A Doença de Gaucher (DG) é a Doença Lisossômica de Depósito mais frequente com prevalência de 1:50.000. Ela é causada pela deficiência da betaglicosidase ácida (GBA), gerando acúmulo de glicosilceramidas (glicocerebrosídio) nos lisossomos. Outra enzima relacionada é a quitotriosidase (QT), onde a atividade está aumentada nos pacientes em até 1000 vezes. Atualmente o diagnóstico é baseado na atividade específica de enzimas em leucócitos ou fibroblastos. O uso de sangue impregnado em papel filtro (SPF) vem sendo ampliado, porém somente como forma de triagem, pois não há forma validada de estabelecer a atividade específica da enzima. A utilização de sangue em papel filtro tem diversas vantagens, tais como: fácil transporte, fácil armazenagem das amostras, menor volume de reação e segurança de manipulação das amostras. Esse estudo tem como objetivo validar técnicas fluorimétricas para o estabelecimento da atividade específica da GBA e QT em sangue impregnado em papel filtro eluido com tampão universal (20 mmol/L de fosfato de sódio, pH 7,0) através da correção do volume amostral pela quantificação de proteínas totais. Além disso, foram miniaturizadas as técnicas padrão para a triagem em SPF e as técnicas confirmatórias padrão ouro em leucócitos da DG. Foram estabelecidos novos valores de referência para todas as técnicas estabelecidas. Foi possível diferenciar os controles saudáveis dos pacientes com DG utilizando as técnicas miniaturizadas em SPF e leucócitos. A atividade específica em SPF se mostrou valida e os coeficientes de variação foram considerados aceitáveis. A estabilidade enzimática foi analisada por 21 dias de armazenamento a 4°C e foi observado que a há um decaimento da atividade da GBA, mas não da QT. Deste modo a atividade específica em SPF pode ser utilizada de forma confiável como método de triagem e confirmação do diagnóstico de DG. / Gaucher disease (GD) is the most frequent Lysosomal Storage Disorder with a prevalence of 1:50,000. It is caused by the deficiency of acid betaglucosidase (GBA), generating glucosylceramide (glucocerebroside) accumulation in the lysosomes. Another enzyme related to GD is chitotriosidase (CT), which activity is increased in patients up to 1000 times. Currently the diagnosis is based on the specific activity of enzymes in leukocytes or fibroblasts. The use of dried blood spots (DBS) has been extended, but only as screening, because there is no validated specific activity of the enzymes. The use of DBS has several advantages, such as easy transport and easy storage of samples, smaller reaction volume and safety of handling samples. This study aims to validate fluorometric techniques for establishing specific activity of the GBA and CT in DBS eluted with universal buffer (20 mmol/L sodium phosphate, pH7.0) by correcting for sample volume quantification of total protein. Moreover, standard screening techniques in DBS and standard diagnosis techniques in leukocytes for GD have been miniaturized. New reference values and cut-off points were established for all techniques. It was possible to differentiate healthy controls from patients with GD using miniaturized techniques in DBS and leukocytes. The specific activity of DBS proved valid and coefficients of variation were acceptable. The enzymatic stability was analyzed by 21 days of storage at 4° C and it was observed that there is a decrease of the activity of GBA, but not of CT. Thus the specific activity on DBS can be used as a reliable screening method and as diagnosis of GD.

