme complexes. Complex I is the first and largest of these, containing 46 subunits, seven encoded by mitochondrial DNA (mtDNA) and the rest by nuclear DNA. Isolated complex I deficiency is a major cause of metabolic errors in infancy and childhood, presenting as encephalomyopathies or multisystem disorders. Due to the bigenomic origin of complex I, the genetic causes of these defects can be either mitochondrial or nuclear. The object of the present work was to identify the underlying genetic cause in cases of children with complex I deficiency and to obtain more information on the structurally and functionally important sites of complex I subunits. The complete coding region of mtDNA was analysed by conformation-sensitive gel electrophoresis and subsequent sequencing. In addition, nine nuclear genes encoding conserved subunits of complex I were sequenced. The structural and functional consequences of the new sequence variants were further elucidated using mutagenesis of homologous residue in bacterial NDH-1 or by studying complex I assembly and expression in patient cell lines. Analysis of the mtDNA coding region in 50 children revealed four definitely pathogenic mutations, 3460G>A, 10191T>C, 11778G>A and 14487T>C, in seven patients. In addition, two novel mtDNA base pair substitutions were identified, 3866T>C in a patient with muscle weakness and short stature and 4681T>C in a patient with Leigh syndrome. The latter mutation causes a Leu71Pro amino acid exchange in the ND2 subunit. Cybrid clones harbouring this mutation retained the complex I defect, and reduced amounts of fully assembled complex I were detected in patient cell lines. The 3866T>C mutation leads to a Ile187Thr amino acid substitution in the ND1 subunit, and functional studies of the homologous amino acid substitution in E. coli showed that this had an effect on the assembly or stability of the NDH-1 holoenzyme. Sequencing of the nine nuclear-encoded complex I genes revealed only one novel base pair substitution with pathogenic potential. Further studies are needed, however, to establish the role of the Arg18Cys substitution in the mitochondrial leading peptide of the TYKY subunit. The above findings emphasize the contribution of mtDNA mutations to the aetiology of pediatric patients with complex I deficiency. Furthermore, two LHON primary mutations were identified in the present cohort of patients, although the clinical signs differed considerably from the classical symptoms of LHON. This suggests that the phenotype caused by primary LHON mutations is more variable than has so far been thought.

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46

Sim, Keow Giak. "Quantitative Fibroblast Acylcarnitine Profiling In The Diagnostic and Prognostic Assessment of Mitochondrial Fatty Acid �-Oxidation Disorders." University of Sydney. Paediatrics and Child Health, 2002. http://hdl.handle.net/2123/801.

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Mitochondrial fatty acid �-oxidation disorders are a group of clinically and biochemically heterogeneous defects mainly associated with intolerance to catabolic stress. The diseases are potentially fatal, but treatable and the prognosis for most diagnosed disorders is generally favourable. Early diagnosis is thus important to prevent morbidity and mortality. This project describes an improved and validated quantitative fibroblast acylcarnitine profile assay for the investigation of suspected fatty acid �-oxidation disorders. Intact cells were incubated with deuterium-labelled hexadecanoate and L-carnitine, and the accumulated acylcarnitines in the medium analysed using electrospray tandem mass spectrometry. This modified procedure is less demanding technically, requires fewer cells and better reflects the in vivo acylcarnitine status than previously published methods. Mitochondrial fatty acid �-oxidation is coupled to the respiratory chain. Functional defects of one pathway may lead to secondary alterations in flux through the other. The diagnostic specificity and the prognostic potential of the in vitro acylcarnitine profile assay were investigated in fibroblasts from 14 normal controls, 38 patients with eight enzyme deficiencies of fatty acid �-oxidation presenting with various phenotypes, and 16 patients with primary respiratory chain defects including both isolated and multiple enzyme complex defects. All fatty acid �-oxidation deficient cell lines revealed disease-specific acylcarnitine profiles related to the sites of defects irrespective of the severity of symptoms or of different mutation. Preliminary studies suggested a correlation between severity of symptoms and higher concentrations of long-chain acylcarnitine species. However, the fibroblast acylcarnitine profiles from some patients with respiratory chain defects were similar to those of controls, whereas others had abnormal profiles resembling those found in fatty acid �-oxidation disorders. In vitro acylcarnitine profiling is useful for the detection of fatty acid �-oxidation deficiencies, and perhaps the prediction of disease severity and prognostic evaluation facilitating decisions of therapeutic intervention and genetic counselling. However, abnormal profiles do not exclusively indicate these disorders, and primary defects of the respiratory chain remain a possibility. Awareness of this diagnostic pitfall will aid in the selection of subsequent confirmatory tests and therapeutic options.

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47

Lemonde, Hugh Arnold. "A study using tandem mass spectrometry and molecular biological techniques to facilitate the diagnoses of inborn errors of bile acid synthesis : with particular reference to #delta#'4-3-oxostero-reductase synthesis deficiency." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342301.

