BACKGROUND: The lysosomal storage disorder, Niemann Pick type C1 (NPC1), presents a variable phenotype including neurovisceral and neurological symptoms. 2-Hydroxypropyl-ss-cyclodextrin (HPssCD)-based therapies are presently the most promising route of intervention. While severe cerebellar dysfunction remains the main disabling feature of NPC1, sensory functions including auditory and olfactory ones are also affected. Morphological and functional anomalies of Npc1 (-/-) mouse retina have also been observed, although the functional integrity of the visual pathway from retina to visual cortex is still unsettled. We have addressed this issue by characterizing the visual evoked potential (VEP) response of Npc1 (-/-) mice and determining if/how HPssCD administration influences the VEPs of both Npc1 (-/-) and Npc1 (+/+) mice. METHODS: VEP elicited by a brief visual stimulus were recorded from the scalp overlying the visual cortex of adult (PN, postnatal days 60, 75, 85 and 100) Npc1 (+/+) and Npc1 (-/-) mice that had received repeated injections of either HPssCD or plain vehicle. The first injection was given at PN4 and was followed by a second one at PN7 and thereafter by weekly injections up to PN49. Cholesterol accumulation and myelin loss were finally assessed by filipin staining and myelin basic protein immunohistochemistry, respectively. RESULTS AND DISCUSSION: We have found that the transmission of visual signals from retina to visual cortex is negatively influenced by the loss of Npc1 function. In fact, the VEP response of Npc1 (-/-) mice displayed a highly significant increase in the latency compared to that of Npc1 (+/+) mice. HPssCD administration fully rescued this defect and counteracted the cholesterol accumulation in retinal ganglion cells and dorsal lateral geniculate nucleus neurons, as well as the myelin loss in optic nerve fibers and axons projecting to the visual cortex observed in of Npc1 (-/-) mice. By contrast, HPssCD administration had no effect on the VEP response of Npc1 (+/+) mice, further strengthening the treatment efficacy. CONCLUSIONS: This study pinpoints the analysis of VEP response as a potentially accurate and non-invasive approach to assess neural activity and visual information processing in NPC1 patients, as well as for monitoring the progression of the disease and assessing the efficacy of potential therapies.

Introdução: As mucopolissacaridoses (MPS) são um conjunto de sete doenças genéticas incluídas dentro das Doenças Lisossômicas que por sua vez fazem parte dos Erros Inatos do Metabolismo (EIM). São doenças multissistêmicas que afetam todo o organismo, com variações conforme o tipo de MPS, sendo que algumas delas possuem tratamento específico. Quase todas comprometem, em graus variados, o sistema osteoarticular, e praticamente todos os pacientes apresentam excreção alterada de glicosaminoglicanos (GAGs) na urina. As MPS são doenças raras que podem ser subdiagnosticadas em função do pouco conhecimento dos profissionais de saúde sobre elas, do pouco acesso aos métodos de triagem e diagnóstico e da sua ampla heterogeneidade clínica, podendo ocorrer formas atenuadas nas quais pode ser difícil de levantar a suspeita clínica de MPS. Material e métodos: o presente estudo foi realizado no período de março de 2012 à janeiro de 2014, tendo incluído 55 pacientes atendidos em serviços de Reumatologia e/ou Ortopedia de Porto Alegre, RS, Brasil e que, apresentavam como principal queixa, manifestações articulares sem etiologia definida. Esses pacientes foram inicialmente investigados através da avaliação quantitativa e qualitativa dos GAGs urinários. Resultados e Discussão: entre os 55 casos investigados, em 1 paciente de 15 anos de idade foi observada na análise dos GAGs urinários excreção aumentada e alteração do padrão qualitativo, sendo posteriormente confirmado o diagnóstico de uma forma atenuada de MPS II, a qual não havia sido suspeitada anteriormente. Conclusão: embora a proporção de pacientes identificados com MPS na amostra estudada tenha sido pequena (1/55), este estudo mostra que ocorre subdiagnóstico dessas doenças e que a triagem sistemática pode contribuir para a identificação de pacientes, os quais podem se beneficiar das medidas de tratamento disponíveis. / Introduction: Mucopolysaccharidoses (MPS) are a set of 7genetic diseases including Lysosomal Diseases , which in turn are part of Inborn Errors of Metabolism (IEM). The MPS are multisystemic conditions that affect the entire body, with variations depending on the type, some of which have specific treatment. Almost all affect, in variable degrees, the osteo-articular system, and virtually all patients have abnormal excretion of glycosaminoglycans (GAGs) in urine. The MPS are rare diseases that are being underdiagnosed due to the little knowledge of health professionals about them, the poor access to screening and diagnostic methods and their extensive clinical heterogeneity. Also, attenuated forms may occur in which it may be difficult to raise the clinical suspicion of MPS. Material and Methods: The present study was conducted from March 2012 to January 2014 and included 55 patients from Rheumatology and/or Orthopedics services of Porto Alegre, RS, Brazil and which had, as main complaint, articular manifestations without defined etiology. These patients were screened by quantitative and qualitative assessment of urinary GAGs. Results and Discussion: Among the 55 cases investigated, in 1 patient 15 years of age was observed in the analysis of urinary GAG excretion and increased change in qualitative standard and subsequently confirmed the diagnosis of an attenuated form of MPS II, which had not previously been suspected. Conclusion: Although the proportion of patients with MPS identified in the study sample was small (1/55), this study shows that occurs underdiagnosis of these diseases and that systematic screening can help to identify patients who may benefit from measures treatment available.