A Doença de Gaucher (DG) é a Doença Lisossômica de Depósito mais frequente com prevalência de 1:50.000. Ela é causada pela deficiência da betaglicosidase ácida (GBA), gerando acúmulo de glicosilceramidas (glicocerebrosídio) nos lisossomos. Outra enzima relacionada é a quitotriosidase (QT), onde a atividade está aumentada nos pacientes em até 1000 vezes. Atualmente o diagnóstico é baseado na atividade específica de enzimas em leucócitos ou fibroblastos. O uso de sangue impregnado em papel filtro (SPF) vem sendo ampliado, porém somente como forma de triagem, pois não há forma validada de estabelecer a atividade específica da enzima. A utilização de sangue em papel filtro tem diversas vantagens, tais como: fácil transporte, fácil armazenagem das amostras, menor volume de reação e segurança de manipulação das amostras. Esse estudo tem como objetivo validar técnicas fluorimétricas para o estabelecimento da atividade específica da GBA e QT em sangue impregnado em papel filtro eluido com tampão universal (20 mmol/L de fosfato de sódio, pH 7,0) através da correção do volume amostral pela quantificação de proteínas totais. Além disso, foram miniaturizadas as técnicas padrão para a triagem em SPF e as técnicas confirmatórias padrão ouro em leucócitos da DG. Foram estabelecidos novos valores de referência para todas as técnicas estabelecidas. Foi possível diferenciar os controles saudáveis dos pacientes com DG utilizando as técnicas miniaturizadas em SPF e leucócitos. A atividade específica em SPF se mostrou valida e os coeficientes de variação foram considerados aceitáveis. A estabilidade enzimática foi analisada por 21 dias de armazenamento a 4°C e foi observado que a há um decaimento da atividade da GBA, mas não da QT. Deste modo a atividade específica em SPF pode ser utilizada de forma confiável como método de triagem e confirmação do diagnóstico de DG. / Gaucher disease (GD) is the most frequent Lysosomal Storage Disorder with a prevalence of 1:50,000. It is caused by the deficiency of acid betaglucosidase (GBA), generating glucosylceramide (glucocerebroside) accumulation in the lysosomes. Another enzyme related to GD is chitotriosidase (CT), which activity is increased in patients up to 1000 times. Currently the diagnosis is based on the specific activity of enzymes in leukocytes or fibroblasts. The use of dried blood spots (DBS) has been extended, but only as screening, because there is no validated specific activity of the enzymes. The use of DBS has several advantages, such as easy transport and easy storage of samples, smaller reaction volume and safety of handling samples. This study aims to validate fluorometric techniques for establishing specific activity of the GBA and CT in DBS eluted with universal buffer (20 mmol/L sodium phosphate, pH7.0) by correcting for sample volume quantification of total protein. Moreover, standard screening techniques in DBS and standard diagnosis techniques in leukocytes for GD have been miniaturized. New reference values and cut-off points were established for all techniques. It was possible to differentiate healthy controls from patients with GD using miniaturized techniques in DBS and leukocytes. The specific activity of DBS proved valid and coefficients of variation were acceptable. The enzymatic stability was analyzed by 21 days of storage at 4° C and it was observed that there is a decrease of the activity of GBA, but not of CT. Thus the specific activity on DBS can be used as a reliable screening method and as diagnosis of GD.

A produção de proteínas recombinantes, principalmente para uso farmacêutico, tem sido intensamente estudada, juntamente com suas propriedades físico-químicas, o que possibilita uma melhor escolha da técnica de purificação, e assim, evitar perdas no rendimento e custos elevados. A glicocerebrosidase (GCR) é uma enzima lisossomal, e sua deficiência ocasiona um distúrbio autossômico recessivo denominado doença de Gaucher. Atualmente o tratamento para essa patologia é por meio da Terapia de Reposição Enzimática (TRE), a qual tem sido realizada com grande êxito. Tendo em vista fornecer dados que possam auxiliar na redução do número de etapas cromatográficas no processo de downstream, este trabalho teve como objetivo, a purificação da GCR expressa por células de Ovário de Hamster Chinês (CHO), em meio livre de soro fetal bovino (SFB). Para se alcançar tais resultados, realizou-se o cultivo das células CHO-GCR em meio quimicamente definido