Rozprawy doktorskie na temat „Gauchers disease”

Gaucher disease (GD) is one of the most prevalent lysosomal storage diseases.A mutation on gene gba1 generates a misfolded enzyme acid β-glucosidase. Due to this, this enzyme is not properly transported from the endoplasmic reticulum (ER) to the lysosome, with the consequent reduction of its lysosomal activity. This leads to the accumulation of the substrate glucosylceramide (GluCer) in the lysosomes of macrophages that originate the clinical symptoms. Besides the expensive Enzymatic Replacement Therapy (ERT), different approaches could be useful to minimize this accumulation of GluCer. One option could be the inhibition of Glucosylceramide Synthase (CGS) in order to reduce the formation of GluCer. Another approach is the use of Pharmacologic Chaperones (PC) that bind to the misfolded GCase in the ER, assisting the correct protein folding and allowing its transport to the lysosome, where the enzymatic degradation of GluCer takes place. In order to facilitate the correct folding of GCase without blocking its hydrolase activity, an ideal PC should show high affinity for GCase in the ER environment (neutral pH) and lower affinity in the lysosome (pH 5.2). For this purpose, we have designed and synthesized two small libraries of compounds in order to study the influence on their affinity to bind GCase of the pKas of the substituents in the side chain of several DNJ derivatives, as well as the pKas of the nitrogen in the sugar mimetic cores, was modulated by introduction of different β- substituents. The synthesized compounds were evaluated as imiglucerase (recombinant Gcase) inhibitors at pH 5.2 and pH 7.0 in presence of detergents that emulate the lipids naturally presents in the cell. In this assay conditions, most of the compounds showed better imiglucerase inhibition at neutral pH than at pH 5.2, but no correlation could be established between the affinity for GCase and their pKas. Even though, it has been discovered the DNJ derivative with higher potency as GCase inhibitor described so far. After analyzing the some representative compounds as GBA1 inhibitors in a detergent-free assay with cell homogenates it has been noticed the importance of the assay conditions when studying the inhibitory potency of different compounds. In this way, the IC50 values obtained in the assay with imiglucerase and detergents drive to totally different conclusions than when the assay was performed with cell homogenates without addition of exogenous detergents. Even so, neither direct correlation between the pKas of the inhibitors and their affinity for GCase could be established at this point. Moreover, the capacity of GCS inhibition of the synthesized compounds was also analyzed, revealing a family of compounds that act as GCS inhibitors more potent that NB-DNJ, a drug that is actually administered as GCS inhibitor for treatment of GD. Going one step further, a family of compounds with potential dual behavior GCS inhibitors and PC for GCase was discovered. On the other hand, GBA2 inhibition studies of the synthesized compounds revealed some nanomolar inhibitors of this enzyme. Finally, a new GBA inhibition assay in intact cell has been developed for the analysis of GBA1 or GBA2 inhibition without lysate cells, and allowing the study of the posterior recovery of GBA activity after inhibitor removal.
La malaltia de Gaucher és una de les malalties d’emmagatzematge lisosomal més freqüent. Una mutació al gen gba1 provoca un incorrecte plegament de l’enzim Glucocerebrosidasa (GCase) que impedeix el seu transport del reticle endoplasmàtic (ER) fins al lisosoma, on té lloc la hidròlisi de la glucosilceramida (GluCer). Això provoca una acumulació d’aquest substrat, originant els símptomes clínics. En aquesta tesi s’ha sintetitzat una petita col·lecció de compostos per tal estudiar la influència del pKa de diferents inhibidors en la seva potencial activitat chaperona, és a dir, estudiar la seva capacitat de unir-se a la GCase mal plegada del reticle per facilitar-ne el correcte plegament i permetre el seu transport cap a lisosoma. Per aquest motiu, es buscaven compostos que presentessin una alta afinitat per la GCase en les condicions neutres del reticle endoplasmàtic, però que tinguessin baixa afinitat per aquest enzim a pHs lleugerament àcids com el del lisosoma (pH 5.2), per tal que no bloquegés l’activitat hidrolasa d’aquest enzim al lisosoma. Desprès d’analitzar els compostos amb diferents assajos, es va posar de manifest la importància de les condicions d’assaig a l’hora d’estudiar la inhibició d’un enzim, ja que diferents assajos poden conduir a conclusions diferents. Malgrat que no es va poder establir cap correlació directa entre el pKa dels inhibidors i la diferència d’activitat segons el pH de l’assaig, es va descobrir un dels derivats de DNJ amb millor potència inhibitòria de imiglucerasa (Gcase recombinant) descrit fins el moment. Per altra banda, es va analitzar la capacitat d’inhibició de GCS dels compostos sintetitzats, descobrint-se alguns inhibidors d’aquest enzim més potents que la NB-DNJ, compost que s’utilitza actualment com a inhibidor de GCS per al tractament de la malaltia de Gaucher, i possible comportament dual (inhibidors de GCS i chaperones per GCase).

Fabry (FD) and Gaucher (GD) disease are lysosomal storage disorders, caused by single enzyme deficiencies on the glycosphingolipid degradation pathway as a result of genetic mutations in the GLA and GBA genes respectively. These result in a functional enzyme deficiency within the lysosome and accumulation of un-degraded substrate. GD is characterised by a bleeding tendency and bone infarction. Patients with FD suffer from a vasculopathy with strokes, proteinuric renal failure and cardiac conduction defects, but both disorders are highly heterogeneous. Abnormal cytokine profiles and a pro-inflammatory state have been found in both FD and GD, leading to the hypothesis that abnormalities at the blood-endothelial interface affecting coagulation and leucocyte adhesion contribute to the pathology of these disorders. This thesis demonstrates the importance of vascular manifestations in the presentation of both GD and FD with failure to identify the underlying cause of these manifestations resulting in delays between the onset of clinical manifestations and arrival at the correct diagnosis. Abnormalities at the blood-endothelial interface identified in GD include up-regulation of adhesion molecules on lymphocytes that may be of importance in the pathogenesis of bone disease, and increased thrombin generation in an endothelial cell model of GD. In FD, whilst cardiac and renal manifestations occur at earlier onset and with greater severity in men, cerebrovascular disease seems to affect both sexes to a similar degree. Monocytes from females with FD exhibit an age-dependent increase in adhesion mimicking the age-dependent increase in cardiac and renal disease seen in these patients but the mechanisms underlying cerebrovascular disease remain uncertain. Initial investigations of platelet prothrombinase activity suggest this may be enhanced in FD. Further investigation of these abnormalities at the cellular level may shed new insights on, and open up new therapeutic options, for the management of the vascular complications of these disorders.

Gaucher disease is a lysosomal storage disorder caused by mutations in the GBA1 gene encoding the enzyme glucocerebrosidase (GCase). Deficiency of GCase causes the accumulation of its substrate glucosylceramide in both visceral organs and the brain. Enzyme replacement therapy is successfully used to ameliorate the visceral pathology, however there is no treatment available for the lethal neurodegeneration. This research focuses on Gaucher disease type II, the most acute neuronopathic form, in which the neuropathology results in death during early infancy. The aim of this project is to intravenously administer adeno-associated viral vector (AAV) based gene therapy to a GCase-deficient mouse model of acute neuronopathic Gaucher disease and assess improvement in lifespan, behaviour, brain and visceral pathology. The untreated Gba1 knock-out mice die 12-14 days after birth following severe neurodegeneration. The AAV vector carrying the functional human GBA1 gene under control of a ubiquitous promoter was intravenously administered to neonatal knock-out mice, with treated animals showing a significant increase in their lifespan (p=0.0081). Since the animals did not develop any evident pathological symptoms, they were sacrificed at 55 days of age for a short-term study. The neuropathology was ameliorated and several of the most affected areas of the brain were partially rescued. The analysis of liver, spleen, lung and heart tissues revealed promising improvements in the visceral pathology. A consequent long-term study was performed on 180-day-old treated mice, with the aim to compare intravenous and intracranial administration of the viral vector. In order to enhance the therapeutic effects of the treatment and improve gene expression in the central nervous system, a novel construct where the GBA1 gene is controlled by a neuron-specific promoter was administered to neonatal knock-out mice. The severe neurodegeneration was further rescued and the life span of treated animals increased. Together, these encouraging results demonstrate that gene therapy could provide an effective treatment for the neuronopathic form of Gaucher disease, for which therapeutic needs are currently unmet.

Gaucher disease (GD), a recessive disorder characterised by hepatosplenomegaly, pancytopenia and skeletal complications, is caused by deficiency of the enzyme glucocerebrosidase (GC). GD leads to the accumulation of glucocerebrosides within macrophages, particularly in the liver and spleen. Current treatment is limited to enzyme replacement therapy (ERT) which is effective for most symptoms however skeletal problems are slow to respond. Treatment also has significant cost and impact on quality of life as infusions must be administered every two weeks. GD is a candidate for gene therapy as bone marrow transplantation has been shown to be curative which serves as a proof-of-concept that correction of haematopoietic stem cells (HSCs) can alleviate disease. This project produced lentiviral vectors carrying a range of constructs. GC was modified to contain a protein transduction domain (PTD) which could facilitate cross-correction of untreated cells in vivo. Recombinant vectors carrying PTD-GBA cDNA corrected the metabolic defect in patient-derived fibroblasts with levels of enzyme activity restored to within the healthy range. Transduced cells secreted active protein, uptake of which by untransduced cells was mediated by fusion of a PTD to the C- but not the N-terminus of the enzyme. The skeletal complications of GD are likely to be caused by enzyme deficiency in the osteoclast, a cell of haematopoietic origin. Therefore it is possible that by transducing HSCs we will be able to alleviate skeletal symptoms. To this end it is shown that modification of HSCs does not affect their ability to generate osteoclasts. It is also demonstrated that osteoclasts derived in vitro from the neuronopathic GD mouse model have increased activity and this could be a useful model for osteoclast correction when treating GD. In conclusion, this project generated lentiviral vectors for use in treating Gaucher disease. Further work should include correction of the osteoclast phenotype and further investigation of the potential for cross-correction in vivo.

Gaucher disease is a rare inherited lysosomal disorder caused by deficiency of the enzyme glucocerebrosidase. Classically, three forms of the disease are recognised: type I or nonneuronopathic, type 2 or acute neuronopathic, and type 3 or subacute or chronic neuronopathic. Neuronopathic Gaucher disease (NGD) is defined as a confirmed diagnosis of Gaucher disease in the presence of neurological symptoms and signs, for which there is no other cause. Horizontal gaze palsy is the clinical hallmark of NGD. Other neurological manifestations include seizures, cerebellar ataxia and pyramidal tract involvements. However, NGD is very heterogeneous and the neurological features vary greatly from patient to patient, not only in terms of manifestations involved but also in terms of severity. The emergence of enzyme replacement therapy has changed the ‘natural history’ of the disease, and patients are now living longer where previously they would have succumbed to the visceral complications of the disease. New emerging therapies are being developed for NGD, however a suitable surrogate marker to monitor neurological disease is lacking. In this study, three different assessment tools were explored to examine their value and sensitivity to assess neurological involvement in NGD. A Severity Scoring Tool developed specifically for NGD was modified and validated to offer a robust assessment tool, with demonstrated sensitivity to track disease progression and distinguish between phenotypes. Additional assessments utilised were gait analysis and diffusion tensor imaging, both of which were sensitive enough to distinguish between the NGD and Type I cohort studied. This is the largest cohort of NGD patients (recruited across three European countries) to be studied prospectively and systematically. It is also the first study to describe the gait pattern of NGD children, and to provide an in-vivo insight of the Gaucher brain utilising diffusion tensor imaging.

Introduzione: La malattia di Gaucher (GD) è la più frequente patologia da accumulo lisosomiale, causata dalla ridotta attività dell’enzima β-glucosidasi acida, che determina un progressivo accumulo di glucosilceramide nelle cellule del sistema reticolo-endoteliale. L’epatosplenomegalia e la citopenia sono le manifestazioni cliniche più comuni, ma i pazienti affetti presentano anche una disregolazione immunologica con alterazione del pattern citochinico e chemochinico e alterazioni funzionali a carico non solo dei monociti-macrofagi, ma anche di linfociti B, T e natural killer (NK). Inoltre, le manifestazioni cliniche sono parzialmente sovrapponibili a quelle della sindrome linfo-proliferativa autoimmune (ALPS) o di altri disordini immunologici ALPS-like. L’obiettivo principale di questo lavoro è l’identificazione di possibili alterazioni immunologiche ALPS-like e di un eventuale difetto di apoptosi nei pazienti con malattia di Gaucher. Pazienti e metodi: Nello studio sono stati arruolati i pazienti con GD valutati presso la UOSD Malattie Rare dell’IRCCS Giannina Gaslini in un periodo di 18 mesi. Per ciascun paziente è stato eseguito uno studio delle sottopopolazioni linfocitarie e dei parametri immunofenotipici e sierologici caratteristicamente alterati nell’ALPS (linfociti T doppi negativi (DNT), B220+DNT, CD27+, T-reg/HLA-DR ratio, IL-10, IL-18, vitamina B12) e uno screening autoanticorpale. Nei pazienti con alterazione significativa di questi parametri è stato eseguito anche uno studio in vitro dell’apoptosi FAS-mediata e dell’attivazione delle caspasi. Risultati: Sono stati inclusi nello studio 41 pazienti, di cui 33 in terapia e 8 non trattati. Un incremento dei linfociti DNT e B220+DNA è stato riscontrato, rispettivamente, in 9/41 (21%) e 7/41 (17%) pazienti. 10/41 (24%) presentavano alterazioni immunologiche suggestive di ALPS, più frequenti nei pazienti non trattati (p=0,003) e nei pazienti con esordio precoce di GD (p=0,01). L’apoptosi indotta da FAS e l’attivazione delle caspasi sono risultate deficitarie in 7 di questi 10 pazienti. Conclusioni: Lo studio dimostra che i pazienti con GD possono presentare delle alterazioni immunologiche ALPS-like e un deficit dell’apoptosi FAS-mediata, più frequenti nei pazienti non trattati e nei pazienti con esordio precoce di malattia. La malattia di Gaucher pertanto dovrebbe essere considerata in diagnosi differenziale nei pazienti con un fenotipo ALPS-like.
BACKGROUND: Gaucher disease (GD) is a rare disorder characterized by defective function of b-glucocerebrosidase, which leads to progressive accumulation of its substrate in various organs, particularly the mononuclear phagocyte system. Hepatosplenomegaly and cytopenia represent the disease’s most common features, but patients with GD also show hyperinflammation, hypergammaglobulinemia, and immune dysregulation involving B, T, and natural killer cells. As clinical phenotype can be underhand, symptoms can overlap with autoimmune lymphoproliferative syndrome (ALPS) or other ALPS-like disorders. OBJECTIVE: To evaluate the ALPS-like immunological pattern and apoptosis function in patients with GD. METHODS: We evaluated lymphocyte subsets and immunophenotypic and serological features of ALPS (double negative T cells [DNTs], B220DDNTs, CD27D, T-reg/HLADR ratio, IL-10, IL-18, vitamin B12) in a population of patients with GD. Moreover, we tested FAS/TRAIL-induced apoptosis and CASP8/CASP10/PARP function in patients showing an immune-dysregulation pattern. RESULTS: A total of 41 patients (33 treated, 8 treatment-naïve) were studied. Nine (21%) and 7 (17%) of 41 patients had high DNT and B220DDNT counts, respectively. Overall, 10 of 41(24%) patients showed immunological features suggestive of ALPS that were more frequent in treatment-naïve subjects (P [ .040 vs P [ .031) and in those with early onset of the disease (P [ .046 vs P [ .011), respectively. FAS-induced apoptosis and caspase activation were further evaluated in these 10 patients and were found to be defective in 7 of them. CONCLUSIONS: We show that patients with GD may have ALPS-like features and FAS-mediated apoptosis defects that are more pronounced in treatment-naïve subjects and in patients with early onset of the disease. Therefore, diagnostic workup of patients with an ALPS-like phenotype should include screening for GD.