Introdução: As mucopolissacaridoses (MPS) são um conjunto de sete doenças genéticas incluídas dentro das Doenças Lisossômicas que por sua vez fazem parte dos Erros Inatos do Metabolismo (EIM). São doenças multissistêmicas que afetam todo o organismo, com variações conforme o tipo de MPS, sendo que algumas delas possuem tratamento específico. Quase todas comprometem, em graus variados, o sistema osteoarticular, e praticamente todos os pacientes apresentam excreção alterada de glicosaminoglicanos (GAGs) na urina. As MPS são doenças raras que podem ser subdiagnosticadas em função do pouco conhecimento dos profissionais de saúde sobre elas, do pouco acesso aos métodos de triagem e diagnóstico e da sua ampla heterogeneidade clínica, podendo ocorrer formas atenuadas nas quais pode ser difícil de levantar a suspeita clínica de MPS. Material e métodos: o presente estudo foi realizado no período de março de 2012 à janeiro de 2014, tendo incluído 55 pacientes atendidos em serviços de Reumatologia e/ou Ortopedia de Porto Alegre, RS, Brasil e que, apresentavam como principal queixa, manifestações articulares sem etiologia definida. Esses pacientes foram inicialmente investigados através da avaliação quantitativa e qualitativa dos GAGs urinários. Resultados e Discussão: entre os 55 casos investigados, em 1 paciente de 15 anos de idade foi observada na análise dos GAGs urinários excreção aumentada e alteração do padrão qualitativo, sendo posteriormente confirmado o diagnóstico de uma forma atenuada de MPS II, a qual não havia sido suspeitada anteriormente. Conclusão: embora a proporção de pacientes identificados com MPS na amostra estudada tenha sido pequena (1/55), este estudo mostra que ocorre subdiagnóstico dessas doenças e que a triagem sistemática pode contribuir para a identificação de pacientes, os quais podem se beneficiar das medidas de tratamento disponíveis. / Introduction: Mucopolysaccharidoses (MPS) are a set of 7genetic diseases including Lysosomal Diseases , which in turn are part of Inborn Errors of Metabolism (IEM). The MPS are multisystemic conditions that affect the entire body, with variations depending on the type, some of which have specific treatment. Almost all affect, in variable degrees, the osteo-articular system, and virtually all patients have abnormal excretion of glycosaminoglycans (GAGs) in urine. The MPS are rare diseases that are being underdiagnosed due to the little knowledge of health professionals about them, the poor access to screening and diagnostic methods and their extensive clinical heterogeneity. Also, attenuated forms may occur in which it may be difficult to raise the clinical suspicion of MPS. Material and Methods: The present study was conducted from March 2012 to January 2014 and included 55 patients from Rheumatology and/or Orthopedics services of Porto Alegre, RS, Brazil and which had, as main complaint, articular manifestations without defined etiology. These patients were screened by quantitative and qualitative assessment of urinary GAGs. Results and Discussion: Among the 55 cases investigated, in 1 patient 15 years of age was observed in the analysis of urinary GAG excretion and increased change in qualitative standard and subsequently confirmed the diagnosis of an attenuated form of MPS II, which had not previously been suspected. Conclusion: Although the proportion of patients with MPS identified in the study sample was small (1/55), this study shows that occurs underdiagnosis of these diseases and that systematic screening can help to identify patients who may benefit from measures treatment available.