livre de SFB (CHO-S-SFM II). Foram realizadas três técnicas cromatográficas (troca iônica, interação hidrofóbica e afinidade), com base em suas propriedades físico-químicas. E para o ensaio da atividade enzimática, foi utilizado o substrato fluorogênico 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G). As células CHO-GCR cultivadas em meio CHO-S-SFM II, apresentando viabilidade maior que 95%, e produção da GCR ativa, durante todo o período de cultivo. Os protocolos aplicados para a purificação da GCR, não apresentaram resultados significativos. Com o volume não retido após cromatografia por interação hidrofóbica, se estimou os valores de KM 2,13 e VMAX 0,0295 UFR/h para as constantes cinéticas da GCR. A diálise no processo de purificação mostrou ser uma etapa necessária para a atividade enzimática da GCR. No cultivo das células CHO-GCR para a formação do banco de trabalho, nos meios RPMI 1640 e α-MEM, ambos com a adição de 10% SFB, não houve diferença significativa no crescimento entre eles, e apresentaram 100% de viabilidade durante todo o período de cultivo. Porém, ao analisar de forma isolada a fase exponencial de cada curva, notou-se que às células cultivadas no meio RPMI 1640, apresentaram taxa de crescimento superior, às cultivadas em meio α-MEM. Concluiu-se que a expressão da GCR em meio livre de SFB, proporciona amostras menos complexas, em relação aos meios de cultura que necessitam de suplementação com SFB, o que pode reduzir o número de etapas cromatográficas, melhorando o rendimento e a redução da perda da atividade da GCR. O meio basal RPMI 1640 com a adição de SFB foi uma alternativa satisfatória para o cultivo das células CHO-GCR. Este estudo forneceu dados que podem contribuir para a melhoria do processo de purificação da GCR. Novas pesquisas podem ser desenvolvidas a fim de melhorar o processo de purificação da GCR. / The production of recombinant proteins, mainly for pharmaceutical use, has been intensively studied along with its physico-chemical properties, which allows a better choice of the purification technique, and thus avoid losses in yield and high costs. Glucocerebrosidase (GCR) is a lysosomal enzyme, and its deficiency causes an autosomal recessive disorder called Gaucher\'s disease. Currently the treatment for this pathology is through Enzymatic Replacement Therapy (ERT), which has been successfully performed. In order to provide data that may help reducing the number of chromatographic steps in the downstream process, this work aimed to purify GCR expressed by Chinese Hamster Ovary (CHO) cells in fetal bovine serum free (FBS). To achieve such results, the CHO-GCR cells were cultured in chemically defined Serum-free medium (CHO-S-SFM II). Three chromatographic techniques (ion exchange, hydrophobic interaction and affinity) were performed, based on their physicochemical properties. And for the enzymatic activity assay, the fluorogenic substrate 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G) was used. CHO-GCR cells cultured in CHO-S-SFM II medium, presenting viability greater than 95% and GCR production active, throughout the culture period. The protocols applied for GCR purification did not present significant results. With the volume not retained after chromatography by hydrophobic interaction, KM values of 2.13 and VMAX 0.0295 UFR/h were estimated for GCR kinetic constants. Dialysis in the purification process was shown to be a step necessary for the enzymatic activity of GCR. In the culture of the CHO-GCR cells for the formation of the working bank, in the media RPMI 1640 and α-MEM, both with the addition of 10% FBS, there was no significant growth difference between them, and they showed 100% viability during all the growing period. However, when analyzing in isolation, the exponential phase of each curve, it was observed that the cells grown in RPMI 1640 medium showed higher growth rates, tham grown in α-MEM medium. It was concluded that the expression of GCR in serum-free medium provides less complex samples, relative to the culture media requiring FBS supplementation, which may reduce the number of chromatographic steps, improving yield and loss reduction of GCR activity. The basal medium RPMI 1640 with the addition of FBS was a satisfactory alternative for culturing the CHO-GCR cells. This study provided data that may contribute to the improvement of the GCR purification process. New research can be developed to improve the GCR purification process.