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.

La malaltia de Gaucher (GD) forma part d'un conjunt de malalties que es coneixen amb el nom de "malalties d'acumulació lisosómica". La GD n'és la més prevalent i en la majoria de casos és causada per mutacions en el gen GBA1, que codifica l'enzim glucocerebrosidasa (GBA1) la qual hidrolitza, principalment, l'esfingolipid glucosilceramida (GlcCer) en glucosa i ceramida. En la GD aquest enzim presenta una activitat deficient i la GlcCer s'acumula en els lisosomes dels macròfags que formen les anomenades "cèl. lules Gaucher". La GD és autosòmica recessiva i compren 3 subtipus clínics, en funció de la presència i el grau de les manifestacions neurològiques: la de tipus 1 (GD1; no neuronopàtica), la de tipus 2 (GD2; neuronopàtica aguda), i la de tipus 3 (GD3; neuronopàtica subaguda). La GD1 es caracteritza per presentar problemes viscerals, hematològics i ossis. La patofisiologia de les manifestacions òssies d'aquesta és poc coneguda però tot apunta a que les cèl. lules de Gaucher, el sistema immunològic i les cèl• lules òssies hi juguen un paper important. De totes les aproximacions terapèutiques pel tractament de la malaltia, cal destacar que la teràpia de substitució enzimàtica (ERT) és la més utilitzada, però en els últims 10 anys la teràpia amb xaperones farmacològiques ha esdevingut una opció atractiva per millorar el plegament i transport d'aquells enzims mutats que ho requereixin. El treball que es presenta en aquesta tesi doctoral ha permès identificar algunes xaperones que presenten una bona capacitat d'augmentar l'activitat enzimàtica d'algunes glucocerebrosidases mutades. A més, s'ha elaborat un model mesenquimal i osteoblàstic de la GD1 en el que s'està avaluant la implicació de la deficient activitat GBA1 en la diferenciació osteoblàstica i, indirectament, en l'activació de la formació dels osteoclasts.
Gaucher disease (GD) is the most prevalent lysosomal storage disorder and in most cases is caused by mutations in the GBA1 gene, which encodes the enzyme glucocerebrosidase (GBA1). This protein mainly hydrolyzes the sphingolipid glucosylceramide (GlcCer) into glucose and ceramide. Gaucher patients have a deficient GBA1 enzyme activity which causes that GlcCer accumulates in lysosomes of macrophages forming the so-called "Gaucher cells". GD is an autosomal recessive disorder and can be classified in three clinical subtypes, depending on the presence and degree of neurological manifestations: type 1 (GD1, no neuronopathic), type 2 (GD2; acute neuronopathic) and type 3 (GD3; subacute neuronopathic). The GD1 is characterized by visceral, haematological and bone manifestations. The pathological mechanisms of bone alterations in GD are still poorly understood. But it seems that Gaucher cells, immunitary system and bone cells play an important role. Of the current therapeutic approaches for treatment of the disease, enzyme replacement therapy (ERT) is the most commonly used. However, in the last 10 years pharmacological chaperones therapy has become an attractive option to improve the folding and transport of some mutated enzymes. The aim of this work has been the identification and characterization of compounds with positive effect on the activity of some mutant glucocerebrosidases to act as chaperones. Also, a model of mesenchymal and osteoblastic GD1 has been generated. In this model is being assessed the involvement of deficient GBA1 activity in the osteoblast differentiation process and, indirectly, in the capacity of osteoclasts formation.
La enfermedad de Gaucher (GD) forma parte de un conjunto de enfermedades que reciben el nombre de "enfermedades de acumulo lisosómico". La GD es la más prevalente y en la mayoría de los casos es debida a mutaciones en el gen GBA1 que codifica la enzima glucocerebrosidasa (GBA1) la cual hidroliza, principalmente el esfingolípido glucosilceramida (GlcCer) en glucosa y ceramida. En la GD ésta enzima presenta una actividad deficiente y la GlcCer se acumula en los lisosomas de los macrófagos formando las células Gaucher. La GD es una patología autosómica recesiva e incluye 3 subtipos clínicos, en función de la presencia y el grado de las manifestaciones neurológicas: tipo 1 (GD1; no neurológica), tipo 2 (GD2; neurológica aguda), y tipo 3 (GD3; neurológica subaguda). La GD1 se caracteriza por presentar problemas viscerales, hematológicos y óseos. La patofisiología de las manifestaciones óseas de la GD1 es poco conocida pero parece ser que las células Gaucher, el sistema inmunológico y las células óseas juegan un papel importante. De todas las aproximaciones terapéuticas para el tratamiento de la GD, la terapia de substitución enzimática (ERT) es la más habitual, pero en los últimos 10 años la terapia con chaperones farmacológicas ha resultado ser una buena opción para mejorar el plegamiento y el transporte de aquellos enzimas mutados que lo requieren. El trabajo que se presenta en ésta tesis doctoral ha permitido identificar algunos productos que presentan una buena capacidad de aumentar la actividad de algunas glucerebrosidasas mutadas y actuar por lo tanto como chaperonas farmacológicas. Además, se ha elaborado un modelo mesenquimal y osteoblástico de la GD1 en el que se está evaluando la implicación de la deficiente actividad de la enzima GBA1 en la diferenciación osteoblástica y, indirectamente, en la activación de la formación de los osteoclastos.

IPSC are potent tools in the creation of disease models for both basic studies on the disease and testing of potential therapeutical drugs. In this context, it has been developed the derivation of iPSC from patient fibroblasts suffering from Gaucher Disease (GD) or Tay Sachs disease (TS). GD is a recessive autosomic disease which is characterized by the deficiency of the enzyme called glucocerebrosidase (GBA), which leads to the accumulation of its substrate glucosylceramide in macrophages and neurons. This disease has 3 forms of clinical presentation: type I, which is systemic; type II, the most severe with a neuronopathic acute presentation; type III, which is a combination of the former two, but without the severity of the type II. Tay Sachs is an autosomic recessive disease which is characterized by the Hexosaminidase A (HexA) deficiency, which leads to GM2 accumulation on the lysosomes of neurons. Patients present neurodegeneration and severe impairment of the brain which ultimately leads to their death. In this project iPSC derived from GD and TS patient fibroblasts. Pluripotent state of the derived iPSC has been characterized. Later, iPSC have been differentiated to neurons in order to confirm the disease phenotype on the in vitro differentiated tissue. In GD the phenotype was corroborated by enzymatic assays and GBA detection by Western blot. A lower GBA activity on GD neurons compared to WT was found, consistent with the minor GBA levels in GD neurons detected in the western blot. In TS, derived neurons were analyzed by immunofluorescence for Lamp2 (lysosome marker), observing an increase in size and number on the TS neurons in contrast to WT. Also, TS neurons were analyzed by transmission electron microscopy, presenting membranous lamellar bodies in the cytosol of TS. Both iPSC diseases have been used as a platform for testing therapeutical drugs efficiency on the iPSC derived neurons.
Las iPS (células pluripotentes inducidas) se han revelado como potentes herramientas en la creación de modelos de enfermedades humanas para su estudio y el testeo de potenciales drogas. En este marco hemos desarrollado un proyecto para derivar iPS de fibroblastos de pacientes de Gaucher y Tay Sachs, ambas enfermedades monogénicas recesivas. La enfermedad de Gaucher se caracteriza por la deficiencia de la glucocerebrosidasa (GBA), lo que conlleva la acumulación de su substrato, la glucosilceramida, en macrófagos y neuronas. Esta enfermedad tiene tres presentaciones I, que es sistémica; II, que es una forma neuronopática aguda, tiene efectos fatales ya que los pacientes rara vez sobreviven a los dos años de edad; y III, que es una mezcla de las dos anteriores, siendo neuronopática crónica, sin llegar a la severidad del tipo II. Tay Sachs es una enfermedad que se caracteriza por la deficiencia de la Hexosaminidasa A (HexA) lo que conlleva el almacenamiento en el lisosoma del gangliósido GM2. Los pacientes de esta enfermedad presentan daños neurológicos, provocando la muerte en la mayoría de los casos. En este proyecto se han desarrollado las iPS derivadas de fibroblastos de un paciente de Gaucher tipo II, y de otro de Tay Sachs. Las iPS resultantes de ambas enfermedades han sido caracterizadas para constatar su estado pluripotente y diferenciadas a neuronas para comprobar que presentan el fenotipo característico de las enfermedades. En el caso de Gaucher, mediante ensayos enzimáticos y detección de la GBA1 por western blot, detectando una menor actividad en las neuronas gaucher que en las WT, lo que es consecuente con la menor cantidad de GBA1 detectada. En el caso de Tay Sachs, las neuronas se han analizado mediante inmunohistoquímica, marcando Lamp2, típico de lisosomas y se ha observado un aumento de tamaño y cantidad respecto de las células WT diferenciadas en paralelo. También han sido analizadas por microscopía electrónica, presentando una acumulación de cuerpos laminares en los lisosomas y aumento de número y tamaño de éstos. Ambas enfermedades han sido utilizadas como modelos para probar compuestos en las neuronas derivadas de las iPS derivadas de fibroblastos del paciente y comprobar su eficacia.

Introdução: A doença de Parkinson é uma enfermidade neurodegenerativa decorrente da perda de neurônios dopaminérgicos na substância negra, principalmente, e em outras regiões cerebrais. Caracteriza-se clinicamente por tremor, rigidez, bradicinesia e instabilidade postural. O tratamento é sintomático e consiste essencialmente na reposição da dopamina deficiente. A etiologia da doença de Parkinson ainda não é conhecida, mas os recentes avanços da Neurologia trouxeram novos conhecimentos acerca dos mecanismos fisiopatológicos envolvidos. Disfunção mitocondrial, estresse oxidativo e degradação de proteínas são alguns dos processos celulares que foram relacionados à degeneração dos neurônios dopaminérgicos. O campo da genética da doença de Parkinson tem recebido atenção especial na última década, graças à descoberta de vários genes associados ao desenvolvimento da doença. Um fator de risco genético recentemente descrito é a presença de mutações no gene da glicocerebrosidase, uma enzima lisossomal cuja deficiência resulta na doença de Gaucher. Apesar de a maioria dos estudos já publicados terem confirmado esta associação, um trabalho mais recente da Noruega não encontrou significância estatística ao analisar a presença destas mutações em pacientes com doença de Parkinson, tornando o assunto ainda controverso. Objetivo: Pesquisar a presença de mutações da glicocerebrosidase em pacientes com diagnóstico de doença de Parkinson no Brasil, acompanhados no ambulatório de Distúrbios do Movimento do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo e correlacionar tais achados com estudos recém-publicados que analisaram esta associação em outras populações em âmbito mundial, além de descrever possíveis características dos pacientes portadores de mutações que os diferenciem de não portadores. Métodos: Foram incluídos no estudo 65 pacientes com o diagnóstico de doença de Parkinson e idade de início da doença inferior ou igual a 55 anos e 267 controles sem a doença, emparelhados para sexo e idade. Foi realizada análise genética de material obtido a partir de raspagem da mucosa oral destes indivíduos, tendo sido pesquisadas as três mutações da glicocerebrosidase mais comuns na população brasileira: N370S, L444P e G377S. Resultados: Em dois dos 65 pacientes e em nenhum dos 267 controles foram identificadas mutações no gene da glicocerebrosidase. Os dois pacientes carreadores de mutações (L444P em um e L444P + E326K em outro) apresentavam quadro clínico indistinguível dos demais pacientes com doença de Parkinson não portadores das mutações. Conclusões: Foi observada uma associação estatisticamente significativa (P=0,0379, teste exato de Fisher) entre doença de Parkinson e mutações da glicocerebrosidase na nossa população. A prevalência de mutações da glicocerebrosidase neste grupo de pacientes foi maior do que a esperada para a população geral, porém menor do que a encontrada em estudos internacionais previamente publicados. Espera-se que a identificação desta nova associação permita uma maior compreensão dos mecanismos subjacentes à doença de Parkinson e que em um futuro próximo possa propiciar o desenvolvimento de novas estratégias terapêuticas.
Introduction: Parkinson\'s disease is a neurodegenerative disorder due to the loss of dopaminergic neurons in the substantia nigra, primarily, and in other brain regions. It is clinically characterized by tremor, rigidity, bradykinesia and postural instability. Treatment is symptomatic and consists essentially in replacing the deficient dopamine. The etiology of Parkinson\'s disease remains unknown, but recent advances in Neurology have provided data concerning the pathophysiological mechanisms involved. Mithocondrial dysfunction, oxidative stress and protein degradation are some of the cellular processes that have been linked to dopaminergic neurons degeneration. The field of genetics in Parkinson\'s disease has gained special attention in the past decade, thanks to the discovery of several genes associated with the development of the disease. A recently described genetic risk factor for Parkinson\'s disease is the presence of glucocerebrosidase gene mutations. Glucocerebrosidase is a lysosomal enzyme which is deficient in Gaucher disease. Although most studies published to date have confirmed such association, a recent article from Norway could not find statistical significance when Parkinson\'s disease patients were analyzed for glucocerebrosidase mutations, generating controversy. Objective: To search for glucocerebrosidase mutations in Parkinson\'s disease patients in Brazil, followed at the Movement Disorders Division at Hospital das Clínicas, University of São Paulo Medical School, and correlate these findings with recently published studies which evaluated this association in other populations worldwide, besides describing possible features of patients carrying the mutations that may help differentiating them from non-carriers. Methods: Sixty five patients diagnosed with Parkinson’s disease, with disease onset before age 55, and 267 age and sex-matched controls were included in the study. DNA analysis of the three most common glucocerebrosidase mutations in the Brazilian population, N370S, L444P and G377S, was performed utilizing samples obtained from mouth mucus. Results: Glucocerebrosidase gene mutations were identified in two of the 65 Parkinson\'s disease patients and in none of the 267 controls. The two patients who were carriers of mutations (one of them had L444P and the other L444P+E326K) had a clinical picture indistinguishable from the other Parkinson\'s disease non-carriers patients. Conclusion: A statistically significant association (P=0,0379, Fisher\'s exact test) between Parkinson\'s disease and glucocerebrosidase mutations was observed in our population. The prevalence of glucocerebrosidase mutations was higher than expected for the general population, though lower than reported in previous international studies. It is expected that the finding of this association will allow a better understanding of Parkinson\'s disease mechanisms and that in a near future it may help providing the development of new therapeutic strategies.

