A Mucopolissacaridose tipo I (MPS I) é causada pela deficiência da α-Liduronidase (IDUA), uma hidrolase lisosomal responsável pela degradação dos glicosaminoglicanos (GAG) heparan e dermatan sulfato. Diversos processos bioquímicos e fisiológicos são afetados pelo acúmulo desses substratos nas células, levando a uma condição patológica multisistêmica. Apesar dos benefícios clínicos obtidos com os tratamentos atualmente disponíveis, várias limitações têm sido relatadas, sendo necessária a busca de novas estratégias terapêuticas. Uma abordagem promissora de terapia gênica/celular para o tratamento da MPS I é a microencapsulação de células geneticamente modificadas. Neste trabalho, produzimos microcápsulas com células recombinantes superexpressando IDUA, as quais foram implantadas em camundongos MPS I a fim de avaliar sua eficiência como uma terapia. No primeiro estudo, as cápsulas foram implantadas no tecido subcutâneo para um tratamento de 120 dias. A atividade de IDUA no soro teve um leve aumento nos primeiros 45 dias. Depois de 120 dias, a atividade de IDUA foi detectada no fígado, rim e coração. A dosagem bioquímica do acúmulo de GAG nos tecidos mostrou níveis reduzidos no rim. A análise histológica confirmou esses resultados e, de modo interessante, mostrou uma reorganização no parênquima hepático com menos células vacuolizadas. Além disso, as microcápsulas foram recuperadas para análise histológica e foi observada a presença de células inflamatórias e uma camada fibrótica em torno das cápsulas. O segundo estudo foi um tratamento de 60 dias com as microcápsulas implantadas no epíplon. Os níveis de IDUA no soro foram transitórios durante o tratamento e 60 dias depois da implantação a atividade enzimática foi detectada apenas no coração. A avaliação do acúmulo de GAG não apresentou diferenças entre os camundongos MPS I tratados e não tratados. Em ambos os estudos, alguns animais foram utilizados para um experimento em curto prazo e um aumento nos níveis de IDUA foi observado nos tecidos 24 horas após a implantação das cápsulas. Concluindo, as células microencapsuladas foram capazes de corrigir alguns aspectos da doença. No entanto, fatores como uma resposta imune contra a enzima e ao biomaterial, ou a dose de células nas cápsulas, podem ter prejudicado a eficiência do tratamento, sugerindo que modificações na técnica são necessárias para obter um melhor desempenho. / Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of α-Liduronidase (IDUA), a lysosomal hydrolase responsible for the degradation of the glycosaminoglycans (GAG) heparan and dermatan sulfate. The consequent accumulation of these substrates throughout the cells affects several biochemical and physiological processes, leading to multisystemic pathological condition. Although the clinical benefits of the treatments currently available, several limitations have been noted and the search for alternative therapeutic strategies are necessary. A promising gene/cell therapy approach for treating MPS I is the microencapsulation of genetically modified cells. In this work, we produced microcapsules containing recombinant cells overexpressing IDUA, which were implanted in MPS I mice in order to evaluate their efficiency as a treatment. In the first study, capsules were implanted in the subcutaneous tissue for a 120-days treatment. Serum IDUA activity was slightly increased in the first 45 days. After 120 days, IDUA activity was detected in the liver, kidney and heart. The biochemical measurement of GAG accumulation in the tissues revealed decreased levels in the kidney. The histological analysis confirmed these results and, interestingly, showed a reorganization of the hepatic parenchyma with less cell vacuolization. In addition, microcapsules were retrieved for histological analysis and it was observed the presence of inflammatory cells and a fibrotic layer around the capsules. The second study was a 60- day treatment with the microcapsules implanted in the omentum. Serum IDUA levels were transient over treatment and 60 days post-implantation the enzyme activity was detected only in the heart. The evaluation of GAG storage did not reveal differences between treated and untreated MPS I mice. In both studies, some animals were used for a shortterm experiment and increased IDUA levels were observed in the tissues 24 h after capsules implantation. In conclusion, microencapsulated cells were able to correct some aspects of the disease. However, factors such as immune response against the enzyme and the biomaterial or the dose of cells in the capsules could be impairing the efficiency of the treatment, suggesting that modifications on the technique are necessary to achieve a better performance.