A produção de proteínas recombinantes, principalmente para uso farmacêutico, tem sido intensamente estudada, juntamente com suas propriedades físico-químicas, o que possibilita uma melhor escolha da técnica de purificação, e assim, evitar perdas no rendimento e custos elevados. A glicocerebrosidase (GCR) é uma enzima lisossomal, e sua deficiência ocasiona um distúrbio autossômico recessivo denominado doença de Gaucher. Atualmente o tratamento para essa patologia é por meio da Terapia de Reposição Enzimática (TRE), a qual tem sido realizada com grande êxito. Tendo em vista fornecer dados que possam auxiliar na redução do número de etapas cromatográficas no processo de downstream, este trabalho teve como objetivo, a purificação da GCR expressa por células de Ovário de Hamster Chinês (CHO), em meio livre de soro fetal bovino (SFB). Para se alcançar tais resultados, realizou-se o cultivo das células CHO-GCR em meio quimicamente definido livre de SFB (CHO-S-SFM II). Foram realizadas três técnicas cromatográficas (troca iônica, interação hidrofóbica e afinidade), com base em suas propriedades físico-químicas. E para o ensaio da atividade enzimática, foi utilizado o substrato fluorogênico 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G). As células CHO-GCR cultivadas em meio CHO-S-SFM II, apresentando viabilidade maior que 95%, e produção da GCR ativa, durante todo o período de cultivo. Os protocolos aplicados para a purificação da GCR, não apresentaram resultados significativos. Com o volume não retido após cromatografia por interação hidrofóbica, se estimou os valores de KM 2,13 e VMAX 0,0295 UFR/h para as constantes cinéticas da GCR. A diálise no processo de purificação mostrou ser uma etapa necessária para a atividade enzimática da GCR. No cultivo das células CHO-GCR para a formação do banco de trabalho, nos meios RPMI 1640 e α-MEM, ambos com a adição de 10% SFB, não houve diferença significativa no crescimento entre eles, e apresentaram 100% de viabilidade durante todo o período de cultivo. Porém, ao analisar de forma isolada a fase exponencial de cada curva, notou-se que às células cultivadas no meio RPMI 1640, apresentaram taxa de crescimento superior, às cultivadas em meio α-MEM. Concluiu-se que a expressão da GCR em meio livre de SFB, proporciona amostras menos complexas, em relação aos meios de cultura que necessitam de suplementação com SFB, o que pode reduzir o número de etapas cromatográficas, melhorando o rendimento e a redução da perda da atividade da GCR. O meio basal RPMI 1640 com a adição de SFB foi uma alternativa satisfatória para o cultivo das células CHO-GCR. Este estudo forneceu dados que podem contribuir para a melhoria do processo de purificação da GCR. Novas pesquisas podem ser desenvolvidas a fim de melhorar o processo de purificação da GCR. / The production of recombinant proteins, mainly for pharmaceutical use, has been intensively studied along with its physico-chemical properties, which allows a better choice of the purification technique, and thus avoid losses in yield and high costs. Glucocerebrosidase (GCR) is a lysosomal enzyme, and its deficiency causes an autosomal recessive disorder called Gaucher\'s disease. Currently the treatment for this pathology is through Enzymatic Replacement Therapy (ERT), which has been successfully performed. In order to provide data that may help reducing the number of chromatographic steps in the downstream process, this work aimed to purify GCR expressed by Chinese Hamster Ovary (CHO) cells in fetal bovine serum free (FBS). To achieve such results, the CHO-GCR cells were cultured in chemically defined Serum-free medium (CHO-S-SFM II). Three chromatographic techniques (ion exchange, hydrophobic interaction and affinity) were performed, based on their physicochemical properties. And for the enzymatic activity assay, the fluorogenic substrate 4-Methylumbeliferyl β-D-glucopyranoside (4MU-G) was used. CHO-GCR cells cultured in CHO-S-SFM II medium, presenting viability greater than 95% and GCR production active, throughout the culture period. The protocols applied for GCR purification did not present significant results. With the volume not retained after chromatography by hydrophobic interaction, KM values of 2.13 and VMAX 0.0295 UFR/h were estimated for GCR kinetic constants. Dialysis in the purification process was shown to be a step necessary for the enzymatic activity of GCR. In the culture of the CHO-GCR cells for the formation of the working bank, in the media RPMI 1640 and α-MEM, both with the addition of 10% FBS, there was no significant growth difference between them, and they showed 100% viability during all the growing period. However, when analyzing in isolation, the exponential phase of each curve, it was observed that the cells grown in RPMI 1640 medium showed higher growth rates, tham grown in α-MEM medium. It was concluded that the expression of GCR in serum-free medium provides less complex samples, relative to the culture media requiring FBS supplementation, which may reduce the number of chromatographic steps, improving yield and loss reduction of GCR activity. The basal medium RPMI 1640 with the addition of FBS was a satisfactory alternative for culturing the CHO-GCR cells. This study provided data that may contribute to the improvement of the GCR purification process. New research can be developed to improve the GCR purification process.