Este estudo descreve a análise molecular de pacientes da doença de Gaucher (DG) e Tay-Sachs (DTS) no Brasil. Foram estudados nove casos de formas clássicas da DTS que mostraram uma prevalência da mutação IVS7+1g>c, já descrita em pacientes Portugueses e dez casos das variantes de início juvenil e tardio da DTS, mostrando heterogeneidade genética. Nos casos da variante B1, percebeu-se uma maior incidência da mutação R178H, também descrita previamente em pacientes Portugueses. A presença das mesmas mutações nos casos Brasileiros e Portugueses se deve provavelmente à ancestralidade comum. Uma família com quatro pacientes da variante de início tardio da DTS mostrou uma extensa variabilidade clínica intrafamilial e identificou relevantes aspectos do diagnóstico e das implicações dos programas de triagem populacional. A análise por RFLP de nove mutações causadoras da DG, em 262 pacientes permitiu detectar 76% das alterações e mostrou uma prevalência das mutações N370S e L444P, similar à descrita em diversas outras populações. Os pacientes com variantes neuronopáticas da doença apresentaram uma alta freqüência da mutação G377S, que também é encontrada em pacientes Portugueses. Os pacientes apresentando a G377S indicaram a existência de um provável mecanismo de efeito de dose alélica para essa mutação. Foi observada uma alta freqüência de alelos resultantes da recombinação do gene GBA com seu pseudogene. Diversas outras relações genótipo-fenótipo puderam ser verificadas, mostrando uma baixa penetrância do genótipo N370S/N370S e corroborando a importância do diagnóstico molecular da DG, devido a seu valor preditivo. A análise de mutações raras no gene GBA usando as técnicas de RFLP, dHPLC e seqüenciamento de DNA possibilitou detectar mutações em 84% dos alelos de 54 pacientes. Foram identificadas 14 novas mutações causadoras da DG. Diversas relações genótipo-fenótipo puderam ser verificadas, conferindo valor preditivo para a detecção dessas mutações. Por fim, a análise da associação da DG e da DP permitiu encontrar uma freqüência significativamente maior de portadores das principais mutações no gene GBA em pacientes parkinsonianos (2/65=3%), com aparecimento precoce da doença, comparados a um grupo controle de 267 indivíduos. Esse trabalho fornece nova evidência de que mutações no gene GBA são um raro, mas consistente fator de risco para a DP.
This study describes the molecular diagnosis of Gaucher (GD) and Tay-Sachs disease (TSD) patients in Brazil. Nine cases of the classic infantile form of TSD were analyzed disclosing a prevalence of the IVS7+1g>c mutation, described previously in Portuguese patients. Ten cases of juvenile and late-onset TSD forms were diagnosed showing genetic heterogeneity. Among the B1 variant cases, there was a predomenance of mutation R178H that was also associated to Portuguese ancestry. The presence of the same mutations in Brazilian and Portuguese cases are probably due to common ancestry. A family with 4 affected patients of late onset TSD showed and extensive intrafamilial clinical variability, highlighting relevant characteristics of diagnosis and implications of heterozygote screening programs. Among 263 GD patients, the detection of nine mutations by RFLP revealed 76% of the mutant alleles and a preponderance of N370S and L444P, similar to other populations. The type 3 neuronopathic patients presented a high frequency of mutation G377S, that is also described among Portuguese cases. The patients with G377S indicated the possibility of an allele dose effect for this mutation. Recombinant alleles, presenting pseudogene mutations were detected at a high frequency. Several genotype-phenotype correlations could be verified, highlighting a low penetrance of genotype N370S/N370S and corroborating the importance of molecular diagnosis in GD cases, due to its predictive value. The search for rare mutations at the GBA gene, using dHPLC and DNA sequencing after RFLP analysis, allowed the detection of 84% of the alleles among 54 patients. Fourteen new GD causing mutations were described. Several genotype-phenotype correlations could be established, confering prective value to the identification of these mutations. Finally, the study of the association of GD and Parkinson disease (PD) lead to the detection of a significant increase in the frequency of GBA mutations carriers, among 65 PD patients (2/65=3%) with earlier disease onset compared to a control group (n=267 individuals). This work confers further evidence for the fact that GBA mutations are a rare but consistent risk factor for PD.

La degenerazione dopaminergica che caratterizza il morbo di Parkinson antecede la manifestazione dei segni clinici. Identificare gli eventi molecolari responsabili dell'insorgenza della malattia di Parkinson rappresenta un traguardo fondamentale sia per poter sviluppare marcatori diagnostici che trattamenti preventivi; tuttavia, lo studio della patogenesi è limitato dalla carenza di modelli appropriati e dalla difficoltà di studiare la malattia umana che evolve lungo un arco temporale di decenni. Le indicazioni che provengono dall’analisi dei fattori di rischio, sono in questo contesto fondamentali per comprendere l’eziopatogenesi. Tra di esse, particolarmente rilevanti sono le mutazioni nel gene GBA1, che codifica per l'enzima lisosomiale β-glucocerebrosidasi (GCasi), essendo considerate il maggiore fattore di rischio per lo sviluppo della malattia di Parkinson; questa proteina risulta essere fondamentale anche per la malattia di Gaucher, in quanto individui portatori di mutazioni in omozigosi sviluppano tale patologia che, in un sottogruppo di pazienti, è caratterizzata anche dalla presenza di neurodegenerazione. Le mutazioni in tale gene sono quindi considerate al crocevia di due malattie; nel caso della malattia di Gaucher l’alterazione dell’attività della GCasi induce nei macrofagi un disturbo lisosomiale considerato il segno distintivo della malattia; nel morbo di Parkinson, invece, si pensa che il difetto enzimatico produca i suoi effetti negativi soprattutto sulla componente neuronale. Allo scopo di studiare gli eventi molecolari caratterizzanti l’incipit della malattia di Parkinson, durante il corso di Dottorato, ho generato sistemi reporter innovativi che permettono di misurare la modulazione di segnali neuroprotettivi in colture cellulari e in vivo, nel cervello di modelli animali, rappresentativi delle fasi prodromiche della malattia di Parkinson, attraverso approcci basati sull’imaging ottico. In particolare, lo studio si è concentrato sul fattore di trascrizione nucleare eritroide-2 (NRF2), che regola la risposta citoprotettiva allo stress ossidativo e agli xenobiotici, e sul fattore di trascrizione EB (TFEB), il principale regolatore delle vie autofagiche e lisosomiali, poiché è noto che la loro deregolazione può comportare danno cellulare. Attraverso l’imaging ottico, esperimenti di immunocitochimica ed immunoistochimica ho dimostrato che nella linea di neuroblastoma dopaminergico SK-N-BE e nell’area celebrale murina corrispondente alla substantia nigra par compacta l'attivazione di Nrf2, ma non quella di Tfeb, precede la neurodegenerazione dopaminergica indotta dalla neurotossina MPP+. Lo studio è stato quindi focalizzato sulla dinamica di attivazione del fattore Nrf2 durante le fasi iniziali del processo neurodegenerativo indotto dall’inibizione dell’enzima GCasi, un evento che, come citato sopra, rappresenta il principale fattore di rischio di contrarre la malattia di Parkinson. Gli esperimenti condotti su linee immortalizzate, su cellule primarie e modelli murini hanno permesso di identificare un nuovo meccanismo di comunicazione tra la microglia ed il neurone, basato sul contatto diretto tra le due tipologie cellulari che avviene attraverso strutture actina-dipendenti presenti nella microglia e che richiede un metabolismo energetico cellulare funzionale. Sorprendentemente, l'inibizione farmacologica della GCasi microgliale è sufficiente per interferire con la capacità della microglia di stimolare una comunicazione neuroprotettiva attraverso l’induzione del segnale NRF2. Lo studio ha infine fornito evidenze che tale disfunzione indotta dall’inibizione della GCasi potrebbe essere ripristinata attraverso un effetto compensatorio sul metabolismo energetico, suggerendo una nuova strategia terapeutica preventiva per la malattia di Parkinson. In conclusione, la mia tesi ha dimostrato che i sistemi reporter sviluppati possono essere strumenti utili per lo studio della dinamica di segnali neuroprotettivi; questa tecnologia è stata sviluppata in particolare per quantificare la risposta allo stress ossidativo e delle vie autofagico/lisosomiali. In particolare, l’applicazione di tali sistemi ha permesso di dimostrare che l'inibizione della GCasi altera la comunicazione citoprotettiva microglia/neurone, aumentando la suscettibilità dei neuroni agli insulti neurotossici. In tal modo abbiamo identificato un nuovo meccanismo che potrebbe spiegare l'aumentato rischio di neurodegenerazione osservato nei portatori di mutazioni GBA1 fornendo un nuovo potenziale bersaglio per possibili strategie terapeutiche preventive.
The Parkinson's disease (PD) evolves over an extended period of time with the onset occurring long before clinical signs begin to manifest. Characterization of the molecular events underlying the PD onset is instrumental for the development of diagnostic markers and preventive treatments but progress in this field is hindered by technical limitations. Risk factors are the obvious target of prevention; mutations in the GBA1 gene, encoding for the lysosomal -glucocerebrosidase (GCase) enzyme, have been associated with the highest risk of PD, while homozygous individuals invariably develop Gaucher’s disease (GD), a pathology characterized by several deficits including neuronal damages. GBA1 mutations impair GCase activity and generate a lysosomal disorder in macrophages considered the hallmark of GD, while in the brain most of the effects of GBA1 mutations have been so far attributed to the GCase deficit in neuronal cells. During my Ph.D. project, I have generated innovative reporter tools and applied different optical imaging approaches to study the activation of brain-protective response in models representative of prodromal phases of PD in vitro and in vivo. The study was focused on nuclear factor erythroid 2-related factor (NRF2), regulating antioxidant/detoxifying response, and the transcription factor EB (TFEB), the master regulator of autophagy and lysosomal pathways, as key signals underlying the early stage of neurodegeneration. In dopaminergic SK-N-BE neuroblastoma cells and in the substantia nigra pars compacta area of the mouse brain, we detected that Nrf2 activation precedes dopaminergic neurodegeneration driven by neurotoxin as demonstrated by cellular, in vivo and ex vivo optical imaging, a finding confirmed by co-localization experiments carried out by immunohistochemistry. Since activation of this transcription factor anticipates dopaminergic neurodegeneration, I focused the study on the cross-talk between a potential PD triggering signal, namely inhibition of the GCase enzyme, and the Nrf2 signaling pathway. Remarkably, I discovered a novel mechanism of microglia-to-neuron communication mediated by cell-to-cell contact, supported by functional actin structures and by an efficient energetic metabolism. Surprisingly, the experiments performed in microglia/neuron co-cultures and in the brain of living mice provided compelling evidence that inhibition of microglial GCase interferes with the ability of these cells to stimulate the NRF2-mediated response in neurons resulting in increased neuronal susceptibility to a xenobiotic. Assays performed in co-culture suggest that counteracting such dysfunction by metabolic compensation could represent a potential therapeutic approach to decrease the risk of neurodegeneration. In conclusion, my thesis demonstrated that the developed reporter systems can be used to study the dynamic modulation of molecular pathways in the living brain; this technology has been developed in particular for the dynamic measurement of oxidative stress and lysosomal/autophagy pathways. Application of these systems allowed to demonstrate that GCase inhibition impairs microglia/neuron communication, thus increasing the susceptibility of neurons to detrimental insults. Taken together, the data identified a novel mechanism underlying the increased risk of neurodegeneration observed in carriers of GBA1 mutations and suggest novel therapeutic strategies for prevention.

Gaucher disease (GD) is a lysosomal storage disorder characterized by β-glucocerebrosidase enzyme deficiency, due to mutations in the GBA1 gene. GD patients, even if affected by a monogenic disease, exhibit a wide range of symptoms, ranging from very mild hematological and visceral phenotypes, to severe neurological involvement. The absence of a clear genotype-phenotype correlation supports the idea that other players act together with GBA1 mutations to give rise to the final clinical picture. To identify new pathway alterations occurring in the pathological condition, GD patient derived induced pluripotent stem cell (iPSC) lines have been generated, characterized and isogenic gene corrected in the mutation site. iPSC differentiation towards the macrophage fate has been employed to investigate the hematopoietic compartment. While GD iPSCs are able to efficiently give rise to CD43+/CD45+ progenitors and mature CD14+/CD163+ monocyte/macrophages, they showed a decreased proliferative potential compared to healthy donor cells. The activation of necroptosis pathway, emerged in both pluripotent and differentiated GD cells, that showed a significant upregulation of the effectors, RIPK3 and MLKL. To dissect brain inflammatory mechanisms, iPSC have been differentiated to neural precursor cells (NPC) and dopaminergic neurons. GD cells presented also at the NPC state both a growth deficit and an increased cell death rate. Since the Hippo pathway is involved in the control of many cellular mechanisms as proliferation, differentiation and cell death, we evaluated its activation in GD lines. Interestingly, many of the pathway transcriptional targets were downregulated in GD neurons and the nuclear localization of YAP, the main pathway effector, resulted reduced, suggesting that the Hippo pathway core is highly activated in the pathogenic context. The identification of altered signalling pathways that may contribute to the cellular metabolism imbalance, are instrumental in the development of new pharmacological approaches to GD that are especially required for neuronopathic patients.