A Mucopolissacaridose tipo I (MPS I) é causada pela deficiência da α-Liduronidase (IDUA), uma hidrolase lisosomal responsável pela degradação dos glicosaminoglicanos (GAG) heparan e dermatan sulfato. Diversos processos bioquímicos e fisiológicos são afetados pelo acúmulo desses substratos nas células, levando a uma condição patológica multisistêmica. Apesar dos benefícios clínicos obtidos com os tratamentos atualmente disponíveis, várias limitações têm sido relatadas, sendo necessária a busca de novas estratégias terapêuticas. Uma abordagem promissora de terapia gênica/celular para o tratamento da MPS I é a microencapsulação de células geneticamente modificadas. Neste trabalho, produzimos microcápsulas com células recombinantes superexpressando IDUA, as quais foram implantadas em camundongos MPS I a fim de avaliar sua eficiência como uma terapia. No primeiro estudo, as cápsulas foram implantadas no tecido subcutâneo para um tratamento de 120 dias. A atividade de IDUA no soro teve um leve aumento nos primeiros 45 dias. Depois de 120 dias, a atividade de IDUA foi detectada no fígado, rim e coração. A dosagem bioquímica do acúmulo de GAG nos tecidos mostrou níveis reduzidos no rim. A análise histológica confirmou esses resultados e, de modo interessante, mostrou uma reorganização no parênquima hepático com menos células vacuolizadas. Além disso, as microcápsulas foram recuperadas para análise histológica e foi observada a presença de células inflamatórias e uma camada fibrótica em torno das cápsulas. O segundo estudo foi um tratamento de 60 dias com as microcápsulas implantadas no epíplon. Os níveis de IDUA no soro foram transitórios durante o tratamento e 60 dias depois da implantação a atividade enzimática foi detectada apenas no coração. A avaliação do acúmulo de GAG não apresentou diferenças entre os camundongos MPS I tratados e não tratados. Em ambos os estudos, alguns animais foram utilizados para um experimento em curto prazo e um aumento nos níveis de IDUA foi observado nos tecidos 24 horas após a implantação das cápsulas. Concluindo, as células microencapsuladas foram capazes de corrigir alguns aspectos da doença. No entanto, fatores como uma resposta imune contra a enzima e ao biomaterial, ou a dose de células nas cápsulas, podem ter prejudicado a eficiência do tratamento, sugerindo que modificações na técnica são necessárias para obter um melhor desempenho. / Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of α-Liduronidase (IDUA), a lysosomal hydrolase responsible for the degradation of the glycosaminoglycans (GAG) heparan and dermatan sulfate. The consequent accumulation of these substrates throughout the cells affects several biochemical and physiological processes, leading to multisystemic pathological condition. Although the clinical benefits of the treatments currently available, several limitations have been noted and the search for alternative therapeutic strategies are necessary. A promising gene/cell therapy approach for treating MPS I is the microencapsulation of genetically modified cells. In this work, we produced microcapsules containing recombinant cells overexpressing IDUA, which were implanted in MPS I mice in order to evaluate their efficiency as a treatment. In the first study, capsules were implanted in the subcutaneous tissue for a 120-days treatment. Serum IDUA activity was slightly increased in the first 45 days. After 120 days, IDUA activity was detected in the liver, kidney and heart. The biochemical measurement of GAG accumulation in the tissues revealed decreased levels in the kidney. The histological analysis confirmed these results and, interestingly, showed a reorganization of the hepatic parenchyma with less cell vacuolization. In addition, microcapsules were retrieved for histological analysis and it was observed the presence of inflammatory cells and a fibrotic layer around the capsules. The second study was a 60- day treatment with the microcapsules implanted in the omentum. Serum IDUA levels were transient over treatment and 60 days post-implantation the enzyme activity was detected only in the heart. The evaluation of GAG storage did not reveal differences between treated and untreated MPS I mice. In both studies, some animals were used for a shortterm experiment and increased IDUA levels were observed in the tissues 24 h after capsules implantation. In conclusion, microencapsulated cells were able to correct some aspects of the disease. However, factors such as immune response against the enzyme and the biomaterial or the dose of cells in the capsules could be impairing the efficiency of the treatment, suggesting that modifications on the technique are necessary to achieve a better performance.