The inherited form of primary open angle glaucoma, a disorder characterized by increased intraocular pressure and retina degeneration, is linked to mutations in the olfactomedin (OLF) domain of the myocilin gene. Disease-causing myocilin variants accumulate within trabecular meshwork cells instead of being secreted to the trabecular extracellular matrix thought to regulate aqueous humor flow and control intraocular pressure. Like other diseases of protein misfolding, we hypothesize myocilin toxicity originates from defects in protein biophysical properties. In this thesis, the first preparative recombinant high-yield expression and purification system for the C-terminal OLF domain of myocilin (myoc-OLF) is described. To determine the relative stability of wild-type (WT) and mutant OLF domains, a fluorescence thermal stability assay was adapted to provide the first direct evidence that mutated OLF is folded but less thermally stable than WT. In addition, mutant myocilin can be stabilized by chemical chaperones. Together, this work provides the first quantitative demonstration of compromised stability among identified OLF variants and placing myocilin glaucoma in the context of other complex diseases of protein misfolding. Subsequent investigations into the biophysical properties of WT myoc-OLF provide insight into its structure and function. In particular, myoc-OLF is stable in the presence of glycosaminoglycans (GAGs), as well as over a wide pH range in buffers with functional groups reminiscent of such GAGs. Myoc-OLF contains significant â-sheet and â-turn secondary structure as revealed by circular dichroism analysis. At neutral pH, thermal melts indicate a highly cooperative transition with a melting temperature of ~55°C. A compact core structural domain of OLF was identified by limited proteolysis and consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. This construct also is capable of generating 3D crystals for structure determination. This data, presented in Chapter 3, inform new testable hypotheses for interactions with specific trabecular extracellular matrix components. To gain further insight into the biological function of myoc-OLF, a facile fluorescence chemical stability assay was designed to identify possible ligands and drug candidates. In the assay described in Chapter 4, the target protein is initially destabilized with a chemical denaturant and is tested for re-stabilization upon the addition of small molecules. The assay requires no prior knowledge of the structure and/or function of the target protein, and it is amendable to high-throughput screening. Application of the assay using a library of 1,280 compounds revealed 14 possible ligands and drug candidates for myoc-OLF that may also generate insights into myoc-OLF function. Due to the high â-sheet content of monomeric myoc-OLF and presence of an aggregated species upon myoc-OLF purification, the ability of myoc-OLF to form amyloid fibrils was suspected and verified. The fibril forming region was confirmed to reside in the OLF domain of myocilin. Kinetic analyses of fibril formation reveal a self-propagating process common to amyloid. The presence of an aggregated species was confirmed in cells transfected with WT myocilin, but to a greater extent in cells transfected with P370L mutant myocilin. Both cell lines stained positive for amyloid. Taken together, these results provide further insights into the structure of myocilin and suggest a new hypothesis for glaucoma pathogenesis. Finally, in a related study, small molecule drug candidates were investigated to treat acid â-glucosidase (GCase), the deficient lysosomal enzyme in Gaucher disease, another protein conformational disorder. Three new GCase active-site directed 3,4,5,6-tetrahydroxylazepane inhibitors were synthesized that exhibit half inhibitory concentrations (IC50) in the low millimolar to low micromolar range. Although the compounds thermally stabilize GCase at pH 7.4, only one of the synthesized analogs exhibits chaperoning activity under typical assay conditions. This successful pharmacological chaperone is also one in which GCase is in its proposed active conformation as revealed by X-ray crystallography. Probing the plasticity of the active-site of GCase offers additional insight into possible molecular determinants for an effective small molecule therapy for GD.