Aim. To investigate the association between Gaucher disease (GD) and gammopathy. Introduction. Gaucher disease is a disorder characterised by deficiency in lysosomal glucocerebrosidase, foamy macrophages, cytopenias, bony lesions, organomegaly and a high incidence of monoclonal/polyclonal gammopathy. Abnormalities in the bone marrow microenvironment, including pseudo-Gaucher cells, have been reported in non-GD patients with myeloma. Methods. Gaucher gene analysis, co-culture, drug assays, western blotting, enzymatic assays, flow cytometry, immunofluorescence and cytotoxic assays. Results. GD patients had a high incidence of gammopathy (polyclonal/monoclonal). Serum biomarkers of macrophage burden were predictive of gammopathy and the Zimran severity score was higher in those with monoclonal than polyclonal gammopathy. Enzyme replacement therapy ameliorated polyclonal gammopathy and stabilised paraprotein levels. Gaucher mutations were not prevalent in Jewish patients with paraproteinaemia (8/77 patients). Non-GD patients with paraproteinaemia had normal serum chitotriosidase and monocyte glucocerebrosidase activity. GD monolayers (osteoclasts/macrophages) did not confer a proliferative or survival advantage on co-cultured myeloma cell lines. GD monolayers, compared to control cultures, reduced sensitivity to melphalan and this was contact dependent. Western blotting identified differences in the levels of Bim and Bcl-xL between myeloma cells harvested from control and GD monolayers. When cultured alone, GD monolayers generated more osteoclasts and this was enhanced by plasma cell coculture (contact independent). GD monolayers did not preferentially rescue myeloma cells pre-treated with doxorubicin from cell death. Patients with GD have decreased lymphocyte glucocerebrosidase activity, abnormal lipid trafficking and low peripheral NK (natural-killer), invariant NK and CD4+ve T-cell numbers. Invariant NK cells, GD derived, displayed impaired proliferation to α-galactosylceramide. Cytotoxicity assays, derived from the GD peripheral blood mononuclear cells, displayed inferior killing compared to control assays. NK killing assays were equivalent between controls and GD patients. Conclusion. This thesis presents novel data, which may confer an increased risk of gammopathy in GD.

Les sphingolipides représentent une classe majeure de lipides qui sont des constituants de toutes les cellules eucaryotes. Ils sont des effecteurs bioactifs impliqués dans les fonctions clés cellulaires comme la différenciation, la prolifération, la motilité, l'apoptose et la sénescence. De plus, leur rôle dans le cancer est maintenant bien établi pour de nombreux sphingolipides. La maladie de Gaucher (MG) est une lipidose héréditaire, à transmission autosomique récessive, due à un déficit enzymatique en glucocérébrosidase (béta-glucosidase acide, codée par le gène GBA1). Cette enzyme est une des différentes enzymes participant au métabolisme des sphingolipides : elle permet la dégradation du glucosylcéramide (GlcCer) en céramide. La MG est caractérisée par une surcharge en GlcCer dans les cellules du système réticulo-endothélial du foie, de la rate et de la moelle osseuse (cellules de Gaucher). Ces dernières années ont permis de montrer qu'il existe une surreprésentation de divers cancers,tels que le myélome multiple et le mélanome, dans la MG. Les mécanismes impliqués ne sont cependant pas connus. L'objectif de ces travaux de thèse est de comprendre la survenue des cancers dans la MG.Nous avons pour cela développé un modèle de cancérogenèse dans la MG en utilisant des modèles murins pour rechercher un effet favorisant du micro environnement de la MG sur la survenue de cancers dans cette maladie et de pouvoir étudier les éventuels mécanisme simpliqués.Nos résultats montrent que dans deux modèles murins différents (D409V et D409/null) deMG, la croissance tumorale des cellules de mélanome est augmentée comparée aux contrôles,chez les femelles mais aussi chez les mâles. Nous avons étudié également au niveau cellulaire quels pouvaient être les types cellulaires impliqués. Nous montrons que les lymphocytes NKTne sont pas diminués dans le foie et dans la rate; de plus, les lymphocytes T CD8+ et les cellules NK ne sont pas diminués dans les souris Gaucher par rapport aux contrôles.En conclusion, nous décrivons le premier modèle murin de développement du mélanome dans la maladie de Gaucher. Dans ce modèle, les mécanismes à l'origine d'une croissance accrue du mélanome demeurent à préciser
Sphingolipids represent a major class of lipids, and are important constituents of alleukaryotic cells. They comprise bioactive effectors implicated in key cellular functions,including cell differentiation, proliferation, motility, apoptosis or senescence. Moreover, theirrole in cancer is now well established for numerous sphingolipids. Gaucher disease (GD) is an inherited lipidosis, with autosomal recessive transmission, due tothe deficient activity of glucocerebrosidase (acid beta-glucosidase, encoded by the GBA1gene). This enzyme is one of multiple enzymes of the metabolic pathway of sphingolipids. Itallows the degradation of glucosylceramide (GlcCer) to ceramide. GD is characterised byGlcCer storage of the reticulo-endothelial cells of the liver, spleen and bone marrow (Gauchercells). Evidence has recently been provided for an overrepresentation of various cancers, inparticular multiple myeloma, in GD. However, the underlying mechanisms remainspeculative.The goal of the present thesis work was to approach the bases for the occurrence of cancers inGD. We developed a model of cancerogenesis in GD, using murine models to study thepossible role of microenvironment in cancer development in this disease and to investigate thepossible cellular mechanisms implicated. Our results showed that, in two different murine models (D409V et D409/null) of GD thetumor growth of melanoma cells is increased as compared to controls, both in males andfemales. We have also studied what cell types could be implicated. NKT lymphocytes are notdecreased in the liver and spleen. In addition, the proportion of T CD8+ cells and NK cells isnot reduced in GD mice as compared to controls. In conclusion, we describe the first murine model of melanoma development in Gaucherdisease. The underlying mechanisms for the facilitated growth of melanoma in this mousemodel remain to be further investigated

Introdução: As doenças de Gaucher (DG), Fabry (DF) e Mucopolissacaridoses (MPS) constituem-se nos tipos mais frequentes de doenças lisossômicas (DL) tratados por terapia de reposição enzimática (TRE). No Brasil, até o presente momento, não há dados de avaliação da qualidade de vida de pacientes com DL, nem instrumento específico validado paratanto. Objetivo: Avaliar a qualidade de vida (QV) de uma amostra de pacientes brasileiros com DG, DF e MPS por meio da aplicação do questionário SF-36. Método: Estudo transversal observacional. O SF-36 é composto por 8 domínios: capacidade funcional, aspecto físico, dor e estado geral de saúde; vitalidade, aspecto social e saúde mental. O questionário foi aplicado, em uma consulta de rotina no ano de 2008, aos pacientes atendidos no ambulatório de DL do Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre, Brasil (SGM/HCPA), com idade igual ou superior a 12 anos e com habilidade cognitiva para preenchimento do mesmo. Resultados: Quarenta e quatro pacientes foram incluídos no estudo (DG= 21, DF=14, MPS= 9; média de idade=27,5±13,7 anos; sexo masculino=27), sendo que 34 (77,2%) estavam em TRE. Considerando a amostra total de pacientes tratados, houve diferença significativa em relação aos domínios capacidade funcional (p=0,036) e estado geral de saúde (p=0,030), sugestiva de melhor QV na DG. Doença de Gaucher: quinze pacientes estavam recebendo TRE com imiglucerase (DG tipo I= 14; DG tipo III= 1; μ tratamento= 8,5±2,5 anos). O domínio com maior pontuação foi saúde mental (μ= 77,3±9,7 para o grupo com TRE, e 72,0±10,8, para o grupo sem TRE); o domínio com menor pontuação foi vitalidade (μ= 67,7±19,9) para o grupo com TRE, e aspectos emocionais (μ= 38,8±44,7) para o grupo sem TRE. A comparação dos pacientes com e sem TRE mostrou que os mesmos diferem somente no domínio “dor” (p= 0,036), cujo escore foi maior entre os pacientes tratados. Doença de Fabry: oito pacientes estavam recebendo TRE com alfagalsidase e 4 com betalgasidase (μ tratamento= 3,5±2,2 anos). Considerando somente os pacientes do sexo masculino (n=10, todos em TRE), o domínio que apresentou maior pontuação foi aspectos emocionais (μ= 66,6±39,4) e o que apresentou menor pontuação foi aspectos físicos (μ= 45,0±40,0. Não foi encontrada diferença estatisticamente significativa entre os grupos recebendo alfagalsidase ou betalgasidase. Para os indivíduos do sexo feminino (n=4, dois em TRE), o domínio que apresentou maior pontuação foi aspectos sociais (μ= 89,8±6,0) e o que apresentou menor pontuação foi estado geral de saúde (μ= 62,2±27,4). Mucopolissacaridoses: nove pacientes foram incluídos (MPS I=3; MPS II=2 MPSIV A= 2, MPS VI=2), sendo que 7 estavam em TRE (MPS I=3; MPS II=2, MPS VI=2; μ tratamento= 4,2 ±2,3 anos). Considerando os pacientes em TRE, a média de pontuação dos domínios variou de 71,4±45,7 (aspectos físicos) a 44,1±21,7 (dor), sendo que em 4 domínios a média foi superior a 60..Entre os pacientes sem TRE, o domínio que apresentou maior pontuação foi aspectos sociais (média= 93,5±6,25) e o que apresentou menor pontuação foi vitalidade (média= 37,5±17,5), sendo que em 5 domínios a média foi superior a 60. Discussão/Conclusão: Embora os nossos achados sejam limitados pelo tamanho amostral, eles sugerem a TRE com imiglucerase tem efeito benéfico na dor apresentada pelos pacientes com DG, e que estes pacientes apresentam melhor QV quando comparados aos pacientes com DF e MPS. Em relação à DF, os escores obtidos foram maiores nas heterozigotas, o que pode ser explicado pela doença ser ligada ao X. Parece existir diferenças tipo-específicas na QV de pacientes com MPS. Estudos adicionais devem ser realizados para confirmar estes achados.
Introduction: Gaucher (GD) and Fabry (FD) disease and mucopolysaccharidosis are the most frequent types of lysosomal storage diseases (LSD) treatable with enzyme replacement therapy (ERT). In Brazil, no data are currently available about the quality of life (QoL) of patients with LSD. Objective: To evaluate QoL in a sample of Brazilian patients with GD, FD and MPS using the SF-36 survey. Method: Observational cross-sectional study. The SF-36 survey was administered to cognitively able patients 12 years or older, who were seen in the Medical Genetics Service of Hospital de Clínicas de Porto Alegre, (SGM/HCPA), Porto Alegre, Brazil. Results: Forty-four patients were included (GD = 21; FD = 14; MPS = 9); mean age was 27.5 ± 13.7 years; and 34 (77.2%) were receiving ERT. The analysis of patients receiving ERT revealed a statistically significant difference in the physical function (p = 0.036) and general health (p = 0.030) domains; results suggests that QoL is better in GD. The comparison of patients with GD on ERT (n= 15) and naïve to ERT (n = 6) revealed differences only in the bodily pain domain (p = 0.036). In the analysis of patients with FD, no significant differences were found in SF-36 scores between male patients receiving agalsidase alpha (n = 7) or agalsidase beta (n = 3); heterozygous women had higher scores than hemizygous individuals. In the group of patients with MPS receiving ERT (MPS I = 3; MPS II = 2; MPS VI = 2), mean domain scores ranged from 71.4 ± 48.7 (role-physical) to 44.1 ± 23.4 (bodily pain), whereas in the group of patients not receiving ERT (MPS IV A = 2), scores ranged from 37.5 ± 24.7 (vitality) to 93.7 ± 8.8 (social functioning). Discussion and Conclusion: Although limited because of the small number of patients included, findings suggest that patients with GD receiving ERT have a better QoL than patients with FD or MPS, and that ERT with imiglucerase has a beneficial effect on pain for patients with GD. In the group of patients with FD, scores were higher in heterozygous women, which may be explained by the fact that FD is an X-linked disorder. There seem to be type-specific QoL differences in the group of patients with MPS. Further studies should be conducted to confirm our findings.