Fu Lai Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 106-110). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Lists of Figures --- p.x / Lists of Tables --- p.xiii / List of Abbreviations --- p.xiv / Amino acid abbreviation --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Human α-L-iduronidase (hIDUA) --- p.2 / Chapter 1.1.1 --- Lysosomal storage disease --- p.2 / Chapter 1.1.2 --- Treatments of MPS 1 --- p.4 / Chapter 1.2 --- Plant cells as bioreactors --- p.5 / Chapter 1.3 --- The Plant secretary pathway --- p.7 / Chapter 1.3.1 --- Transport of soluble proteins --- p.9 / Chapter 1.3.2 --- Transport of integral membrane proteins --- p.10 / Chapter 1.4 --- Differences between plant and human proteins --- p.11 / Chapter 1.5 --- Reducing the differences between plant and human proteins --- p.12 / Chapter 1.6 --- Previous study: Expression of IDUA in transgenic tobacco plant --- p.13 / Chapter 1.7 --- Project objectives --- p.14 / Chapter 1.8 --- Long term significance --- p.14 / Chapter Chapter 2 --- Materials and Methods --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials --- p.18 / Chapter 2.2.1 --- Chemical --- p.18 / Chapter 2.2.2 --- Plant materials --- p.18 / Chapter 2.2.3 --- Plasmid vectors and bacterial strains --- p.18 / Chapter 2.2.4 --- Human a-iduronidase (hIDUA) cDNA --- p.19 / Chapter 2.2.5 --- Primers --- p.20 / Chapter 2.3 --- Methods --- p.22 / Chapter 2.3.1 --- Generation of IDUA antibodies --- p.22 / Chapter 2.3.1.1 --- Synthetic peptide raised IDUA antibodies --- p.23 / Chapter 2.3.1.1.1 --- Design of synthetic peptides --- p.23 / Chapter 2.3.1.1.2 --- Immunization of rabbits --- p.25 / Chapter 2.3.1.2 --- E. coli-derived rhIDUA protein --- p.25 / Chapter 2.3.1.2.1 --- Cloning and expression of rhIDUA --- p.25 / Chapter 2.3.1.2.2 --- Western analysis of E. coli-derived rhIDUA --- p.29 / Chapter 2.3.1.2.3 --- MS/MS analysis of rhIDUA protein --- p.29 / Chapter 2.3.1.2.4 --- Immunization of rabbits --- p.31 / Chapter 2.3.2 --- Affinity-purified antibodies --- p.33 / Chapter 2.3.3 --- Characterization of affinity-purified IDUA antibodies --- p.33 / Chapter 2.3.4 --- Construction of chimeric gene constructs --- p.34 / Chapter 2.3.5 --- Expression of IDUA in tobacco BY-2 cells --- p.39 / Chapter 2.3.5.1 --- Electropoartion of Agrobacteria --- p.39 / Chapter 2.3.5.2 --- Agrobacterium-mediated transformation --- p.39 / Chapter 2.3.5.3 --- Screening of positive trans formants --- p.40 / Chapter 2.3.6 --- Characterization of transgenic BY-2 cell expressing IDUA fusion --- p.40 / Chapter 2.3.6.1 --- Genomic DNA polymerase chain reaction (Genomic DNA PCR) --- p.40 / Chapter 2.3.6.1.1 --- Genomic DNA extraction from BY-2 callus --- p.40 / Chapter 2.3.6.1.2 --- Genomic DNA PCR of tobacco BY-2 callus --- p.41 / Chapter 2.3.6.2 --- Reverse transcription-PCR (RT-PCR) --- p.42 / Chapter 2.3.6.2.1 --- Total RNA extraction from BY-2 cell --- p.42 / Chapter 2.3.6.2.2 --- RT-PCR of BY-2 cell --- p.42 / Chapter 2.3.6.3 --- Western blot analysis of BY-2 cell and medium --- p.43 / Chapter 2.3.6.3.1 --- Protein extraction from tobacco BY-2 cells and culture medium --- p.43 / Chapter 2.3.6.3.