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Pérez, Martí Albert. "The role of FGF21 in the metabolic response to amino acid restriction." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/401895.

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Obesity and associated metabolic diseases have reached epidemic proportions, affecting not only high-income countries but also low- and middle-income ones. In this context, the search for therapeutic approaches to treat obesity is becoming a priority worldwide. In this regard, the metabolic hormone fibroblast growth factor 21 (FGF21) has been identified as a potential candidate for the treatment of obesity and metabolic syndrome. Previous work by our group described that FGF21 is highly induced in liver in response to leucine deprivation and that the transcription factor ATF4 mediates this induction. The present work is the follow-up of this initial observation. To delve deeper into the molecular mechanisms that regulate FGF21 expression during leucine deprivation, we focused on the transcriptional repressor Rev-erbα, which functions both as a core repressive component of the cell autonomous clock and as a regulator of metabolic genes. Our results reveal a consistent negative correlation between Fgf21 and the Pgc-1α/heme/Rev-erbα axis across various nutritional states and that a decrease in Rev-erbα activity enhances the ATF4-mediated upregulation of the human FGF21 promoter. Consequently, we propose a model whereby the induction of Fgf21 upon leucine deprivation is the consequence of the sum of two factors: binding of the activator ATF4 to the promoter and the absence of the repressor Rev-erbα. Given the coincidence between the effects of leucine deprivation and those observed during FGF21 treatment, we analysed the role of FGF21 during leucine deprivation. In the current study, we demonstrate that weight loss, downregulation of key lipogenic genes in liver and WAT, and BAT activation in response to leucine deprivation are partly FGF21-dependent. Given the unfeasibility to translate single amino acid deprivation to humans, we focussed on low-protein diets (LPDs) as a more realistic approach. The LPD increased circulating FGF21 levels with an associated upregulated expression in liver. Analysis of serum human samples from the PREDIMED study extended the correlation between LPD and FGF21 to humans. The ATF4-mediated upregulation of Fgf21 in liver was partially responsible for the weight loss observed in mice fed a LPD, since the liver specific Fgf21 knockout mice (LFgf21KO) mice were partially protected from this loss. Focusing on the effects of FGF21 on scWAT and given the capacity of FGF21 to produce the browning of white fat depots, we examined the activation of the thermogenic programme in this tissue. Accordingly, scWAT browning caused by the LPD did not occur in mice lacking hepatic Fgf21. As UCP1 activity is related to EE, the blunted induction of Ucp1 in the LPD-fed LFgf21KO mice, may contribute to the reduction in weight loss observed in this mouse model under these circumstances. The administration of the b-blocker propranolol to protein-restricted mice allowed us to distinguish between the roles of FGF21 and noradrenaline. While Ucp1 expression was upregulated independently of adrenergic signalling, Dio2 and Pparγ expression was blunted by propranolol treatment. These results point to the induction of Ucp1 as a direct effect of liver-delivered FGF21 on scWAT and discard a CNS-mediated effect. In addition, the LPD improved glucose tolerance, and this improvement was not observed in LFgf21KO mice, indicating a role of FGF21 in glucose metabolism during protein restriction. Our findings show that the effects of a LPD depend, at least in part, on the circulating levels of FGF21 and consequently on the liver production of this growth factor. Given the parallelism between the results of our study in humans and those in mice, we postulate that modulation of dietary protein content can bring about changes in the circulating levels of FGF21 in mice and humans. L’obesitat i les malalties metabòliques que en deriven són un problema de salut mundial. En aquest context, la recerca d’estratègies terapèutiques pel tractament de l’obesitat ha esdevingut una prioritat. En aquest sentit, el factor metabòlic fibroblast growth factor 21 (FGF21), ha estat identificat com a un prometedor candidat pel tractament de l’obesitat i la síndrome metabòlica. El nostre laboratori va descriure que en resposta a la privació de leucina els nivells de FGF21 augmenten dràsticament i que el factor de transcripció activating transcription factor (ATF4) n’és el responsable. Els resultats d’aquesta tesi són la continuació i desenvolupament d’aquesta observació inicial. Aprofundint en els mecanismes de regulació que controlen l’expressió de FGF21 en resposta a la privació de leucina, els nostres resultats indiquen que el repressor transcripcional Rev-erbα participa significativament en aquesta regulació i que la disminució dels nivells de Rev-erbα correlacionen amb una activació del promotor de FGF21. A més a més, en aquest estudi demostrem que la pèrdua de pes, la disminució de l’expressió de gens lipogènics en fetge i teixit adipós blanc, així com l’activació del teixit adipós marró en resposta a la privació de leucina són, al menys parcialment, dependents de FGF21. Finalment, amb la finalitat de fer els nostres resultats traslladables a humans, demostrem que una dieta amb baix contingut proteic augmenta les nivells circulants de FGF21 en ratolins i humans, i que això succeeix a través de l’increment d’ATF4. L’increment dels nivells de FGF21 degut a la restricció proteica provoquen l’increment de l’expressió dels gens termogènics en el teixit adipós blanc subcutani, la pèrdua de pes i la millora la tolerància a la glucosa. El conjunt d’aquest resultats destaquen el paper clau del factor FGF21 com a mediador dels efectes metabòlics que es produeixen durant la restricció d’aminoàcids i suggereixen la disminució del contingut de proteïna de la dieta com a estratègia per incrementar-ne els nivells.