Made available in DSpace on 2014-12-17T14:13:52Z (GMT). No. of bitstreams: 1 RigobertoGC_DISSERT.pdf: 2462609 bytes, checksum: 1753126d41dc7df59645485b6308c3f5 (MD5) Previous issue date: 2011-05-30 / Background. Gaucher Disease (GD) is a hereditary lysosomal storage disorder characterized by the accumulation of glucosylceramide, mainly in the cells of the reticuloendothelial system, due to a deficiency of the enzyme acid &#946;-glucosidase (GBA). Diagnosis is usually based on measurement of GBA activity in peripheral leukocytes. The purpose of this study was to evaluate the ability of screening for GBA and chitotriosidase activity using Dried Blood Spots on Filter Paper (DBS-FP) to identify individuals at high risk for GD in high-risk populations such as that of Tabuleiro do Norte, a small town in Northeastern Brazil. Methods. Between June 1, 2007 and May 31, 2008, 740 consented residents and descendants of traditional families from Tabuleiro do Norte were submitted to screening with DBS-FP. Subjects with GBA activity <2.19 nmol/h/mL were referred to analysis of GBA and chitotriosidase activity in peripheral leukocytes and in plasma, respectively. Subjects at highest risk for GD (GBA activity in peripheral leukocytes <5.6 nmol/h/mg protein) were submitted to molecular analysis to confirm diagnosis. Results. Screening with DBS-FP identified 135 subjects (18.2%) with GBA activity <2.19 nmol/h/mL, 131 of whom remained in the study. In 10 of these (7.6%), GBA activity in leukocytes was 2.6 5.5 nmol/h/mg protein. Subsequent molecular analysis confirmed 6 cases of heterozygosity and 4 normals for GD. Conclusion. DBS-FP assay was shown to be an effective initial GD screening strategy for high-prevalence populations in developing regions. Diagnosis could not be established from GBA activity in leukocytes alone, but required confirmation with molecular analysis / A doen?a de Gaucher (DG) ? uma patologia de dep?sito de gordura nos lisossomos, de heran?a autoss?mica recessiva, caracterizada pelo ac?mulo do substrato glicosilceramida, principalmente nas c?lulas do sistema reticuloendotelial, em raz?o da defici?ncia da enzima &#946;-glicosidase ?cida (GBA). O diagn?stico, comumente, ? feito pela dosagem da atividade da GBA em leuc?citos perif?ricos. Tabuleiro do Norte (TN), Cear?, Brasil, ? um munic?pio com cerca de 28.000 habitantes com a preval?ncia da DG de 1:4.000 habitantes, possivelmente a mais elevada do Brasil. O objetivo da disserta??o ? avaliar o rastreamento para DG realizado em TN com base na an?lise das atividades enzim?ticas da GBA e da quitotriosidase em amostras Sangue Seco em Papel de Filtro (SSPF). Entre 01 de junho de 2007 a 31 de maio de 2008, 740 indiv?duos residentes e descendentes de fam?lias de TN participaram do rastreamento para DG a partir de amostras de SSPF. Indiv?duos com atividade GBA<2,19 nmol/h/mL foram selecionados para an?lise da atividade da GBA e da quitotriosidase em leuc?citos perif?ricos e no plasma, respectivamente. Os indiv?duos com maiores riscos de DG (atividade de GBA em leuc?citos perif?ricos <5,6 nmol/h/mg de prote?na) foram referenciados para an?lise molecular para confirma??o diagn?stica. A triagem com amostras de SSPF identificou 135 indiv?duos (18,2%) com atividade da GBA<2,19 nmol/h/mL, dos quais 131 permaneceram no estudo. Em dez destes (7,6%), a atividade da GBA em leuc?citos variou de 2,6-5,5 nmol/ h/mg de prote?na, considerados suspeitos da DG. A an?lise molecular subsequente revelou, entretanto, que se tratava de seis indiv?duos heterozigotos para a muta??o G377S e, em quatro deles, n?o foram identificadas muta??es da DG. A an?lise enzim?tica de amostras de SSPF mostrou ser uma estrat?gia eficaz de triagem da DG em popula??es com alto risco, mas a medida da atividade da GBA em leuc?citos deve ser realizada para confirma??o diagn?stica. O diagn?stico de DG em indiv?duos assintom?ticos n?o deve ser firmado baseando-se apenas na an?lise da atividade da GBA em leuc?citos, sendo necess?ria, tamb?m, a confirma??o diagn?stica pela an?lise molecular

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.