Introdução: As mucopolissacaridoses (MPS) são um conjunto de sete doenças genéticas incluídas dentro das Doenças Lisossômicas que por sua vez fazem parte dos Erros Inatos do Metabolismo (EIM). São doenças multissistêmicas que afetam todo o organismo, com variações conforme o tipo de MPS, sendo que algumas delas possuem tratamento específico. Quase todas comprometem, em graus variados, o sistema osteoarticular, e praticamente todos os pacientes apresentam excreção alterada de glicosaminoglicanos (GAGs) na urina. As MPS são doenças raras que podem ser subdiagnosticadas em função do pouco conhecimento dos profissionais de saúde sobre elas, do pouco acesso aos métodos de triagem e diagnóstico e da sua ampla heterogeneidade clínica, podendo ocorrer formas atenuadas nas quais pode ser difícil de levantar a suspeita clínica de MPS. Material e métodos: o presente estudo foi realizado no período de março de 2012 à janeiro de 2014, tendo incluído 55 pacientes atendidos em serviços de Reumatologia e/ou Ortopedia de Porto Alegre, RS, Brasil e que, apresentavam como principal queixa, manifestações articulares sem etiologia definida. Esses pacientes foram inicialmente investigados através da avaliação quantitativa e qualitativa dos GAGs urinários. Resultados e Discussão: entre os 55 casos investigados, em 1 paciente de 15 anos de idade foi observada na análise dos GAGs urinários excreção aumentada e alteração do padrão qualitativo, sendo posteriormente confirmado o diagnóstico de uma forma atenuada de MPS II, a qual não havia sido suspeitada anteriormente. Conclusão: embora a proporção de pacientes identificados com MPS na amostra estudada tenha sido pequena (1/55), este estudo mostra que ocorre subdiagnóstico dessas doenças e que a triagem sistemática pode contribuir para a identificação de pacientes, os quais podem se beneficiar das medidas de tratamento disponíveis. / Introduction: Mucopolysaccharidoses (MPS) are a set of 7genetic diseases including Lysosomal Diseases , which in turn are part of Inborn Errors of Metabolism (IEM). The MPS are multisystemic conditions that affect the entire body, with variations depending on the type, some of which have specific treatment. Almost all affect, in variable degrees, the osteo-articular system, and virtually all patients have abnormal excretion of glycosaminoglycans (GAGs) in urine. The MPS are rare diseases that are being underdiagnosed due to the little knowledge of health professionals about them, the poor access to screening and diagnostic methods and their extensive clinical heterogeneity. Also, attenuated forms may occur in which it may be difficult to raise the clinical suspicion of MPS. Material and Methods: The present study was conducted from March 2012 to January 2014 and included 55 patients from Rheumatology and/or Orthopedics services of Porto Alegre, RS, Brazil and which had, as main complaint, articular manifestations without defined etiology. These patients were screened by quantitative and qualitative assessment of urinary GAGs. Results and Discussion: Among the 55 cases investigated, in 1 patient 15 years of age was observed in the analysis of urinary GAG excretion and increased change in qualitative standard and subsequently confirmed the diagnosis of an attenuated form of MPS II, which had not previously been suspected. Conclusion: Although the proportion of patients with MPS identified in the study sample was small (1/55), this study shows that occurs underdiagnosis of these diseases and that systematic screening can help to identify patients who may benefit from measures treatment available.