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49

Chipeaux, Caroline. "Recherche et validation de biomarqueurs lipidiques du globule rouge par chromatographie en phase liquide couplée à la spectrométrie de masse. Application au diagnostic et au suivi thérapeutique de la maladie de Gaucher." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS419.

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Chez l’homme, les erreurs innées du métabolisme des lipides sont dues à des déficits enzymatiques, entraînant une accumulation intracellulaire de substrats lipidiques. Il en résulte un large éventail de symptômes tels que des atteintes viscérales, osseuses et dans certains cas neurologiques. En outre, de nombreux patients atteints de ce type de maladie présentent des anomalies hématologiques et vasculaires attribuées à des anomalies rhéologiques du globule rouge (GR). Ces observations ont conduit à l’hypothèse de l’existence d’un lien entre les propriétés anormales du GR et sa composition lipidique. Or actuellement, le profil lipidique du GR normal reste méconnu. Cependant, le diagnostic précoce de ces troubles est d’une importance capitale pour la prise en charge des patients, notamment dans les cas où un traitement correctif est disponible. La maladie de Gaucher (MG) de type 1, qui est une maladie lysosomale caractérisée par un déficit en β-glucocérébrosidase et pour laquelle un traitement enzymatique substitutif (ERT) est proposé, en est le meilleur exemple. D’où l’intérêt de disposer d’un outil simple et rapide de diagnostic de ce type de maladie.Dans le cas de la MG, le diagnostic repose encore sur la mise en évidence, laborieuse, du déficit enzymatique. Néanmoins, des travaux récents suggèrent que les anomalies rhéologiques du GR pourraient être dues à l’accumulation de quatre sphingolipides, le glucosylcéramide, la glucosylsphingosine, la sphingosine et la sphingosine-1-phosphate, qui seraient de bons candidats biomarqueurs. Or, les méthodes actuelles de dosage de ces sphingolipides nécessitent au moins deux étapes chromatographiques, avec pour chacune une étape longue et fastidieuse de préparation de l’échantillon, ce qui ne facilite guère une approche lipidomique de ce sujet. En outre, seul le glucosylcéramide a été dosé dans le GR tandis que les trois autres sphingolipides n’ont été dosés que dans le plasma. Ces candidats biomarqueurs restent donc à valider.Dans cette thèse, nous avons développé et validé une méthode simple et rapide, par UHPLC-MS/MS, de dosage simultané des 4 sphingolipides impliqués dans la MG. L’application de cette méthode à des GR provenant de patients atteints de la MG, en collaboration avec l’Institut National de Transfusion Sanguine et la société Shire, nous a permis de : 1- valider un biomarqueur parmi les quatre proposés et de montrer que les trois autres n’étaient pas suffisamment spécifiques ; 2- vérifier l’efficacité du traitement ERT actuellement proposé et 3- confirmer l’hypothèse de départ reliant les anomalies rhéologiques du GR à sa composition lipidique.De même, une étude systématique des conditions opératoires nous a permis de généraliser la méthode proposée à l’identification et au dosage de l’ensemble des sphingolipides présents dans un GR ainsi que des phospholipides, constituants majoritaires de sa membrane. Appliquée à la quantification simultanée d’une trentaine de sphingolipides et de phospholipides dans le GR normal et celui de la MG, cette méthode nous a permis de mettre en évidence l’implication d’autres lipides polaires dans la maladie de Gaucher, outre les 4 sphingolipides jusqu’alors proposés. De même, il est prévu de l’adapter à moyen terme pour le profilage total, par classe, de tous les lipides présents dans le GR.Enfin, nous avons évalué d’autres techniques de SM telles que la haute résolution et la mobilité ionique (TWIMS et DIMS) dans le but d’affiner la recherche de nouveaux biomarqueurs, notamment par l’identification des lipides isomères non discriminables par les techniques de MS conventionnelles. Grâce à une collaboration avec le Laboratoire de Chimie Physique (LCP, CNRS UMR 8000) nous avons montré la faisabilité de cette approche en séparant en DIMS deux isomères : la galactosylsphingosine 18:1 et la glucosylsphingosine 18:1 et nous poursuivons actuellement cette étude pour séparer d’autres couples d’isomères In humans, hereditary disorders of lipid metabolism are due to enzyme deficiencies, resulting in intracellular accumulation of lipid substrates. This results in a wide range of symptoms such as visceral, bone and in some cases neurological disorders. Furthermore, many patients suffering such diseases have hematologic and vascular symptoms attributed to red blood cell (RBC) rheological abnormalities. These observations led to a hypothesis linking RBC abnormal properties to its lipid composition. However, the lipid profile of normal RBC remains unknown to date. Early diagnosis of these conditions is of importance notably when a therapy is available. This is the case for Gaucher disease (GD) type 1, a lysosomal disorder characterized by β-glucocerebrosidase deficiency, where an enzyme replacement therapy (ERT) is proposed. Hence, the availability of a simple and rapid tool of diagnosis of such a disorder is of great importance, notably for a better patient care and monitoring.To the best of our knowledge, standard diagnosis procedures and monitoring of GD patients are still based on the tedious evaluation of enzyme deficiency. Nevertheless, recent works suggest that these rheological disorders may be due to the accumulation of four sphingolipids, glucosylceramide, glucosylsphingosine, sphingosine and sphingosine-1-phosphate, which could be considered as relevant biomarkers. However, most of current determination methods of these sphingolipids require at least two liquid chromatographic runs, each with a time-consuming sample preparation step that does not facilitate a lipidomic approach. In addition, only glucosylceramide was quantified in RBC while the other three sphingolipids were quantified only in plasma. Thus, these biomarker candidates remain to be validated.In this PhD, we describe a simple and rapid UHPLC-MS/MS method of simultaneous determination of the 4 sphingolipids involved in GD in both plasma and RBC. The application of this method to RBC from GD patients, in collaboration with the Institut National de Transfusion Sanguine and Shire (USA), allowed us: 1- to validate one biomarker among the four proposed candidates and to show that the other three candidates are not specific; 2- to check the efficiency of the proposed ERT and 3- to confirm the initial hypothesis linking the RBC rheological abnormalities to its lipid composition.Also, a systematic study of the operating conditions allowed us to generalize the proposed method to the determination of not only all the sphingolipids present in RBC but also all phospholipids, which are the major constituents of its membrane. The application of the later method to the simultaneous quantification of thirty sphingolipids and phospholipids in normal and GD RBCs, allowed us to validate it and to unravel the involvement of other candidate biomarkers of GD, different from the 4 previous sphingolipids. Providing appropriate modifications, this method is intended to be used for the profiling of all lipid classes in plasma and RBC. This is our main objective in the medium-term.Finally, we evaluated other modern MS techniques such as high resolution (HRMS) and ion mobility (TWIMS and DIMS) in order to refine the investigation of new biomarker candidates, including the separation of lipid isomers that cannot be discriminated by conventional MS techniques. Indeed, in collaboration with the Laboratoire de Chimie Physique (LCP, CNRS UMR 8000), we here show the feasibility of this approach by achieving the separation of two isomers, by the DIMS technique: galactosylsphingosine 18:1 and glucosylsphingosine 18:1, which cannot be separated by conventional methods. We are currently pursuing these investigations in order to separate other isomers

