Dissertations / Theses on the topic 'Gangliosidosis'

Orientador: Carlos Eduardo Steiner
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A gangliosidose GM1 é uma doença rara causada pela deficiência da enzima ?-galactosidase, decorrente de mutações no gene GLB1, acarretando o acúmulo de gangliosídeos, principalmente o GM1. É classificada em três formas dependendo da idade de início dos sintomas. Em todas ocorrem alterações esqueléticas e deterioração neurológica, sendo que na forma adulta predominam sinais extrapiramidais como distonia. No presente estudo descrevemos as características de 12 pacientes com gangliosidose GM1 nas formas juvenil e crônica de 10 famílias não aparentadas provenientes da região de Campinas, SP, e do sul do estado de Minas Gerais. Foram detalhados a história clínica e o exame físico, em especial o neurológico, bem como de aspectos radiológicos, ultrassonográficos, ecocardiográficos e de neuroimagem. Metade dos casos iniciou com queixas ósteo-articulares e outra metade com sintomas neurológicos, porém com a evolução todos apresentaram uma combinação de disostose múltipla e neurodegeneração. Opacificação de córnea e angioqueratomas foram vistos em um caso, cada. Outros sinais comumente associados às doenças de depósito lisossômico não foram vistos nesta casuística. Todos apresentaram baixa estatura, disostose múltipla, disartria e prejuízo nas atividades de vida diária, 10 tinham distonia e disfagia, nove atrofia muscular e oito sinais piramidais e alterações da movimentação ocular. Barra óssea e os odontoideum foram vistos em dois casos, sendo alterações previamente não descritas nessa condição. Exames de neuroimagem mostraram aumento do sistema ventricular e hipointensidade de sinal em globos pálidos em todos, além de deformidades vertebrais, hiperintensidade de sinal de putâmen e atrofia cortical na maioria. Alterações em tálamo, substância branca ou atrofia cerebelar não foram identificadas nessa série
Abstract: GM1 gangliosidosis is a rare disorder caused by deficiency in ?-galactosidase activity due to mutations in the GLB1 gene, leading to acumulation of gangliosides in multiple organs. Three main clinical forms have been described according to the age of onset. All present with skeletal deformities and neurologic deterioration, and in the adult form extrapyramidal signs including dystonia are frequent. In the present study we describe 12 subjects of 10 unrelated families from the region of Campinas and the southern state of Minas Gerais. Clinical information included detailed history, full neurologic examination, radiologic, ultrasonographic, echocardiographic, and neuroimaging description. Half of subjects presented initially with skeletal deformities, while the remaining opened clinical presentation with neurologic features. However, over time all presented dysostosis multiplex and neurodegeneration. Corneal clouding and angiokeratomas were seen in one individual each. Other features commonly described in lysosomal storage disorders were not found in this series. All subjects presented with short stature, dysostosis multiplex, dysarthria, and impairment of activities of daily living, 10 had extrapyramidal signs, nine had muscular atrophy, and eight had pyramidal signs and mild oculomotor abnormalities. A vertebral bone bar and os odontoideum were found in two patients, being previously undescribed in this condition. Neuroimaging revealed enlargement of the ventricular system and hypointensity of globus pallidus in all, besides vertebral deformities, putaminal hyperintensity, and cortical atrophy in most patients. Thalamic changes, abnormal white matter or cerebellar atrophy were not seen in this series
Mestrado
Genetica Medica
Mestre em Ciências Médicas