Lysosomal storage disorders (LSDs) are rare inherited metabolic disorders due to a deficiency of specific lysosomal enzymes or transporters. Almost 50 genetic disorders caused by deficiencies of lysosomal and non-lysosomal proteins are known nowadays, collectively with an incidence of ~1/7000 newborns in the worldwide population. Dysfunctions of such lysosomal enzymes or transporters can lead to several consequences that include incomplete degradation and/or recycling of intracellular molecules. Despite lysosomal proteins are present in almost all type of cells, the accumulation of undegraded substrates in LSDs affected patients is generally limited to cells, tissues and organs in which substrate turnover is high, leading to a wide spectrum of phenotypes and affected organs for different LSDs. Among these LSDs, the most common is the Gaucher disease (GD) with an incidence of 1 in 200.000 newborn in the worldwide population, a rate that increases to 1 in 850 in the population of Ashkenazi Jewish (Eastern Europen). This disorder is due to a deficiency of the lysosomal enzyme β-glucocerebrosidase (GBA) that in pathological conditions is not able to degrade its substrate, glucosylceramide, into glucose and ceramide. A hallmark of the disease is the presence of “Gaucher cells”, which are lipid laden-macrophages, in different tissues. Together with this characteristic, GD patients manifest hepatosplenomegaly, anemia, thrombocytopenia and severe bone disfunction such as osteonecrosis, osteopenia, bone crisis and fracture of long bones. GD patients can be classified in three different clinical subtype, based upon the presence and severity of neurological defects. Type I GD, also referred to as non-neuronophatic form, is the most frequent subtype, the symptoms manifest in adulthood and patients do not present neurological involvement. Type II GD patients, manifest severe neurological defects since early life stages and death occurs in childhood. Type III GD patients manifest less severe neurological defects when compared to type II GD and generally symptoms occur during childhood. This classification of the disease based on the absence or presence and severity of neurological defects is oversimplified. Nowadays, the broad spectrum of phenotypes and the recognition of overlap among these GD subtypes has led to the concept that this disorder is a continuum of phenotypes, ranging from the less severe (GD 1) to the more severe forms (GD2 and GD3). The most effective and well tolerated treatment available for this disorders is the enzyme replacement therapy (ERT), that consists in the administration of a recombinant enzyme able to restore the β-glucocerebrosidase functions. Despite its effectiveness on hepatosplenomegaly, anemia and thrombocytopenia symptoms, this therapy has a very limited effect on the recovery of the skeletal and neurological defects. Different GD murine model have been developed through the years to understand the pathogenetic mechanism behind these broad spectrum of phenotype. Despite the availability of all mice models mimicking differents Gaucher disease phenotypes, a completely reliable animal model does not exist and the pathogenic alterations occuring during early life stages can not be explored and elucidated yet. The aim of my PhD project was to investigate the bone pathogenetic mechanisms of Gaucher disease using a new animal model. To address this purpose, I’ve used a zebrafish model due to its easy manipulation and the transparency of the embryos that allow to follow all the early developmental stages. Using a morpholino-mediated knockdown approach and a stable genetic mutant line, I could investigate what was the effect of deficiency of the enzyme Gba1 during early stages of embryonic development. Moreover, the defects observed in these zebrafish models resemble the most common GD phenotypes, like hepatosplenomegaly, anemia and skeletal deformity, making them good models to study the molecular mechanisms of the bone phenotype. By analyzing the main molecular markers involved in bone development, as col10a1, runx2b and osx, I could point out that bone defects observed in these models are determined by an alteration in the osteoblasts differentiation process. Also, using zebrafish transgenic lines in which fluorescent proteins such as GFP are expressed under the control of specific promoters for major molecular signaling pathways, allowed me to identify alterations of Wnt and BMP due to deficiency of the enzyme β-glucocerebrosidase. In this work, characterization of a novel animal model for the study of Gaucher disease, highlighted that dysfunction of the lysosomal enzyme β-glucocerebrosidase can lead to alteration of major molecular signaling involved in the embryonic development, such as Wnt and BMP. Both these pathways have an important role in the formation and maintenance of osteoblasts lineage and early defects in these signal during embryogenesis could lead to defect in the differentiation program of mesenchymal stem cells progenitors. The results showed in this doctoral thesis, highlight for the first time the early involvement of two pathways, the Wnt and BMP signaling, behind the bone pathogenesis of Gaucher disease.
Le patologie da accumulo lisosomiale sono malattie metaboliche rare a carattere ereditario determinate da carenze di specifici enzimi o trasportatori lisosomiali, che hanno complessivamente un’incidenza di ~1/7000 nuovi nati nella popolazione mondiale. Al giorno d’oggi, almeno 50 disordini genetici sono causati da difetti in enzimi lisosomiali, che determinano l’incompleta degradazione e/o il riciclaggio di molecole a livello intracellulare con conseguente accumulo all’interno del lisosoma dei substrati enzimatici. Nonostante la presenza di proteine lisosomiali in quasi tutti i tessuti ed organi del corpo, l’accumulo del materiale non digerito è generalmente limitato solo a quelle cellule, tessuti od organi nel quale il ricambio del substrato enzimatico è molto elevato. Questa caratteristica determina differenti fenotipi per le varie patologie da accumulo lisosomiale, in quanto diversi organi o cellule possono essere coinvolti. Tra queste patologie, la malattia di Gaucher è la più frequente con un’incidenza di 1 su 200.000 nati vivi nella popolazione mondiale. La frequenza di questa patologia, aumenta drasticamente a 1 su 850 all’interno della popolazione degli ebrei Ashkenazi (Europa dell’Est). Questa malattia è causata da mutazioni a carico del gene che codifica l’enzima lisosomiale β-glucocerebrosidase (GBA). Tali mutazioni determinano l’incorretto ripiegamento della proteina enzimatica che, di conseguenza, non è in grado di degradare il suo substrato, la glucosilceramide, che si accumula nel lisosoma. Una delle caratteristiche di questa patologia è la presenza delle così dette “cellule di Gaucher”, ovvero macrofagi ad elevato contenuto di substrato non degradato, in differenti tessuti. Insieme alla presenza di questi macrofagi alterati, pazienti affetti dalla malattia di Gaucher presentano ingrossamento di fegato e milza (epatosplenomegalia), anemia, trombocitopenia e gravi disfunzioni a carico del sistema scheletrico quali osteonecrosi, riduzione della densità ossea, dolori cronici e frequenti fratture a carico delle ossa lunghe. Si possono distinguere tre sottocategorie di pazienti affetti da GD, generalmente classificati sulla base della presenza e gravità dei difetti a carico del sistema nervoso centrale (SNC). I pazienti affetti da GD di tipo I sono i più frequenti, hanno un’insorgenza della patologia in età tardiva ma non presentano coinvolgimento del SNC. I pazienti affetti da GD di tipo II, invece, manifestano i primi sintomi della malattia fin nei primi anni di vita e spesso i gravi difetti a carico del sistema nervoso possono portare alla morte del paziente. La terza categoria di pazienti, GD tipo III, manifestano i sintomi durante l’età infantile e i difetti neurologici sono meno gravi rispetto a quelli dei pazienti di tipo II. Al giorno d’oggi, questa classificazione basata sulla presenza di difetti neurologici è poco credibile a causa della presenza di fenotipi diversificati all’interno della stessa sottocategoria di pazienti. Il concetto di uno spettro continuo di fenotipi che variano dal meno grave (GD tipo I) al più severo (GD tipo II e III) è più appropriato per descrivere questa patologia. La terapia maggiormente utilizzata per il trattamento della sintomatologia di questa malattia è la terapia enzimatica sostitutiva (ERT), che consiste nella somministrazione di un enzima ricombinante in grado si sopperire alla mancanza della β-glucocerebrosidasi. Nonostante sia ben tollerata dalla maggioranza dei pazienti e sia in grado di far regredire l’ingrossamento di fegato e milza, l’anemia e la trombocitopenia, tale terapia ha effetti davvero limitati sui difetti scheletrici e neurologici. Nel corso degli anni, diversi modelli murini sono stati sviluppati per cercare di comprendere quali siano i meccanismi patogenetici della malattia che inducono questo ampio spettro di fenotipi. Sfortunatamente, la maggior parte di questi modelli animali non sono vitali o non manifestano tutti i difetti della malattia. Lo scopo del mio progetto di dottorato è stato quello di generare un nuovo modello animale per comprendere i meccanismi patogenetici a monte dei difetti ossei della malattia di Gaucher. A tal fine, mi sono avvalsa dell’uso dello zebrafish per la sua facilità di manipolazione e la trasparenza delle uova che permettono di seguire lo sviluppo embrionale fin dalle prime fasi. Utilizzando la tecnica del morfolino e avvalendomi di un modello genetico mutante stabile in zebrafish, ho potuto studiare quale fosse l’effetto della mancanza dell’enzima Gba1 fin dalle prime fasi dello sviluppo embrionale. Questi modelli, inoltre, manifestano insieme ai principali difetti di questa patologia, come l’ingrossamento di milza e fegato e l’anemia, anche i difetti a carico del sistema scheletrico, rendendoli dei buoni modelli per studiare i meccanismi molecolari a monte del fenotipo osseo. Analizzando i principali marcatori molecolari coinvolti nello sviluppo osseo, come col10a1, runx2b e osx, ho potuto evidenziare che i difetti ossei osservati in questi modelli sono determinati da un difetto nel processo di differenziamento degli osteoblasti. Inoltre, l’utilizzo di linee transgeniche di zebrafish nelle quali proteine fluorescenti, come la GFP, sono espresse sotto il controllo di promotori specifici per le principali vie di segnale molecolari, mi ha permesso di individuare alterazioni a carico delle vie di segnale Wnt e BMP in conseguenza alla carenza dell’enzima β-glucocerebrosidasi. Con questo lavoro di dottorato, la caratterizzazione di un nuovo modello animale per lo studio della malattia di Gaucher ha permesso di evidenziare che, disfunzioni a carico di un enzima lisosomiale come la β-glucocerebrosidasi, può determinare alterazioni in segnali molecolari molto importanti per lo sviluppo embrionale, quali il Wnt ed il BMP. Entrambe queste vie molecolari svolgono ruoli importanti nel processo di formazione e mantenimento degli osteoblasti e alterazioni precoci di questi segnali durante l’embriogenesi possono determinare difetti nel processo di differenziamento cellulare da progenitori mesenchimali staminali. I risultati ottenuti durante questo lavoro di dottorato, hanno evidenziato per la prima volta il precoce coinvolgimento di due vie di segnale molecolari, il Wnt e il BMP, nella patogenesi ossea della malattia di Gaucher.

INTRODUÇÃO: A doença de Gaucher (DG) é um erro inato do metabolismo, do grupo das doenças lisossômicas, causado pela atividade deficiente da enzima glicocerebrosidase. Os tipos mais comuns da DG são o tipo I, que é o mais freqüente e não apresenta comprometimento neurológico; o II, agudo e neuropático; e o III, subagudo e neuropático. Todos os tipos caracterizam-se pela heterogeneidade clínica, com manifestações sintomáticas e de intensidade distintas, tais como hepatoesplenomegalia, alterações ósseas e hematológicas. Alguns estudos descrevem alterações metabólicas como gasto energético basal (GEB) aumentado – hipermetabolismo - em pacientes sem tratamento. A terapia de escolha para a DG é a reposição enzimática (TRE), a qual consegue reverter muitas das manifestações da doença. OBJETIVOS: 1) Avaliar o GEB por meio de calorimetria indireta em pacientes com DG do Centro de Referência do Rio Grande do Sul; 2) Avaliar o estado nutricional dos pacientes incluídos no estudo; 3) Relacionar o GEB com as condições clínicas dos pacientes. METODOLOGIA: Estudo transversal, prospectivo, controlado. Os pacientes atendidos no CRDG foram convidados a participar do estudo (n= 29), sendo que 17 concordaram (média de idade= 30,0 ± 17,2 anos, sexo masculino= 8; DG tipo III= 3 pacientes). Os pacientes com DG tipo I (n= 14; sexo masculino= 6) foram pareados por sexo, idade e índice de massa corporal (IMC) com controles hígidos para avaliação do GEB. Para determinação dos valores de VO2 e VCO2 foi utilizado um ergoespirômetro (MedGraphics Cardiorespiratory Diagnostic Systems, modelo CPX-D). Os pacientes e os controles receberam orientação prévia quanto ao jejum e o repouso e no dia da calorimetria foram pesados e medidos, sendo então calculado o IMC. Os pacientes não apresentavam outras morbidades, nem estavam em uso de medicamentos que poderiam interferir no GEB. Nas análises estatísticas, foi utilizado o GEB em kcal/kg/dia. RESULTADOS: A avaliação do estado nutricional revelou que, no grupo dos pacientes com DG tipo I, cinco estavam com sobrepeso e os demais eutróficos; no grupo com DG tipo III, dois pacientes encontravam-se desnutridos e um eutrófico. Foram realizadas 19 avaliações do GEB em 17 pacientes: dois pacientes a realizaram no período pré e após 6 meses de TRE. A média de idade e de IMC dos pacientes com DG tipo I e dos controles foi de 32,8 ± 17,6 e 32,1 ± 16,6 anos e 23,3 ± 3,1 e 22,4 ± 3,1kg/m2, respectivamente. A idade dos pacientes com DG tipo III foi, respectivamente, 12, 17 e 20 anos. Quatorze pacientes estavam recebendo TRE (média de tempo de TRE= 6,6 ± 5,3 anos; média de dose de enzima= 27,1 ± 11,7 UI/kg/inf. de imiglucerase). A média de GEB dos pacientes com DG tipo I em TRE (n= 12) foi 27,1% maior do que a dos controles (p= 0,007). O GEB de pacientes em TRE (n=12) comparado aos sem TRE (n= 4) não apresentou diferença (p= 0,92). Comparando o GEB dos pacientes em TRE e o de seus controles com o GEB estimado pela equação de Harris-Benedict, observou-se que os pacientes apresentaram GEB 6,3% maior do que o estimado (p= 0,1), enquanto que seus controles tiveram GEB 17,0% menor do que o estimado (p= 0,001). O GEB medido dos pacientes com DG tipo III foi, respectivamente, 14%, 72% e 16% maior do que o estimado pela equação de Harris e Benedict. Não foi encontrada associação significativa entre GEB e as seguintes variáveis: idade, peso, estatura, escore de gravidade, quantidade de enzima recebida, idade de início de TRE, tempo de tratamento e presença ou ausência de megalias. A correlação do GEB com o IMC foi negativa e significativa, conforme esperado. DISCUSSÃO/CONCLUSÕES: O estado nutricional classificado pelo IMC mostrou que a maior parte dos pacientes com DG tipo I estava eutrófica; no entanto, um terço apresentou pré-obesidade. Dois dos três pacientes com DG tipo III encontravam-se desnutridos. Todos os pacientes, mesmo em TRE, apresentaram um GEB significativamente maior do que os controles. A TRE não consegue normalizar o hipermetabolismo desses pacientes.
INTRODUCTION: Gaucher disease (GD) is an inborn error of metabolism of the group of lysosomal diseases, caused by the deficient activity of the glucocerebrosidase enzyme. The most common types of GD are: type I, which is the most frequent and does not present neurological compromise; type II, which is acute and neuropathic; and type III, which is subacute and neuropathic. All types are characterized by clinical heterogeneity and symptomatic manifestations of various intensity, such as hepatoesplenomegaly and bone and hematological alterations. Some studies have described metabolic alterations, such as increased basal metabolic rate (BMR), that is, hypermetabolism, in untreated patients. The therapy of choice for GD is enzyme replacement therapy (ERT), which can stop many manifestations of the disease. OBJECTIVES: 1) To evaluate BMR by means of indirect calorimetry in patients with GD seen at the Reference Center for Gaucher Disease of Rio Grande do Sul (RCGD); 2) To evaluate the nutritional status of patients included in the study; 3) To relate BMR with clinical conditions presented by patients. METHODS: The present was a prospective, controlled, cross-over study. Patients seen at the RCGD were invited to participate in the study (n=29); of these, 17 agreed to participate (mean age=30.0 ± 17.2 years, male= 8; GD type III=3 patients). Patients with GD type I (n=14; male= 6) were paired by gender, age, and body mass index (BMI) to healthy controls to evaluate BMR. To determine the values of VO2 and VCO2 an ergospirometer was used (MedGraphics Cardiorespiratory Diagnostic Systems, model CPX-D). Patients and controls received previous orientation as to fasting and resting and, on the day of the calorimetry, were weighed and measured in order for the BMI to be calculated. Patients did not present any other morbidity, neither were they making use of any medication that could interfere with BMR. In the statistical analyses, BMR in kcal/kg/day was used. RESULTS: The evaluation of the nutritional status showed that, in the group of patients with GD type I, five patients were overweight; the other were eutrophic; in the group of patients with GD type III, two patients were malnourished; one was eutrophic. Nineteen evaluations of BMR were conducted in 17 patients; two patients conducted the evaluation in the period pre-ERT and after 6 months of ERT. Mean age and mean BMI of patients with GD type I and controls were 32.8 ± 17.6 and 32.1 ± 16.6 years and 23.3 ± 3.1 and 22.4 ± 3.1kg/m2, respectively. The age of patients with GD type III was, respectively, 12, 17 and 20 years. Fourteen patients were receiving ERT (mean time of ERT=6.6 ± 5.3 years; mean enzyme dose=27.1 ± 11.7 UI/kg/inf of imiglucerase). The mean BMR of patients with GD type I on TRE (n=12) was 27.1% higher when compared to controls (p=0.007). When compared to patients not on ERT (n=4), the BMR of patients on ERT (n=12) did not show any difference (p=0.92). Comparing the BMR of patients on ERT and that of their controls with the BMR estimated by the Harris-Benedict equation, we observed that patients showed a 6.3% higher BMR than the estimated (p=0.1), while the BMR of their controls was 17.0% lower than the estimated (p=0.001). The BMR of patients with GD type III was, respectively, 14%, 72% and 16% higher than the estimated by the Harris-Benedict equation. No significant association was found between BMR and the following variables: age; weight; height; severity score; amount of enzyme received; age at beginning of ERT; time of treatment; and presence or absence of megalies. The correlation between BMR and BMI was negative and significant, as expected. DISCUSSION/CONCLUSIONS: The nutritional status classified by BMI showed that most patients with GD type I were eutrophic; however, one third of the patients showed pre-obesity. Two of the three patients with GD type III were malnourished. All patients, even on ERT, showed a significantly higher BMR when compared to controls. In conclusion, ERT was not able to normalize the hypermetabolism of these patients.