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 2.3.6.3.3 --- Immunodetection and Coomassie blue stain --- p.44 / Chapter 2.3.7 --- Purification of IDUA from culture media --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Generation of IDUA antibodies --- p.48 / Chapter 3.1.1 --- Cloning and expression of rhIDUA in E. coli --- p.48 / Chapter 3.1.2 --- Characterization of IDUA antibodies --- p.51 / Chapter 3.1.2.1 --- Specificity of IDUA antibodies towards hIDUA protein. --- p.51 / Chapter 3.1.2.2 --- Cross-reactivity of IDUA antibodies with wild type tobacco BY-2 cell --- p.55 / Chapter 3.2 --- Chimeric gene constructs construction and confirmation --- p.58 / Chapter 3.3 --- Screening of transformed tobacco BY-2 callus with kanamycin-resistance --- p.66 / Chapter 3.4 --- Genomic DNA PCR screening of transformed tobacco BY-2 callus . --- p.67 / Chapter 3.5 --- RT-PCR screening of transformed BY-2 cells --- p.70 / Chapter 3.6 --- Western blot analysis of transformed tobacco BY-2 cells and culture media --- p.72 / Chapter 3.6.1 --- Tobacco BY-2 cells --- p.72 / Chapter 3.6.2 --- Tobacco BY-2 cell culture media --- p.76 / Chapter 3.7 --- Purification of IDUA protein in culture media --- p.81 / Chapter Chapter 4 --- Discussion --- p.82 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.89 / Chapter 5.1 --- Summary --- p.90 / Chapter 5.2 --- Future perspectives --- p.92 / Appendix Identification and Characterization of an Unknown Protein by 1B Antibody --- p.93 / Chapter 6.1 --- Introduction --- p.94 / Chapter 6.2 --- Objectives --- p.94 / Chapter 6.3 --- Materials and Methods --- p.95 / Chapter 6.3.1 --- Western blot analysis of different plant species --- p.95 / Chapter 6.3.2 --- Subcellular localization of the unknown protein --- p.95 / Chapter 6.3.3 --- Affinity-purification of the unknown protein --- p.95 / Chapter 6.4 --- Results --- p.97 / Chapter 6.4.1 --- Western blot analysis of different plant species --- p.97 / Chapter 6.4.2 --- Subcellular localization of an unknown protein --- p.98 / Chapter 6.4.3 --- Affinity-purification of 1B protein --- p.104 / Chapter 6.5 --- Summary and Future Perspectives --- p.105 / Chapter 6.5.1 --- Summary --- p.105 / Chapter 6.5.2 --- Future Perspectives --- p.105 / References --- p.106

As Mucopolissacaridoses são erros inatos do metabolismo, fazem parte das doenças lisossômicas de depósito e ocorrem devido à deficiência na atividade de enzimas que catalisam a degradação de glicosaminoglicanos. O objetivo desse estudo foi aperfeiçoar o diagnóstico bioquímico das Mucopolissacaridoses dos tipos I, VI e VII, estabelecendo o uso do tampão fosfato de sódio 20 mmol/L pH 7,0 (tampão universal - TU) e outros parâmetros bioquímicos. Nesse trabalho foi aprimorada a técnica de medida de atividade da beta-glicuronidase (GUSB), enzima deficiente na MPS VII, reduzindo a quantidade de reagentes em 4 vezes e a utilização do tamanho dos picotes de sangue impregnado em papel filtro (SPF) para 1,2 mm. Estudamos a cinética da atividade da GUSB determinando o pH ótimo (4,4), Km (1,25 mM), Vmáx (594,48 nmol/h/mL), termoestabilidade (inativação significante da enzima a partir de 60 min a 60 ºC) e tempo e temperatura de armazenamento (até 30 dias à 4, 25 e 37 ºC, acima de 60 dias à -20 ºC) e estabelecemos um intervalo de referência para a atividade da GUSB em amostras de indivíduos saudáveis nessa metodologia (174,4 nmol/h/mL a 781,9 nmol/h/mL). Estabelecemos o uso do TU para determinação das atividades da alfa-iduronidase (IDUA), arilsulfatase B (ASB) e GUSB medindo a atividade enzimática em SPF eluído nesse tampão e correlacionamos com a técnica espectrofluorimétrica já padronizada para cada enzima em SPF de 1,2 mm em amostras de indivíduos saudáveis As correlações foram positivas e os coeficientes de validação da técnica estavam dentro dos limites aceitáveis. As médias de atividade determinadas para indivíduos saudáveis foram: 14,65 + 4,35 nmol/h/mL (IDUA), 22,51 + 5,09 nmol/h/mL (ASB) e 531,92 + 121,05 nmol/h/mL (GUSB). Foram analisados parâmetros bioquímicos envolvidos em estresse oxidativo no plasma de indivíduos com MPS VI e comparados com MPS I e controles