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Benetó, Gandia Noelia. "iPSCs, CRISPR/Cas9 y protocolos de diferenciación basados en factores de transcripción para generar nuevos modelos neuronales y astrocíticos del síndrome de Sanfilippo." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/673991.

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El objetivo general de esta tesis doctoral ha sido la generación de nuevos modelos celulares para el síndrome de Sanfilippo y su utilización para ensayar una aproximación terapéutica para esta enfermedad basada en el uso de siRNAs como terapia de reducción de sustrato. Los objetivos concretos han sido: 1) Generación de modelos celulares • Obtención y caracterización de líneas isogénicas con mutaciones en el gen HGSNAT derivadas de células madre pluripotentes inducidas de donantes sanos, mediante la tecnología de edición génica CRISPR/Cas9. • Diferenciación de las células madre pluripotentes inducidas con mutaciones en el gen HGNSAT a neuronas y astrocitos, utilizando un protocolo optimizado. • Obtención y caracterización de líneas isogénicas con mutaciones en el gen NAGLU derivadas de células madre pluripotentes inducidas de donantes sanos, mediante la tecnología de edición génica CRISPR/Cas9. 2) Aproximación terapéutica basada en siRNAs • Evaluación del efecto de diferentes siRNAs contra los genes EXTL2 y EXTL3, como terapia de reducción de sustrato, en fibroblastos de pacientes de Sanfilippo C. • Evaluación del efecto terapéutico de siRNAs contra el gen EXTL2, como terapia de reducción de sustrato, en neuronas derivadas de células madre pluripotentes inducidas con mutaciones en HGSNAT.

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Tesis sobre el tema "Inborn errors of"

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