INTRODUÇÃO: A doença de Gaucher (DG) é um erro inato do metabolismo causado pela atividade deficiente da enzima glicocerebrosidase e subdivide-se em três tipos: tipo I (DG tipo I), que é o mais frequente e não apresenta comprometimento do sistema nervoso central; o II (DG tipo II), agudo e neuronopático; e o III (DG tipo III), subagudo e neuropático. Todos os tipos caracterizam-se pela heterogeneidade clínica, com manifestações de intensidade distintas, tais como: hepatoesplenomegalia, alterações hematológicas e dores ósseas. Alterações no metabolismo energético também são descritas. A terapia de escolha para a DG é a reposição enzimática (TRE). OBJETIVO PRINCIPAL: Avaliar o estado nutricional e a homeostase de energia em pacientes com DG em TRE. MÉTODOS: A presente tese contemplou 4 etapas: Etapa 1) Elaboração de revisão sistemática da literatura sobre aspectos nutricionais da DG tipo I. Etapa 2) Avaliação da coorte de pacientes acompanhados no Centro de Referência para DG do Rio Grande do Sul (CRDG/RS; n= 38; DG tipo I=35; DG tipo III= 3) quanto a dados relativos ao estado nutricional. Etapa 3) Avaliação do gasto energético basal por calorimetria indireta dos pacientes com DG tipo III do CRDG/RS. Etapa 4) Avaliação, por meio de estudo transversal controlado, dos níveis de grelina, leptina e adiponectina de pacientes com DG tipo I do CRDG/RS, com idade superior a 18 anos e em TRE há mais de 6 meses (n=15); os pacientes foram pareado por sexo, idade e IMC com controles hígidos. RESULTADOS: Etapa 1) Foram localizados 175 estudos, dos quais 28 preencheram os critérios de inclusão. Etapa 2) Avaliação da coorte de pacientes acompanhados no Centro de Referência para DG do Rio Grande do Sul (CRDG/RS; n= 38; DG tipo I=35; DG tipo III= 3) quanto a dados relativos ao estado nutricional. Etapa 2) Os dados antropométricos dos pacientes adultos com DG tipo I (n=31) revelaram que quatorze apresentavam sobrepeso ou obesidade grau I e todos os pacientes com idade inferior a 18 anos estavam com peso e estatura adequados. A idade dos pacientes apresentou alta correlação com o IMC e com o nível de ferritina. O IMC apresentou correlação com a ferritina e esta com o colesterol total e com o LDL-colesterol. O colesterol total apresentou correlação com o HDL, com o LDL e uma correlação negativa com a quitotriosidase. O subgrupo que iniciou o tratamento com idade superior a 18 anos (n=16) teve um aumento significativo de IMC após a TRE (p=0,001) e o que iniciou o tratamento antes de 16 anos (n=10) teve um aumento significativo no escore-z para estatura e IMC (p=0,004 e p= 0,032, respectivamente). Etapa 3) Os pacientes com DG tipo III apresentaram hipermetabolismo e dois deles estavam desnutridos. Etapa 4) A mediana dos níveis de grelina, leptina e adiponectina dos pacientes não diferiu da dos controles. Os níveis de grelina e adiponectina apresentaram correlação positiva entre si e com o HDL-colesterol; e inversa com o IMC, circunferência de cintura e triglicerídeos. Os níveis de leptina apresentaram correlação inversa com o LDL-colesterol e direta com o IMC, circunferência da cintura, dose de enzima, triglicerídeos, insulina e HOMA-IR. Oito pacientes preenchiam os critérios para síndrome metabólica, quatro dos quais estavam com resistência à insulina pelo índice HOMA-IR. CONCLUSÕES: Os dados da revisão sistemática indicaram que o tratamento com imiglucerase melhora os índices de crescimento de crianças e adolescentes com DG tipo I o que está em consonância com os dados encontrados nesta coorte. Em relação aos pacientes avaliados, o estado nutricional classificado pelo IMC mostrou que quase metade dos pacientes com DG tipo I estava com excesso de peso e que a TRE parece contribuir para esse achado. O hipermetabolismo em pacientes com DG tipo III parece constituir-se num biomarcador da gravidade da doença. A leptina apresentou alta associação com a insulina e com o índice HOMA-IR, podendo tornar-se um biomarcador para avaliar indícios precoces de resistência à insulina em pacientes com DG. Aumento de peso, síndrome metabólica e resistência à insulina parecem ser frequentes em pacientes com DG tipo I. Estudos adicionais são necessários para investigar as associações encontradas.
INTRODUCTION: Gaucher disease (GD) is an inborn error of metabolism, caused by the deficient activity of the glucocerebrosidase enzyme and is divided into three types: type I, which is the most frequent and does not present neurological compromise; type II, which is acute and neuronopathic; and type III, which is subacute and neuronopathic. All types are characterized by clinical heterogeneity and symptomatic manifestations of varied intensity, such as hepatosplenomegaly, hematologic dysfunction, bone pain; energy homeostasis dysfunction is also present. The choice therapy for GD is enzyme replacement therapy (ERT). OBJECTIVE.To assess the nutritional status and the energy homeostasis in patients affected by Gaucher Disease under enzyme replacement therapy. METHODS.This present study is composed of 4 stages. Stage 1) Systematic literature review on GD type I nutritional aspects. Stage 2) Assessment of data revolving around nutritional status of the patients cohort followed at the GD Reference Center in Rio Grande do Sul(CRDG/RS; n= 38; GD type I=35; GD type III= 3. Stage 3) Assessment of basal energetic expenditure by indirect calorimetry in GD type III patients at CRDG/RS. Stage 4) Assessment, by means of controlled transversal study, of ghrelin, leptin and adiponectin levels in GD- I patients, age over 18 yo and under ERT for at least 6 months (n=15); the patients were pair matched with healthy controls for sex, age and BMI. RESULTS: Stage 1) 175 studies were found, of which 28 met the inclusion criteria. These studies have shown ERT is associated to: growth normalization in children and teenagers with delayed development; partial correction of hypermetabolism and glycemic profile dysfunctions; and increase in weight as well as insulin resistance and development of Diabetes mellitus type 2 in adults. Stage 2) The anthropometric data of adult patients with GD type I (n=31) pointed out fourteen showed overweight or obesity level 1, and all patients aged under 18yo showed adequate weight and height. The patient’s age showed high correlation with BMI and ferritin levels. BMI presented correlation with ferritin and the latter with total cholesterol and LDL- cholesterol. Total cholesterol showed correlation with HDL, with LDL and negative correlation with chitotriosidase. The subgroup comprising those who were over 18 years of age (n=16) at the beginning of treatment had a significant increase in BMI after ERT (p=0,001) and those beginning treatment under the age of 16 showed (n=10) significant increase in the z-score for height and BMI (p=0,004 and p= 0,032, respectively). Stage 3) GD type III patients showed hypermetabolism and two of them (2/3) were malnourished. Stage 4) The median of ghrelin, leptin and adiponectin levels of patients did not differ from that of the controls. The ghrelin and adiponectin levels presented positive correlation between themselves, with HDL-cholesterol, and inverse correlation with BMI, waist circumference, and triglycerides. The leptin levels presented inverse correlation with LDL-cholesterol and direct correlation with BMI, waist circumference, enzyme dose, triglycerides, insulin, and HOMA-IR. Eight patients (n=15) met the criteria for metabolic syndrome, four of which had insulin resistance, as measured by the HOMA-IR index. DISCUSSION AND CONCLUSIONS: Data from the systematic review showed the treatment with imiglucerase improves growth in children and adolescents with GD type I, this meets the findings in this cohort. In relation to the patients assessed, the nutritional status measured by BMI showed that almost half of the GD type I patients were overweight and that ERT seems to contribute to this finding. Hypermetabolism in GD type III patients seems to be a biomarker of the severity of this disease. Leptin presented high association with insulin and with the HOMA-IR index, and may eventually become a biomarker to evaluate early evidence of insulin resistance in GD patients. Weight increase, metabolic syndrome and insulin resistance seem to be frequent in GD type I patients. Further research is necessary to investigate the findings herein researched.

This thesis examines the size and distribution of benefits from the use of transgenic tobacco as a production vehicle for pharmaceutical proteins. Ex-ante welfare benefits are estimated for the introduction of two biotech innovations. In both cases economic surplus model with imperfect competition is employed to assess the size and distribution of benefits from these alternative uses of tobacco. An introductory chapter presents an overview of the topic followed by chapters 2 and 3 which contain the two case studies. The first paper (chapter 2) examines the case of Human Serum Albumin production from transgenic tobacco. The second paper (chapter 3) examines the case of Glucocerebrosidase Enzyme from transgenic tobacco. Results demonstrate that new products from bio-pharming applications stand to generate significant social benefits. The introduction of Human Serum Albumin generates average annual gains of $46 million and the introduction of Glucocerebrosidase Enzyme generates average annual gains of $500 to $600 million.
Master of Science

Introdução: As Doenças lisossômicas (DLs) são condições genéticas, herdadas na sua maioria de forma autossômica recessiva, caracterizadas usualmente pela deficiência de enzimas lisossômicas específicas, envolvidas na síntese, degradação, armazenamento ou transporte de macromoléculas necessárias para o funcionamento normal do organismo. Nas situações mais típicas, o substrato não degradado acumula-se progressivamente nos lisossomos, com repercussões estruturais e funcionais, levando a sinais e sintomas característicos. Os pacientes apresentam um amplo espectro de manifestações clínicas, que podem incluir disfunção de órgãos, anormalidades esqueléticas, envolvimento neuronal, entre outras. O diagnóstico é usualmente obtido pela identificação da deficiência enzimática específica em leucócitos obtidos do sangue periférico, usualmente realizado em laboratórios de referência. O transporte da amostra pode ser um obstáculo quando o serviço requisitante está situado longe do centro de referência ou em outro país, situação em que a amostra de sangue pode chegar ao laboratório já sem condições de ser analisada. Objetivo: Este estudo teve como objetivo principal, tornar disponível um método mais simples, seguro e acessível que utiliza amostras de leucócitos impregnados em papel filtro (LIPF) como uma nova ferramenta para o diagnóstico bioquímico de pacientes com DLs. Métodos: O estudo envolveu amostras de pacientes com diagnóstico previamente confirmado de DLs (amostra de conveniência, por se tratarem de doenças raras, com incidências individuais ao redor de 1:100.000 recém-nascidos vivos). Foram incluídos no estudo os pacientes com diagnóstico já estabelecido de DLs selecionadas (MPS IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe), independente do sexo e/ou idade, atendidos no Serviço de Genética Médica do HCPA, que concordaram em participar do estudo. O grupo de referência negativo foi constituído pelas amostras de 50 indivíduos hígidos, adultos, de ambos os sexos. Resultados: Os resultados obtidos nos ensaios enzimáticos de pacientes com MPS IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe, indicaram que as enzimas analisadas em amostras de LIPF permitiram a identificação de todos os pacientes, com sensibilidade de 100%. Os testes de estabilidade realizados nas amostras de LIPF indicaram que as amostras, quando mantidas a 4ºC, se mostram estáveis por pelo menos 30 dias. Conclusões: Nas condições utilizadas, amostras de LIPF se mostraram adequadas para a identificação segura de pacientes com MPS tipo IVA, Doença de Krabbe, Doença de Gaucher e Doença de Pompe. As amostras de leucócitos secos em papel filtro são mais estáveis e seguras para o transporte, indicando que possa ser esta uma importante ferramenta para facilitar a identificação de pacientes com DLDs, especialmente daqueles que vivem em áreas que tem dificuldades para a remessa de amostras líquidas para serviços de referência.
Background: Lysosomal Disorders (LDs) are genetic conditions, mostly inherited in autosomal recessive fashion, usually characterized by a deficiency of specific lysosomal enzymes involved in the synthesis, degradation, storage or transportation of macromolecules necessary for normal functioning of the organism. Typically, the non-degraded substrate is progressively accumulated in lysosomes, with structural and functional repercussions, leading to characteristic signs and symptoms. Affected patients present a wide range of clinical manifestations, which may include organ dysfunction, skeletal anomalies, neuronal involvement, etc. The diagnosis is normally made through identification of the specific enzyme deficiency in white blood cells from a sample of peripheral blood, usually performed in reference laboratories. The transporting of a liquid sample can be a problem when the test orderer is located far from the reference center or in a foreign country, as often the blood sample arrives at the laboratory in poor condition and cannot be properly analyzed. Aim: The main aim of this study was to make available a new technique that is simpler, safer and more accessible, using leukocytes impregnated on filter paper (LIFP) as a new tool for the biochemical diagnosis of patients with LSDs. Methods: This study involved samples of patients with previously confirmed diagnosis of selected LSDs (a convenience sample, as these are rare diseases, with individual incidences around 1:100.000 live newborns). Patients with an established diagnosis of MPS IVA, Krabbe Disease, Gaucher’s disease and Pompe disease regardless of sex and/or age, cared for at the Genetics Service of HCPA and who agreed to participate were included in the study. The negative reference group comprised blood samples from 50 healthy adults of both genders. Results: The results obtained in the enzymatic assays of patients with MPS IVA, Krabbe Disease, Gaucher’s disease, and Pompe disease indicated that the analyzed enzymes in LIFP samples allowed the identification of all patients, with sensitivity of 100%. The stability tests performed in LIFP samples indicated that samples, when maintained at 4ºC, were stable for at least 30 days. Conclusions: In the conditions used, LIFP samples were shown to be adequate for a reliable identification of patients with MPS IVA, Krabbe Disease, Gaucher’s disease, and Pompe disease. Blood samples on filter paper are more stable and reliable for transportation, indicating that this may be an important tool to facilitate the identification of patients with LSDs, particularly those living in areas with difficulties for the shipment of liquid samples to reference cervices.