Orientador: Carlos Eduardo Steiner
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Gangliosidose GM1 é uma doença autossômica recessiva rara, classificada em três formas clínicas de acordo com a idade de apresentação dos sintomas e a gravidade, provocada pela deficiência da enzima lisossômica ?-galactosidase que leva ao acúmulo, principalmente, do gangliosídeo GM1. A forma juvenil geralmente apresenta início entre sete meses e três anos de idade, com progressão lenta dos sinais neurológicos, dimorfismos menos graves que na forma infantil e deformidades ósseas. A forma crônica é caracterizada por apresentações clínicas mais leves e sintomas extrapiramidais. O gene codificador da enzima é o GLB1, no qual mais de 130 mutações foram descritas. No presente estudo foi realizada a caracterização molecular de 10 indivíduos de nove famílias não relacionadas diagnosticados com gangliosidose GM1, nas formas juvenil e crônica. Todas as famílias são originárias do interior do estado de São Paulo ou do sul do estado de Minas Gerais. Para a análise realizada foi possível identificar a mutação anteriormente descrita p.T500A, em sete das nove famílias estudadas, a inserção c.1717- 1722insG e a mutação p.R59H foram encontradas em duas famílias (a última segregou juntamente com o polimorfismo descrito IVS12+8T>C). As demais mutações descritas (p.F107L, p.L173P, p.R201H, p.G311R) foram encontradas em uma família cada. Uma alteração neutra (p.P152P) e duas mutações (p.I354S e p.T384S) são inéditas. Foi possível identificar a ocorrência de uma mutação de novo em uma família. Todas as mutações foram encontradas em heterozigose
Abstract: GM1 gangliosidosis is a rare autosomal recessive, classified in three clinical types according to age of onset and severity. The disease is caused by the deficiency of lysosomal enzyme ?-galactosidase that leads to the accumulation of GM1 ganglioside. The juvenile form usually shows an onset between seven months and three years of age, with slowly progressive neurological signs, less severe dysmorphisms than the infantile form and skeletal changes. The adult form is specified by a milder clinical manifestations and extrapyramidal signs. The lysossomal enzyme is coded by the GLB1 gene which more than 130 mutations have been decribed. In the present study it was genotyped 10 individuals of nine unrelated families originated from the States of São Paulo and Minas Gerais diagnosed with the juvenile and chronic forms of the disease. It was possible to find the previously described mutations p.T500A in seven of the nine families, c.1717-1722insG and p.R59H in two alleles (the latter also segregating with IVS12+8T>C), and p.F107L, p.L173P, p.R201H, and p.G311R in one familie each. One neutral alteration (p.P152P) and two mutations (p.I354S and p.T384S) are described for the first time. The occurrence of a de novo mutation was seen in one family. All patients presented as heterozygous compound
Mestrado
Ciencias Biomedicas
Mestra em Ciências Médicas

Thesis advisor: Thomas N. Seyfried
GM1 gangliosidosis is a glycosphingolipid lysosomal storage disease caused by a genetic deficiency of acid b-galactosidase (β-gal), the enzyme that catabolyzes GM1 within lysosomes. Accumulation of GM1 and its asialo form (GA1) occurs primarily in the brain, leading to progressive neurodegeneration and brain dysfunction. Less information is available on the neurochemical pathology in optic nerve and sciatic nerve of GM1- gangliosidosis. Here we analyzed the lipid content and myelin structure in optic and sciatic nerve in 7 and 10 month old normal β-gal (+/?) and GM1-gangliosidosis β-gal (-/-) mice. Optic nerve weight was lower in the β-gal -/- mice than in unaffected β-gal +/? mice, but no difference was seen between the normal and the β-gal -/- mice for sciatic nerve weight. The concentrations of GM1 and GA1 were significantly higher in optic nerve and sciatic nerve in the β-gal -/- mice than in β-gal +/? mice. The content and composition of myelin-enriched cerebrosides, sulfatides, plasmalogen ethanolamines were significantly lower in optic nerve of β-gal -/- mice than in β-gal +/? mice, however cholesteryl esters were enriched in the β-gal -/- mice. No significant abnormalities in these myelin enriched lipids were detected in sciatic nerve of the β-gal -/- mice. The abnormalities in GM1 and myelin lipids in optic nerve of β-gal -/- mice were also associated with abnormalities in the X-ray diffraction pattern including myelin content in fresh nerves [M/(M +B)] and periodicity (d). With the exception of a slight reduction in myelin content, no abnormalities in the X-ray diffraction pattern were observed in sciatic nerve of β-gal -/- mice. The results indicate that neurochemical pathology is greater in optic nerve than in sciatic nerve of β-gal -/- mice
Thesis (MS) — Boston College, 2014
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Thesis advisor: Thomas N. Seyfried
Thesis advisor: Charles Hoffman
Bis(monoacylglycero)phosphate, BMP, is a negatively charged glycerol-phospholipid with an unusual sn-1;sn-1’ structural configuration. BMP is primarily enriched in endosomal/lysosomal membranes. BMP is thought to play a role in glycosphingolipid degradation and cholesterol transport. It constitutes only about 1-2% of the total phospholipids in most mammalian cells, but is abundant in lung alveolar macrophages where it can comprise up to 16% of the total phospholipids. BMP also accumulates in tissues of humans and animals with lysosomal storage disorders. However, little information is available on BMP levels in gangliosidosis brain tissue. In this work, I found that total BMP content was significantly greater in cells of macrophage/microglial origin than in cells of macroglial origin (astrocyte, oligodendrocyte progenitor), whether normal or tumorigenic. I also observed that BMP in brain was significantly greater in humans and in animals (mice, cats, American black bears) with either GM1 or GM2 ganglioside storage diseases, than in brains of normal subjects. Since BMP is associated with macrophages, I also analyzed the BMP levels in relation to disease-associated inflammation in gangliosidoses. I found that BMP levels were increased due to accumulation of primary storage material gangliosides, rather than an outcome of disease-associated inflammation. In addition, in this thesis I also explored the effect of new ketogenic diet formula from Solace Nutrition (KetoGen) on the growth and metastatic spread of the VM-M3 tumor. Most current drug therapies for cancer are toxic and only marginally effective in providing long-term management. Respiratory insufficiency with compensatory aerobic fermentation (Warburg effect) is the hallmark biochemical phenotype of nearly all neoplastic cells within tumors. Calorie restriction, which lowers blood glucose and elevates ketone bodies, is known to reduce tumor growth to a certain extent, however it does not reduce systemic metastasis. Tumor bearing VM mice were fed either a standard lab chow diet in unrestricted amounts (SD-UR), a standard lab chow restricted to obtain an 18% reduction in body weight (SD-R), or the KetoGen diet restricted (KG-R) to match the body weights of the SD-R group. Tumor size was significantly smaller and organ metastasis was significantly less in the KG-R group than in the SD-UR or SD-R groups. Even though blood glucose was reduced similarly in both the SD-R and KG-R groups, blood ketones were 3-fold higher in the KG-R group than in the SD-R group. These results show that VM-M3 tumor growth and systemic metastasis were managed better with the restricted KetoGen KD than with calorie restriction of a high carbohydrate standard diet. As all human and mouse tumors cells suffer from respiratory insufficiency, my findings suggest that the restricted KetoGen diet should be an effective non-toxic therapy against tumor growth and systemic metastatic cancer
Thesis (PhD) — Boston College, 2015
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