saudáveis. A medida da atividade da SOD não diferiu entre os grupos, a atividade de CAT encontrava-se diminuída tanto em MPS VI quanto em MPS I e a dosagem de TBARS estava aumentada em ambas as MPS em relação aos controles. A partir desse estudo, foi possível padronizarmos e aperfeiçoarmos novas técnicas para o diagnóstico laboratorial para a MPS I, VI e VII além de introduzir o estresse oxidativo como um possível marcador no uso da terapia de reposição enzimática. / Mucopolysaccharidoses are inborn errors of metabolism, being part of lysosomal storage diseases and occuring due to deficiency in the activity of enzymes that catalyze the degradation of glycosaminoglycans. The aim of this study was to improve the biochemical diagnosis of Mucopolysaccharidoses of types I, VI and VII, establishing the use of 20 mmol/L sodium phosphate buffer pH 7.0 (universal extraction buffer - UEB) and other biochemical parameters. In this work, the activity measurement technique of beta-glucuronidase (GUSB), enzyme deficient in MPS VII, has been improved, reducing the amount of reagents in 4 times and using the size of dried blood spots (DBS) for 1.2 mm. We studied the kinetics of GUSB activity by determining the optimum pH (4.4), Km (1.25 mM), Vmax (594.48 nmol/h/mL), thermostability (significant inactivation of the enzyme from 60min at 60 ºC) and storage time and temperature (up to 30 days at 4, 25 and 37 °C, above 60 days at -20 °C) and established a reference range for GUSB activity in samples from healthy subjects in this methodology (174.4 nmol/h/mL at 781.9 nmol/h/ mL). We established the use of TU to determine the activities of alpha-iduronidase (IDUA), arylsulfatase B (ASB) and GUSB by measuring the enzymatic activity in DBS eluted in this buffer and correlated with the standardized spectrofluorometric technique for each enzyme in DBS of 1.2 mm in samples from healthy individuals Correlations were positive and the validation coefficients of the technique were within acceptable limits. The activity means determined for healthy individuals were 14.65 ± 4.35 nmol/h/mL (IDUA), 22.51 ± 5.09 nmol/h/mL (ASB) and 531.92 ± 121.05 nmol/h/mL (GUSB). Biochemical parameters involved in oxidative stress in the plasma of individuals with MPS VI and compared to MPS I and healthy controls were analyzed. Measurement of SOD activity did not differ between groups, CAT activity was decreased in both MPS VI and MPS I and the TBARS dosage was increased in both MPS compared to controls. From this study, it was possible to standardize and improve new techniques for laboratory diagnosis for MPS I, VI and VII, besides introducing oxidative stress as a possible marker in the use of enzyme replacement therapy.

As Mucopolissacaridoses são erros inatos do metabolismo, fazem parte das doenças lisossômicas de depósito e ocorrem devido à deficiência na atividade de enzimas que catalisam a degradação de glicosaminoglicanos. O objetivo desse estudo foi aperfeiçoar o diagnóstico bioquímico das Mucopolissacaridoses dos tipos I, VI e VII, estabelecendo o uso do tampão fosfato de sódio 20 mmol/L pH 7,0 (tampão universal - TU) e outros parâmetros bioquímicos. Nesse trabalho foi aprimorada a técnica de medida de atividade da beta-glicuronidase (GUSB), enzima deficiente na MPS VII, reduzindo a quantidade de reagentes em 4 vezes e a utilização do tamanho dos picotes de sangue impregnado em papel filtro (SPF) para 1,2 mm. Estudamos a cinética da atividade da GUSB determinando o pH ótimo (4,4), Km (1,25 mM), Vmáx (594,48 nmol/h/mL), termoestabilidade (inativação significante da enzima a partir de 60 min a 60 ºC) e tempo e temperatura de armazenamento (até 30 dias à 4, 25 e 37 ºC, acima de 60 dias à -20 ºC) e estabelecemos um intervalo de referência para a atividade da GUSB em amostras de indivíduos saudáveis nessa metodologia (174,4 nmol/h/mL a 781,9 nmol/h/mL). Estabelecemos o uso do TU para determinação das atividades da alfa-iduronidase (IDUA), arilsulfatase B (ASB) e GUSB medindo a atividade enzimática em SPF eluído nesse tampão e correlacionamos com a técnica espectrofluorimétrica já padronizada para cada enzima em SPF de 1,2 mm em amostras de indivíduos saudáveis As correlações foram positivas e os coeficientes de validação da técnica estavam dentro dos limites aceitáveis. As médias de atividade determinadas para indivíduos saudáveis foram: 14,65 + 4,35 nmol/h/mL (IDUA), 22,51 + 5,09 nmol/h/mL (ASB) e 531,92 + 121,05 nmol/h/mL (GUSB). Foram analisados parâmetros bioquímicos envolvidos em estresse oxidativo no plasma de indivíduos com MPS VI e comparados com MPS I e controles saudáveis. A medida da atividade da SOD não diferiu entre os grupos, a atividade de CAT encontrava-se diminuída tanto em MPS VI quanto em MPS I e a dosagem de TBARS estava aumentada em ambas as MPS em relação aos controles. A partir desse estudo, foi possível padronizarmos e aperfeiçoarmos novas técnicas para o diagnóstico laboratorial para a MPS I, VI e VII além de introduzir o estresse oxidativo como um possível marcador no uso da terapia de reposição enzimática. / Mucopolysaccharidoses are inborn errors of metabolism, being part of lysosomal storage diseases and occuring due to deficiency in the activity of enzymes that catalyze the degradation of glycosaminoglycans. The aim of this study was to improve the biochemical diagnosis of Mucopolysaccharidoses of types I, VI and VII, establishing the use of 20 mmol/L sodium phosphate buffer pH 7.0 (universal extraction buffer - UEB) and other biochemical parameters. In this work, the activity measurement technique of beta-glucuronidase (GUSB), enzyme deficient in MPS VII, has been improved, reducing the amount of reagents in 4 times and using the size of dried blood spots (DBS) for 1.2 mm. We studied the kinetics of GUSB activity by determining the optimum pH (4.4), Km (1.25 mM), Vmax (594.48 nmol/h/mL), thermostability (significant inactivation of the enzyme from 60min at 60 ºC) and storage time and temperature (up to 30 days at 4, 25 and 37 °C, above 60 days at -20 °C) and established a reference range for GUSB activity in samples from healthy subjects in this methodology (174.4 nmol/h/mL at 781.9 nmol/h/ mL). We established the use of TU to determine the activities of alpha-iduronidase (IDUA), arylsulfatase B (ASB) and GUSB by measuring the enzymatic activity in DBS eluted in this buffer and correlated with the standardized spectrofluorometric technique for each enzyme in DBS of 1.2 mm in samples from healthy individuals Correlations were positive and the validation coefficients of the technique were within acceptable limits. The activity means determined for healthy individuals were 14.65 ± 4.35 nmol/h/mL (IDUA), 22.51 ± 5.09 nmol/h/mL (ASB) and 531.92 ± 121.05 nmol/h/mL (GUSB). Biochemical parameters involved in oxidative stress in the plasma of individuals with MPS VI and compared to MPS I and healthy controls were analyzed. Measurement of SOD activity did not differ between groups, CAT activity was decreased in both MPS VI and MPS I and the TBARS dosage was increased in both MPS compared to controls. From this study, it was possible to standardize and improve new techniques for laboratory diagnosis for MPS I, VI and VII, besides introducing oxidative stress as a possible marker in the use of enzyme replacement therapy.