Introduction: Gaucher disease is a recessive disorder, mutation of the GBA1 gene leads to a reduction in β-glucocerebrosidase (GBA) activity, accumulation of glucosylceramide and abnormal levels of other sphingolipids. Features include hepatosplenomegaly, cytopenia and bone disease including osteopenia and osteonecrosis. Enzyme replacement is the most common therapy however bone manifestations can be slow to respond, some patients show no improvement or continue to suffer bone events. Gaucher patients are reported to have a higher incidence of multiple myeloma with an estimated risk ratio up to 51.1. Aim: To investigate the bone marrow microenvironment in GD by partial recreation of this environment using co-culture systems. Methods: Co-culture of up to 3 cell types, histochemical staining, immunofluorescence imaging, functional assay, enzyme activity, therapy and lipid assays, cytotoxicity assay, flow cytometry. Results: Gaucher patient (GD) peripheral blood mononuclear cell derived osteoclast cultures (OC) generated more osteoclasts and at earlier time points than control cultures. GD osteoclasts were larger, had more nuclei and resorbed more bone. Addition of GBA inhibitor CBE to control OC increased osteoclast generation, size and nuclei number. Addition of glucosylceramide to OC increased osteoclast numbers in both control and GD cultures. In vitro GD osteoclast numbers correlated with active bone disease, bone pain and anaemia. Addition of GD-specific therapies to GD OC decreased osteoclast generation, also observed in a selection of patients receiving GD-specific therapies. Uncoupling between GD osteoblast precursors and osteoclasts was observed. Culture of control OC with plasma cell line NCI-H929 increased osteoclast generation. CBE inhibition of human osteoblast cell line SaOS-2 reduced calcium deposition. Glucosylceramide increased NCI-H929 cell number after 7 days. NCI-H929 viability increased when in contact with osteoclasts in SaOS-2 co-cultures. Conclusion: Osteoblast and osteoclast dysregulation may contribute to bone disease in GD. The GD bone microenvironment may provide a pro-survival environment for plasma cells.

Gaucher disease (GD) is a lysosomal storage disorder with wide clinical heterogeneity, caused by mutations in the GBA gene leading to low or deficient levels of the lysosomal enzyme Glucocerebrosidase (GBA1). Mutations in GBA1 are also the single most common genetic risk factor for developing Parkinson’s disease (PD). However, the majority of GD patients and carriers do not develop PD, suggesting the possibility of other disease modifying factors. GBA2 is a non-lysosomal enzyme capable of hydrolysing the same substrates as GBA1 and hence may be a potential disease modifying factor. Initially, assays for GBA1 and GBA2 were developed to record the relative activities in a range of tissue types. Marked tissue differences in GBA1 and GBA2 activities were recorded with GBA2 the predominant enzyme in brain. Assessment of GBA1 and GBA2 in GD, PD and dystonia patient leucocytes revealed, as expected, decreased GBA1 activity in GD samples. However, 13% of idiopathic PD and dystonia patients also displayed decreased activity. Leucocyte GBA2 activity was found to be elevated in half of GD patients and in brain from a GD mouse model. One patient with mild GD but profound PD had undetectable GBA2 activity. Cellular models of GBA1 and/or GBA2 inhibition, oxidative stress, and mitochondrial dysfunction were used to further study the interplay between GBA1 and GBA2 using SH-SY5Y cells. Inhibition of GBA1 was not found to be associated with an increase in GBA2 activity or protein expression. Similarly, inhibition of GBA2 was not found to be associated with changes in GBA1 activity. Loss of mitochondrial function or oxidative stress was not found to cause loss of GBA1 activity while GBA1 inhibition did not increase cellular susceptibility to oxidative stress. Measurement of ceramides using mass spectrometry did not reveal any downstream effects of GBA1 or GBA2 inhibition on ceramide levels.

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Fundação Oswaldo Cruz. Instituto Fernandes Figueira. Departamento de Ensino. Programa de Pós-Graduação em Saúde da Criança e da Mulher. Rio de Janeiro, RJ, Brasil
A Doença de Gaucher (DG) é uma Doença de Depósito Lisossômico (DDL) e seu tratamento baseia - se na terapia de reposição enzimática. Tal terapia foi um marco na vida de pacientes e especialistas, pois mudou a história da evolução da doença, caracterizando um a nova era na Genética Médica. Este trabalho tem como objeto de pesquisa as perspectivas trazidas por profissionais, com experiência em trata r a Doença de Gaucher no Sistema Único de Saúde no estado do Rio de Janeiro. Uma vez que a DG é a única condição d o grupo das DDL a ser contemplada por uma Política Ministerial, promovendo acesso a drogas de alto custo através de um Protocolo Clínico e Diretrizes Terapêuticas (PCDT). O o bjetivo geral foi a nalisar a prática da aplicação do protocolo oficial de tratame nto da DG e o seu entendimento a partir da ótica dos médicos tratadores, profissionais de saúde e gestores do Centro de Referência , o Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti (HEMORIO). Os objetivos específicos foram primeiramente id entificar a formaçã o profissional dos envolvidos no programa, analisar a ótica desses profissionais sob re as recomendações do PCDT e como estes situam o Centro de Referência (CR) e m seu atual funcionamento , e d iscutir de maneira crítica a visão dos profiss i onais a respeito dos benefícios e de possíveis falhas do programa. Foram realizadas entrevistas temáticas semiestruturadas e a elas aplicou - se a análise de conteúdo. No que tange ao entendimento sobre o PCDT - DG e o seu viii funcionamento, os resultados apontam a importância da existência de um balizador, um programa robusto governamental, revisado por especialistas bem capacitados no tema. O PCDT - DG foi um avanço na saúde, oficializando e garantindo o acesso à medicação de maneira embasada, controlada por câmar as técnicas estaduais, permitindo a efetuação de pregões públicos, uma maneira transparente de aquisiçã o de drogas de alto custo comparada a medidas judiciais . Os sujeitos da pesquisa são favoráveis ao programa, no entanto possuem uma abordagem crítica ao sistema de saúde no que diz respeit o a entraves na rede de assistência cirúrgica e de reabilitação. Um grande gargalo atualmente no SUS não é exclusivo ao programa da DG: certos questionamentos éticos na fomentação do diagnóstico laboratorial por parte da indústria farmacêutica, apesar de haver relações amigáveis entre esses dois atores no CR. Concluímos que muitos avanços foram conquistados a partir da implementação do protocolo e que talvez este possa servir como modelo para garantir acesso ao tratamento de outras DDL. Algumas incongruências do siste ma são questionáveis e discutida s entre gestores, médicos e usuários, entretanto ainda são muito poucos os estudos publicados no Brasil sobre o tema .
Gaucher disease (GD) is a Lysosomal Storage Disease (LSD) and its treatment is based on enzyme replacement therapy. Such therapy was a milestone in patients `s lives and experts in the field, changing the disease natural history . This work aims at present ing the treatment of GD in the Unified Health System i n the state of Rio de Janeiro, as it is the only LSD to be covered by a Ministerial policy , which promotes access to high cost drugs through a Clinical Gui deline (CG) . The overall objective was to analyze the practical application of the CG protocol in the treatment of GD, and how this guideline was interpreted and used by the medical c haracters , health professionals and ma nagers of the Reference Center and the State Institute of Hematology Arthur de Siquei ra Cavalcanti (HEMORIO ). The specific objectives were to identify the training of those involved in the program, to analyze how professionals viewed the recommendations included in the CP, what they thought about the Reference Center for GD and to critical ly discuss the benefits and possi ble shortcomings of the program . Thematic semi - structured interviews were conducted, and the content analysis was applied. Regarding the understanding of the CP - GD and its op eration, the results point the importance of t he existence of a robust government program, reviewed by well - trained experts in the subject. The CP - GD was a health`s breakthrough , ensuring access to medication, controlled by state technical chambers, a llowing the practice of public auctions, a transpar ent way of purchasing high - cost drugs when compared to individual litigation. The steakeholder`s research were x favo rable to the program, although they criticized the health network constraints for specialized care, such as surgical services and rehabilitat ion. Another major bottleneck in the health system, not exclusive for G D is ethical issues regarding laboratory diagnosis by the pharmaceutical industry. We conclude d that many advances have been achieved from the implementation of the CP, and that hopeful ly this can serve as a model to ensure access t o treatment for other LSD. Managers, physicians and users point out some inconsistencies in the system although there is still limited published data on this subject in Brazil.

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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 β-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 β-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

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

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.

Introduzione La malattia di Gaucher (MG) è una malattia lisosomiale ereditaria caratterizzata da accumulo di glicosfingolipidi nelle cellule del sistema reticolo-endoteliale causato da deficit dell’enzima glucocerebrosidasi. L’epato-splenomegalia, piastrinopenia e alterazioni ossee sono le principali manifestazioni della MG tipo 1, insieme ad un profilo metabolico con aumentato dispendio energetico, stato infiammatorio, insulino-resistenza ed ipolipemia. Il coinvolgimento epatico è frequente e spazia da forme benigne fino alla fibrosi epatica e cirrosi. La terapia enzimatica sostitutiva e di riduzione del substrato sono risultate efficaci sulle complicanze viscerali ed ossee e sul miglioramento della spettanza e qualità di vita. I pazienti risultano quindi esposti alle possibili complicanze di uno stile di vita inadeguato e dei fattori di rischio cardio-metabolici con potenziale ricaduta sulle complicanze metaboliche ed epatiche. Descrizione degli studi Lo studio è stato progettato per caratterizzare il profilo metabolico e l’entità della malattia epatica in una coorte di pazienti adulti con MG tipo 1 e per valutare la loro relazione con le variabili MG-relate e con lo stile di vita, in quanto le conoscenze attuali sono molto limitate. I pazienti sono stati valutati al basale e alle visite di controllo, raccogliendo dati di severità di malattia e terapia; parametri antropometrici e metabolici; lo stile di vita è stato valutato con questionari validati; la composizione corporea mediante DEXA e la malattia epatica mediante ecografia addominale, risonanza magnetica ed elastometria epatica (Fibroscan®). Studio preliminare – In una coorte di 37 pazienti con MG, la fibrosi epatica significativa, definita da valori di rigidità > 7 kPa, è stata evidenziata in una parte rilevante di pazienti (19%) ed è risultata associata con la severità di malattia e con una minore durata di terapia. Tra i pazienti stabili in terapia, la fibrosi epatica è risultata significativamente associata anche alle variabili della sindrome metabolica (MetS), suggerendo un potenziale ruolo delle comorbidità metaboliche nella progressione della malattia epatica. Studio 1 – In un sottogruppo di 20 pazienti, le comorbidità metaboliche sono ampiamente rappresentate e la steatosi epatica, definita da un CAP ≥ 250 dB/min, è stata riscontrata nel 40% dei pazienti. La steatosi è risultata associata alla MetS ma non alle variabili di severità di malattia e alla terapia. Inoltre, i pazienti con steatosi presentavano un profilo metabolico peggiore rispetto al gruppo senza steatosi. Studio 2 – Valutazione delle comorbidità metaboliche ed epatiche in relazione allo stile di vita e alla composizione corporea. Dall’analisi dei questionari sulla dieta ed attività fisica, è emersa un’elevata prevalenza di dieta non equilibrata ed inattività fisica. Lo studio della composizione corporea ha mostrato un’aumentata massa grassa e ridotta massa magra ed ossea. La dieta non equilibrata e la sedentarietà sono associate agli indici di adiposità e alle variabili della MetS, suggerendo che lo stile di vita inadeguato possa avere un ruolo sulle complicanze epato-metaboliche nei pazienti in terapia stabile. Studio 3 – Infine, è stata valutata la modifica del profilo metabolico nel tempo e gli outcomes epatici in uno studio longitudinale di follow-up. Conclusioni I fattori di rischio cardio-metabolici, la steatosi e la fibrosi epatica sono altamente prevalenti e sembrano relati allo stile di vita inadeguato nella nostra coorte di pazienti adulti con MG. Pertanto, il controllo dello stile di vita e dei fattori di rischio metabolici dovrebbero essere parte integrante della terapia, preferibilmente in ambito multidisciplinare, con lo scopo di prevenire complicanze epato-metaboliche
Background Gaucher disease (GD) is a rare inherited lysosomal storage disorder, characterized by glycosphingolipids accumulation in cells of the reticulo-endothelial system due to deficiency of the lysosomal enzyme acid beta-glucosidase. Hepato-splenomegaly, cytopenia and bone disease represent the main features of type 1 GD. GD patients present a peculiar metabolic profile characterized by increased energy expenditure, systemic inflammation, peripheral insulin resistance and hypolipidemia. Liver disease is frequent and variable, ranging from benign hepatomegaly to fibrosis or cirrhosis. Enzyme replacement therapy and substrate reduction therapy have proven to be very effective on visceral and bone disease and to improve patients’ morbidity and quality of life. Aging GD patients, as the general population, may develop lifestyle-related metabolic risk factors with a potential impact on morbidity and mortality. Description of the studies Limited data are available about body composition, metabolic features, liver disease and lifestyle in GD. This project was designed to characterize the metabolic profile and the liver disease burden in a cohort of adult type 1 GD patients and to evaluate their relationship with GD variables and lifestyle. Patients were evaluated at baseline and follow-up visits, collecting data about GD severity and therapy, anthropometric and metabolic parameters; lifestyle habits were evaluated by validated questionnaires; body composition was assessed by DEXA and liver disease by abdominal ultrasound, magnetic resonance and liver transient elastometry (Fibroscan®). Preliminary study - In a cohort of 37 adult type 1 GD patients, a notable proportion (19%) showed significant liver fibrosis, defined as liver stiffness > 7 kPa, which was associated with GD severity features and a short therapy duration. Among patients on stable ERT, liver fibrosis was also significantly associated with the metabolic syndrome components, suggesting a potential role of metabolic comorbidities in liver disease progression. Study 1 – Within a subgroup of 20 patients of the cohort, metabolic comorbidities were widely represented and liver steatosis, defined as CAP value ≥ 250 dB/min, was detected in 40% of them. Significant steatosis was associated with metabolic syndrome, but not with GD severity variables and therapy duration. Moreover, patients with liver steatosis showed a worse metabolic profile and higher liver stiffness than the counterpart without steatosis. Study 2 – Then, we aimed at evaluating metabolic and liver-related comorbidities in relation to lifestyle and body composition. Analysis of the dietary and physical activity questionnaires showed, respectively, a high prevalence of unbalanced diet and physical inactivity. Body composition evaluation showed an increased fat mass and decreased bone and lean mass. Unbalanced diet and sedentariness were positively associated with indices of adiposity and metabolic syndrome features, suggesting that unhealthy lifestyle had a potential role on metabolic and liver complications in GD patients on stable ERT. Study 3 – Finally, we aimed at evaluating changes in metabolic profile over time and at identifying predictors of liver-related outcome in a longitudinal follow-up study. Conclusions Cardiometabolic risk factors, liver steatosis and fibrosis are highly prevalent and seem to be mainly related to unhealthy lifestyle in our adult GD cohort on stable therapy. For that reason, a cardiometabolic and lifestyle assessment in GD patients is advisable. Moreover, lifestyle changes and control of metabolic risk factors should be part of the therapeutic approach for GD patients, preferably by a multidisciplinary team, aimed at preventing metabolic and liver-related complications