Metachromatic leukodystrophy (MLD) and Gaucher disease (GD) are caused by a deficiency of arylsulphatase A (ASA) and b-glucocerebrosidase (GBA), respectively. They are lysosomal storage disorders with a heterogeneous clinical spectrum encompassing visceral, skeletal and neurologic involvement resulting in high morbidity and mortality. The overall aim of this study is to elucidate the genetic component/s of high ASA and GBA enzyme activity in normal healthy individuals with the ultimate goal of using this information to produce greater protein activity from a recombinant protein. A wide variation in ASA and GBA enzyme activity levels has been observed in the normal population. The first objective of this project was to identify and characterise single nucleotide polymorphisms (SNPs) in the arylsulphatase A (ARSA) and glucocerebrosidase (GBA) genes that are responsible for determining the levels of expressed enzyme activity in the normal population. The second objective was to assess the contribution of transcriptional regulation and TCP80 mediated translational control to normal enzyme variation. TCP80, a translational control protein that interacts with the GBA coding region, is a splice variant of the interleukin binding factor 3 (ILF3) gene. Ten samples from individuals with high ASA activity and twenty samples from individuals with high GBA activity were screened for polymorphisms via denaturing high pressure liquid chromatography (dHPLC) and sequencing. The frequency of these polymorphisms in the normal population was determined using dot-blot hybridisation. Fifteen ARSA polymorphisms (4 promoter, 5 coding, 5 intronic and 1 poly(A) signal) and two GBA polymorphisms (1 intronic and 1 in 3¢-UTR) were identified. Two low frequency ASA polymorphisms (2723A > G, W193C) were found to be correlated with low activity, while another low frequency ASA polymorphism (1101+123C > T) was found to be correlated with high activity in a population of 113 individuals. Real time PCR was used to measure mRNA levels of GBA, ASA and LF3 along with enzyme activity levels of GBA and ASA in two cell types (leucocytes and skin fibroblasts) from four healthy individuals and seven cell lines (HL60, THP1, Huh7, U118, SW1353, Hep G2, and B-cells). Transcriptional control was evident for all three genes with GBA mRNA levels varying over 30 fold, ASA mRNA levels varying over seven fold and ILF3 levels varying more than 24 fold. The 5¢-flanking region of GBA was investigated for the cis-elements responsible for tissue-specific expression. However, it was not possible to demonstrate that the cis-element region was influencing GBA expression. Translational efficiency was measured using the magnitude of the mRNA:enzyme activity ratio as an indicator. GBA translational inefficiency was most pronounced in B cells which require four times more mRNA molecules than hepatocytes (Hep G2) and over 25 times more mRNA molecules than chondrocytes (SW1353) to produce one unit of GBA enzyme activity. Except in B-cells, GBA translational efficiency appears to increase as ILF3 mRNA levels decrease. The tissue-specific variation observed in the protein levels of the ILF3 splice variants, TCP80 and DRBP76, may play a role. The correlation of several low frequency SNPs with low ASA enzyme activity or high ASA activity indicates a role in determining the distribution of enzyme activity levels in the normal population. However, there do not appear to be any common high activity polymorphisms. Knowledge of the exact mechanisms responsible for the observed transcriptional and translational control of these lysosomal genes will greatly enhance the understanding of genotype-phenotype correlation and the contribution of genetic variants to natural variation.

Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...

5 Abstract Definitive diagnosis of inherited metabolic disorders commonly depends on the measurement of enzyme activity (which is often complicated) and/or molecular genetic testing. Yet even the standard mutation analysis can bring false negative results in the case of gross chromosomal rearrangements or incorrect regulation of gene expression due to the mutations in regulatory regions. In the present study I focused on characterization of complex mutations affecting the gene encoding ornithin transcarbamylase (OTC) followed by studies of regulatory regions of OTC and GBA (the gene encoding β-glucocerebrosidase). In the first study we identified 14 novel mutations including three large deletions in a cohort of 37 patients with OTC deficiency (OTCD). Subsequently we evaluated clinical significance of all these mutations. We also found a heterozygote carrying a hypomorphic mutation and manifesting OTCD most likely due to unfavorable X-inactivation which was observed independently in three different peripheral tissues. In order to evaluate the clinical significance of a promoter variation c.-366A>G found in a family with mild OTCD we identified three alternative transcription start sites (TSSs) of human OTC and delimited the promoter. We also found a distal enhancer and performed functional analysis of both...