Le développement de thérapies ciblées est un enjeu majeur en santé et l’essor des nanovecteurs permet de répondre à ces besoins cliniques. Le premier axe de cette thèse est consacré à l’étude du potentiel thérapeutique de nanoparticules multifonctionnelles pour l’imagerie médicale, la thérapie photothermique et la délivrance de drogue pour le traitement du cancer. Le deuxième axe de recherche s’oriente vers le ciblage thérapeutique actif. L’entreprise NanoMedSyn a pour objectif de développer un ciblage actif du récepteur du mannose 6-phosphate, permettant un meilleur adressage des médicaments et des traitements plus efficaces. Ce type de ciblage peut avoir des retombées multiples pour la thérapie anticancéreuse mais également pour la thérapie des maladies lysosomales qui sont des maladies génétiques rares. NanoMedSyn développe des dérivés synthétiques de glycovecteurs innovants, appelés AMFA, qu’elle exploite en exclusivité. Les AMFA ont une bonne affinité pour le récepteur du mannose 6-phosphate et ont été greffés sur des nanoparticules multifonctionnelles dans le but d’améliorer l’adressage et la thérapie photodynamique biphotonique d’un cancer pédiatrique : le rhabdomyosarcome ; et sur des enzymes lysosomales pour le traitement de maladies lysosomales telle que la maladie de Pompe. / The development of targeted therapies is a major health issue and the rise of nanovectors makes it possible to meet these clinical needs. The first approach of this thesis is dedicated to the study of the therapeutic potential of multifunctional nanoparticles for medical imaging, photothermal therapy and drug delivery in cancer treatment. The second line of research focuses on active therapeutic targeting. The NanoMedSyn company aims to develop an active targeting of the mannose 6-phosphate receptor, allowing a better addressing of drugs and more effective treatments. This type of targeting may have multiple benefits for cancer therapy but also for the treatment of the lysosomal diseases which are rare genetic diseases. NanoMedSyn develops innovative synthetic derivatives of glycovectors, called AMFA, which it exploits exclusively. AMFA have a good affinity for the mannose 6-phosphate receptor and have been grafted on multifunctional nanoparticles in order to improve addressing and two-photon photodynamic therapy of a pediatric cancer: the rhabdomyosarcoma; and on lysosomal enzymes for the lysosomal diseases treatment such as for Pompe disease.

Récemment, le concept de chaperon pharmacologique a émergé pour le traitement des maladies lysosomales. Comme inhibiteurs réversibles de glycosidases mutantes impliquées dans ces maladies, les chaperons pharmacologiques sont capables, à des concentrations sub-inhibitrices, de sauver ces enzymes des mécanismes de destruction du réticulum endoplasmique (RE). Ainsi, une partie de l’activité enzymatique est restaurée. Les iminosucres sont connus pour être une classe importante de chaperons pharmacologiques. Au cours de ce travail de thèse, de nouvelles classes d’iminosucres mono- et multivalents ont été conçues et synthétisées. Nos objectifs étaient de mettre en évidence de nouveaux chaperons pour la β-glucocérébrosidase, impliquée dans la maladie de Gaucher, mais également d’identifier de nouveaux inhibiteurs des α-glucosidases du RE impliquées dans la destruction de la protéine déficiente chez les malades atteints de la mucoviscidose. Plusieurs stratégies ont été mises en œuvre: l’utilisation d’une méthodologie de diamination d’alcènes pallado-catalysée, d’une méthodologie permettant la synthèse rapide d’une bibliothèque de composés iminosucres par chimie click ou encore de la multivalence. Une étude poussée sur la multivalence et l’inhibition de glycosidases a également été réalisée en faisant varier des paramètres clés de la multivalence tels que la valence, la charpente, le linker, ou encore la nature des ligands iminosucres. Le premier exemple d’un effet multivalent puissant jusqu’à quatre ordre de grandeur sur l’inhibition de glycosidases a été mis en évidence avec des systèmes iminosucres multivalents basés sur des charpentes de type β-cyclodextrine et fullerène C60. / Recently an innovative concept for the treatment of lysosomal diseases as emerged called pharmacological chaperone. Pharmacological chaperones are reversible inhibitors of the deficient glycosidases involved in these diseases. These molecules are able, at sub-inhibitory concentrations, to stabilize the enzymes and rescue them from the destruction by the quality control system of the endoplasmic reticulum. A part of the catalytic activity of the enzyme could be restored. Iminosugars are known to be an important class of pharmaceutical chaperones. During this PhD work, novel classes of mono- and multivalent iminosugars were designed and synthesized in order to identify novel pharmacological chaperones for the glycosidase: β-glucocerebrosidase involved in Gaucher’s disease and novel inhibitors of the α-glucosidases involved in the destruction of the defective protein delF508CFTR in cystic fibrosis. Several strategies were applied to achieve this aim. These strategies consist in the use of a synthetic methodology of palladium catalyzed alkenes diamination, the use of an efficient methodology to synthesize a library of novel iminosugars by click chemistry and the use of multivalency. A full study on the impact of multivalency on glycosidases inhibition was also completed by changing crucial structural parameters including valency, scaffold, linker and ligand. The first strong multivalent effect on glycosidases inhibition up to four orders of magnitude was reported with multivalent iminosugars based on β-cyclodextrin or C60 fullerene cores.