As Mucopolissacaridoses são erros inatos do metabolismo, fazem parte das doenças lisossômicas de depósito e ocorrem devido à deficiência na atividade de enzimas que catalisam a degradação de glicosaminoglicanos. O objetivo desse estudo foi aperfeiçoar o diagnóstico bioquímico das Mucopolissacaridoses dos tipos I, VI e VII, estabelecendo o uso do tampão fosfato de sódio 20 mmol/L pH 7,0 (tampão universal - TU) e outros parâmetros bioquímicos. Nesse trabalho foi aprimorada a técnica de medida de atividade da beta-glicuronidase (GUSB), enzima deficiente na MPS VII, reduzindo a quantidade de reagentes em 4 vezes e a utilização do tamanho dos picotes de sangue impregnado em papel filtro (SPF) para 1,2 mm. Estudamos a cinética da atividade da GUSB determinando o pH ótimo (4,4), Km (1,25 mM), Vmáx (594,48 nmol/h/mL), termoestabilidade (inativação significante da enzima a partir de 60 min a 60 ºC) e tempo e temperatura de armazenamento (até 30 dias à 4, 25 e 37 ºC, acima de 60 dias à -20 ºC) e estabelecemos um intervalo de referência para a atividade da GUSB em amostras de indivíduos saudáveis nessa metodologia (174,4 nmol/h/mL a 781,9 nmol/h/mL). Estabelecemos o uso do TU para determinação das atividades da alfa-iduronidase (IDUA), arilsulfatase B (ASB) e GUSB medindo a atividade enzimática em SPF eluído nesse tampão e correlacionamos com a técnica espectrofluorimétrica já padronizada para cada enzima em SPF de 1,2 mm em amostras de indivíduos saudáveis As correlações foram positivas e os coeficientes de validação da técnica estavam dentro dos limites aceitáveis. As médias de atividade determinadas para indivíduos saudáveis foram: 14,65 + 4,35 nmol/h/mL (IDUA), 22,51 + 5,09 nmol/h/mL (ASB) e 531,92 + 121,05 nmol/h/mL (GUSB). Foram analisados parâmetros bioquímicos envolvidos em estresse oxidativo no plasma de indivíduos com MPS VI e comparados com MPS I e controles saudáveis. A medida da atividade da SOD não diferiu entre os grupos, a atividade de CAT encontrava-se diminuída tanto em MPS VI quanto em MPS I e a dosagem de TBARS estava aumentada em ambas as MPS em relação aos controles. A partir desse estudo, foi possível padronizarmos e aperfeiçoarmos novas técnicas para o diagnóstico laboratorial para a MPS I, VI e VII além de introduzir o estresse oxidativo como um possível marcador no uso da terapia de reposição enzimática. / Mucopolysaccharidoses are inborn errors of metabolism, being part of lysosomal storage diseases and occuring due to deficiency in the activity of enzymes that catalyze the degradation of glycosaminoglycans. The aim of this study was to improve the biochemical diagnosis of Mucopolysaccharidoses of types I, VI and VII, establishing the use of 20 mmol/L sodium phosphate buffer pH 7.0 (universal extraction buffer - UEB) and other biochemical parameters. In this work, the activity measurement technique of beta-glucuronidase (GUSB), enzyme deficient in MPS VII, has been improved, reducing the amount of reagents in 4 times and using the size of dried blood spots (DBS) for 1.2 mm. We studied the kinetics of GUSB activity by determining the optimum pH (4.4), Km (1.25 mM), Vmax (594.48 nmol/h/mL), thermostability (significant inactivation of the enzyme from 60min at 60 ºC) and storage time and temperature (up to 30 days at 4, 25 and 37 °C, above 60 days at -20 °C) and established a reference range for GUSB activity in samples from healthy subjects in this methodology (174.4 nmol/h/mL at 781.9 nmol/h/ mL). We established the use of TU to determine the activities of alpha-iduronidase (IDUA), arylsulfatase B (ASB) and GUSB by measuring the enzymatic activity in DBS eluted in this buffer and correlated with the standardized spectrofluorometric technique for each enzyme in DBS of 1.2 mm in samples from healthy individuals Correlations were positive and the validation coefficients of the technique were within acceptable limits. The activity means determined for healthy individuals were 14.65 ± 4.35 nmol/h/mL (IDUA), 22.51 ± 5.09 nmol/h/mL (ASB) and 531.92 ± 121.05 nmol/h/mL (GUSB). Biochemical parameters involved in oxidative stress in the plasma of individuals with MPS VI and compared to MPS I and healthy controls were analyzed. Measurement of SOD activity did not differ between groups, CAT activity was decreased in both MPS VI and MPS I and the TBARS dosage was increased in both MPS compared to controls. From this study, it was possible to standardize and improve new techniques for laboratory diagnosis for MPS I, VI and VII, besides introducing oxidative stress as a possible marker in the use of enzyme replacement therapy.