A doença de Gaucher (DG) é uma doença de armazenamento lisossômico causada por uma mutação no gene que codifica a enzima β-glicosidase, a deficiência dessa enzima provoca o acúmulo de glicosilceramidas nos lisossomos do sistema retículo endotelial. A DG é divididas em três tipos, o tipo I é a forma mais branda e mais prevalente da doença. As consequências desta doença incluem hepatoesplenomegalia, deficiências ósseas, manifestações hematológicas e neurodegeneração, porém os mecanismos fisiopatológicos ainda não estão totalmente esclarecidos. Portanto, a fim de esclarecer a fisiopatologia envolvida na DG, o presente estudo avaliou a produção de espécies reativas de oxigênio, as atividades da superóxido dismutase, catalase e glutationa peroxidase, os níveis de nitritos, os imunoconteúdos de iNOS e de pNF-kB, os danos ao DNA e o conteúdo sulfidrilas em diferentes componentes da sangue de indivíduos afetados. Os pacientes foram divididos em dois grupos: controles com diagnóstico negativo para DG e pacientes diagnosticados com DG tipo I, tratados com terapia de reposição enzimática. O sangue foi coletado 5 minutos antes do tratamento. Os resultados mostraram que a atividade de superóxido dismutase foi reduzida em eritrócitos, enquanto atividade da glutationa peroxidase foi aumentada no plasma de pacientes com DG. Os níveis de nitritos e o imunoconteúdo pNF-kB estavam significativamente aumentados no plasma e leucócitos, respectivamente. Ensaio do cometa foi realizada no sangue total e indicou danos no DNA. Observou-se também um dano oxidativo a proteínas, devido a redução do conteúdo sulfidrilas em plasma e eritrócitos. Nossos resultados sugerem que pacientes com DG, mesmo em tratamento, apresentam alteração no status oxidativo/nitrativo e parâmetros inflamatórios, bem como evidências de danos ao DNA no sangue, o que poderia ser, pelo menos em parte, associado à fisiopatologia da DG.
Gaucher disease (GD) is a lysosomal storage disorder caused by a mutation in the gene encoding β-glucosidase enzyme, this enzyme deficiency leads to glucosylceramides accumulation in the lysosomes of the reticulum endothelial system. GD divided into three types, where in type I is the mildest and most prevalent form of disease. The consequences of this disease include hepatosplenomegaly, bone impairments, hematologic manifestations and neurodegeneration, but pathophysiology mechanisms are still not well elucidated. Therefore, in order to clarify the pathophysiology involved in GD, the present study evaluated reactive oxygen species production, superoxide dismutase, catalase and gluthatione peroxidase activities, nitrite levels, immunocontent of iNOS and pNF-κB, DNA damage and sulfhydryl content in differents components of blood from affected individuals.Patients were divided into two groups: controls with negative diagnosis to GD and patients diagnosed to GD type I treated with enzyme replacement therapy. Blood were collected 5 minutes before the treatment. Results showed that superoxide dismutase activity was reduced in erythrocytes while gluthatione peroxidase activity was increased in plasma of GD patients. Nitrite levels and pNF-κB immunocontent were significantly increased in plasma and leukocytes, respectively. Comet assay was performed in whole blood and indicated DNA damage. We also observed an oxidative damage to proteins elicited by decreased sulfhydryl content in plasma and erythrocytes. Our findings suggest that patients with GD, even in treatment, have altered oxidative/nitrative status and inflammatory parameters, as well as evidence of DNA damage in blood, what could be at least in part, associated with pathophysiology of GD.

Glucosidases são enzimas que catalisam a hidrólise de ligações glicosídicas liberando unidades monossacarídicas de um terminal não redutor de um oligossacarídeo ou glicoconjugado. Iminoaçúcares são alcalóides piperidínicos polihidroxilados isolados de plantas (gênero Morus) e microrganismos (Bacillus), como nojirimicina (NJ) (1) e 1-desoxinojirimicina (DNJ) (2), os quais são descritos como inibidores de glucosidase. O potencial uso destes inibidores no tratamento de infecções virais, crescimento tumoral, metástases, diabetes, doença de Gaucher e osteoartrite tem motivado a comunidade científica na busca por novos derivados iminoaçúcares. Desse modo, a síntese de pseudodissacarídeos, contendo ambos resíduos de iminoaçúcar e glicopiranose, constitui uma estratégia interessante de obtenção desses derivados, apesar dos desafios envolvidos na geração da ligação entre estes dois açúcares. Por esta razão, foi utilizada a estratégia de click chemistry como uma ferramenta para introduzir uma ponte de grupo 1,2,3-triazol entre os açúcares a partir do acoplamento de azido-glicosídeo com N-propargil-iminoaçúcar. Desta forma, a síntese do iminoaçúcar N-propargílico (73), com função acetileno terminal, foi realizada em cinco etapas e foi usado na reação de cicloadição 1,3- dipolar com três derivados glicosídicos contendo grupo azido nas posições anomérica (C-1), C-3 ou C-6. A partir desta reação CuAAC (Copper(I)-catalyzed Azide-Alkyne Cycloaddition), três novos pseudo-dissacarídeos (77, 81 e 85) foram sintetizados em rendimentos moderados e foram, então, avaliados em ensaios de - D-glucosidase isolada de Sacharomyces cerevisiae. Nestes testes preliminares, o composto 77 foi o mais ativo, o qual foi capaz de inibir a atividade da enzima em 40% a 1mM. Esses resultados encorajam a realização de novos experimentos, principalmente, a determinação de Ki e avaliação da atividade relativa à replicação do vírus HIV. Portanto, a obtenção destes pseudodissacarídeos trouxe uma contribuição importante no que diz respeito à química de carboidratos e também ao tratamento das doenças citadas.
Glucosidases are enzymes that catalyze the hydrolysis of glycosidic bonds releasing monosaccharide units from a non-reducing end of an oligosaccharide or glycoconjugate. Iminosugars are polihydroxilate piperidinic alkaloids isolated from plants (Morus alba) and microorganisms (Bacillus), such as nojirimicin (NJ) (1) and 1- deoxynojirimicin (2), which are described as glucosidase inhibitors. The potential use of these inhibitors in the treatment of viral infection, tumoral growing, metastasis, diabetes, Gaucher´s disease and osteoarthritis has stimulated the scientific community on the search for novel iminosugar derivatives. Thereby, the synthesis of pseudodisaccharides, having both iminosugar and glycopyranose residues, represents an interesting strategy to obtain these derivatives, despite the challenges involved in generating the link between these two sugars. For this reason, we have used click chemistry as a tool to introduce a 1,2,3-triazole bridge between the sugars from the coupling of azide-glycosides with N-propargyl-iminosugar. Thus, the synthesis of N-propargyl-iminosugar (73), containing the terminal acetylene function, was performed in five steps, and was used in the 1,3-dipolar cycloaddition reaction with three glycosidic derivatives containing the azide group at anomeric (C-1), C-3 or C-6 positions. By applying this CuAAC (Copper(I)-catalyzed Azide-Alkyne Cycloaddition), three novel pseudo-disaccharides (77, 81 and 85) were synthesized in moderate yields and then, evaluated in -D-glucosidase assays isolated from Sacharomyces cerevisiae. In these preliminary test, compound 77 was the most active from the series, which was able to inhibit 40% of the enzyme activity at 1 mM. These results encourage us to perform new experiments, notably the determination of Ki and evaluation towards HIV replication. Thus, a contribution regarding carbohydrate chemistry and treatment of the supracited diseases was achieved by the synthesis of these pseudodisaccharides.

Dans un contexte où les iminosucres multivalents représentent, en tant qu’inhibiteurs puissants de glycosidases, des structures privilégiées pour le développement de nouveaux agents thérapeutiques, nous nous sommes intéressés à ce type de composés pour le traitement de deux maladies génétiques rares. Le premier axe de recherche a consisté à synthétiser des iminosucres di- à tétravalents en série 1-désoxymannojirimycine dans le but d’inhiber l’α1,2-mannosidase I du réticulum endoplasmique qui est impliquée dans la destruction de la protéine delF508-CFTR chez les malades atteints de la mucoviscidose. Un effet multivalent fort sur la correction de cette protéine mutée a alors été mis en évidence avec un composé trivalent basé sur le pentaérythritol. Efficace à des concentrations submicromolaires, ce dernier s’est montré 140 fois plus efficace que le modèle monovalent correspondant. Le second axe de recherche a consisté à identifier de nouveaux chaperons pharmacologiques de la β-glucocérébrosidase, l’enzyme lysosomale impliquée dans la maladie de Gaucher. Pour cela, nous avons préparé une série d’iminosucres hétérodivalents conçus pour cibler simultanément le site actif et un site secondaire de cette enzyme. Même si cet objectif n’a pas encore été atteint, nous avons malgré tout mis en évidence des chaperons monovalents capables de quasiment quadrupler l’activité de la β-glucocérébrosidase portant la mutation G202R. En marge de ces deux axes principaux, une sonde mécanistique basée sur un C-glycoside multivalent a également été développée dans le but de préciser les mécanismes à l’origine des effets multivalents puissants observés pour l’inhibition des glycosidases
Because multivalent iminosugars represent, as potent glycosidase inhibitors, privileged structures for the design of novel drugs, we took a particular interest in this class of compounds for the treatment of two rare genetic diseases. The first research topic was dedicated to the synthesis of di- to tetravalent iminosugars in the 1-deoxymannojirimycin series in order to inhibit the endoplasmic reticulum α1,2-mannosidase I involved in the destruction of delF508-CFTR, the mutant protein responsible of cystic fibrosis. A strong multivalent effect for restoring its activity in cells was reported with a trivalent analogue based on pentaerythritol. This submicromolar corrector was found to be 140-fold more potent than the corresponding monovalent model. The second research topic focused on the identification of novel pharmacological chaperones of the β-glucocerebrosidase, the lysosomal enzyme involved in Gaucher’s disease. For this purpose, we developed a series of heterodivalent iminosugars designed to both bind to the active site and a secondary site of the enzyme. This goal could not be reached yet, nevertheless we identified monovalent chaperones which were able to fourfold increase β-glucocerebrosidase activity in G202R cell lines. Next to these main research topics, a mechanistic probe based on a multivalent C-glycoside was also developed to investigate the multivalent effect of iminosugar clusters in glycosidase inhibition

L’altération de la réserve fonctionnelle cardiovasculaire (CV) à l’exercice est un signe précoce d’insuffisance cardiaque (IC). Nous avons étudié l’hypothèse d’une altération de la réserve CV dans deux modèles physiopathologiques : -une étude des pressions de remplissages du ventricule gauche (VG) lors d’un exercice de faible intensité avait pour finalité de démasquer une anomalie cardiaque latente au repos; cette approche a été appliquée à des patients à risque élevé d’IC à fraction d’éjection préservée.-une étude plus approfondie de la réponse CV au cours d’un exercice maximal avait pour double objectif, de démasquer, mais aussi d’analyser en détail les composantes physiopathologiques de l’altération de la fonction cardiaque. Cette approche a été appliquée à l’étude de la drépanocytose, un modèle de cardiopathie à haut débit. Dans la première partie de ce travail, nous avons montré qu’il était possible de démasquer une altération du remplissage VG dès une faible intensité d’exercice. Ces résultats observés initialement en utilisant des outils non-invasifs ont été confirmés en utilisant le cathétérisme cardiaque comme gold standard. Dans la seconde partie de ce travail, l’évaluation de patients drépanocytaires au cours d’un exercice maximal a permis de mettre en évidence une altération de la réserve fonctionnelle CV liée à un défaut de la réserve chronotrope et de la réserve en pré-charge du VG.Nos travaux constituent une contribution à l’étude de la physiopathologie CV. Les différentes modalités d’exploration qui ont été utilisées, et qui ont pour certaines été mises au point pour la réalisation de cette thèse, pourraient servir à l’étude d’autres cardiopathies
Despite normal investigations at rest, latent heart failure can be observed in a number of patients during exercise. We investigated the hypothesis of an impairment of cardiovascular reserve in two pathophysiological models: - in patients at high risk of heart failure with preserved ejection fraction, we measured left ventricular filling pressures at low exercise level in order to unmask early stages of left ventricular diastolic dysfunction;- moreover, using combined echocardiography and cardiopulmonary exercise testing, a comprehensive study of cardiovascular response to maximal exercise was performed in patients with sickle cell anemia which was chosen as a model of chronic high-output states. We showed that very low exercise level could unmask early stage heart failure with preserved ejection fraction. These results, initially observed using Doppler echocardiography, were confirmed by direct invasive measurement of left ventricular filling pressure during exercise. In patients with sickle cell anemia, we found a decrease of cardiac output response to exercise. This alteration of cardiovascular reserve was characterized by a chronotropic incompetence and a blunted left ventricular preload reserve. The pathophysiological data provided by our studies may help improve and optimize management of patients with heart failure. The stress protocols which have been developed and used in our investigations could be useful to study other models of heart diseases

L’objectiu d’estudi de la tesi és el desenvolupament de nous mètodes sintètics per a la l’obtenció d’oligosacàrids i tio-anàlegs mitjançant processos polimèrics d’obertura d’anell. Aquestes estructures es poden trobar en una gran varietat de productes naturals on formen part de substàncies biològicament actives. D’aquesta manera, s’han desenvolupen rutes sintètiques per l’obtenció de carbohidrats epóxidats, episulfurats, carbonatats i tiocarbonatats. Els monòmers sintetitzats han estat sotmesos a diferents reaccions d’oligomerització. Curiosament, la cerca de nous mètodes de síntesi d’epitio-carbohidrats va donar lloc al descobriment de sucres 1,3-oxazolin-2-tioderivats. Aquests nous compostos han esdevingut inhibidors altament específics de β-glucosidases amb un gran potencial contra la malaltia de Gauche. Els diferents estudis d’inhibició es troben recollits en la memòria.
The final goal of this thesis is the development of strategic methods fro the synthesis of well-defined 1,2-linked oligosaccharides and S-linked thio-analogues through different polymerization techniques. These structures form a complex group of biomolecules with an unsurpassed structural diversity, performing a variety of biological functions. In this context, the present work aimed to develop new procedures in carbohydrate chemistry, focusing in the oligomerization reactions. Thus, efficient syntheses have been studied for accessing the suitable carbohydrate based monomers (including epoxides, episulfides, carbonates and tiocarbonates). Unexpectedly, the synthesis of epithiocarbohydrates afforded carbohydrate-derived 1,3-oxazolidine-2-thiones. These compounds have proved to be quite attractive as new enzyme inhibitors for Gaucher disease. All enzyme inhibition studies can be found in the manuscript.