GM1 gangliosidosis is an autosomal recessive lysosomal storage disease, caused by a deficiency in the enzyme β-galactosidase. The disease affects the CNS, liver, kidney, heart and skeletal system, leading to severe neurodegeneration and death. We propose to treat this disorder using ex vivo hematopoietic stem cell therapy. The effectiveness of this therapy requires the recruitment of transduced donor cells to the CNS. This is only found to occur after mice are conditioned with total body irradiation, due to the increase in CNS cytokine production and blood brain barrier permeability that occurs. As the use of total body irradiation in pediatric patients has been linked to future developmental problems, this myeloablation approach is often avoided in younger patients in favor of a conditioning regimen using the chemotherapy drugs, busulfan and cyclophosphamide. Whether donor cells can enter the CNS when a busulfan and cyclophosphamide conditioning regimen is used has not been determined. In this study we plan to quantify the cytokine and blood-brain barrier permeability increases necessary for donor cells to be recruited to the CNS after total body irradiation. We will then investigate whether busulfan and cyclophosphamide conditioning and/or the chronic neuroinflammation present in GM1 mice can produce similar conditions and facilitate the recruitment of donor hematopoietic stem cells to the CNS. Finally we will assess whether ex vivo hematopoietic stem cell gene therapy is still an effective therapy when busulfan and cyclophosphamide are used for myeloablative conditioning.

GM1 gangliosidosis is a lysosomal storage disorder caused by a deficiency in the catabolizing enzyme β-galactosidase (βgal). This leads to accumulation of GM1-ganglioside (GM1) in the lysosome inducing ER stress and cell death. GM1 gangliosidosis is primarily a disorder of the central nervous system (CNS) with peripheral organ involvement. In this work we report two major findings, 1) systemic treatment of GM1 gangliosidosis with an adenoassociated virus (AAV9) encoding mouse-βgal (mβgal) in a GM1 gangliosidosis mouse model (βGal-/-), and 2) an investigation into an intracranial injection of a therapeutic AAVrh8 encoding mβgal. Systemic treatment of GM1 gangliosidosis with AAV9 resulted in a moderate expression of enzyme in the CNS, reduction of GM1 storage, significant retention of motor function and a significant increase in lifespan. Interestingly, the therapeutic effect was more robust in females. Intracranial injections of AAVrh8 vector expressing high levels of βgal resulted in enzyme spread throughout the brain, significant retention of motor function and a significant increase in lifespan. Histological alterations were also found at the injection site in both βGal-/- and normal animals. We constructed a series of vectors with a range of decreasing enzyme expression levels to investigate the cause for the unanticipated result. Microarrays were performed on the injection site and we showed that a lower expressing AAVrh8-mβgal vector mitigated the negative response. Intracranial injection of this newly developed vector was shown to clear lysosomal storage throughout the CNS of βGal-/- mice. Taken together, these studies indicate that a combined systemic and fine-tuned intracranial approach may be the most effective in clearing lysosomal storage completely in the CNS while providing therapeutic benefit to the periphery.

GM2 gangliosidoses are a family of lysosomal storage disorders that include both Tay-Sachs and Sandhoff diseases. These disorders result from deficiencies in the lysosomal enzyme β-N-acetylhexosaminidase (HexA). Impairment of HexA leads to accumulation of its substrate, GM2 ganglioside, in cells resulting in cellular dysfunction and death. There is currently no treatment for GM2 gangliosidoses. Patients primarily present with neurological dysfunction and degeneration. Here we developed a central nervous system gene therapy through direct injection that leads to long-term survival in the Sandhoff disease mouse model. We deliver an equal mixture of AAVrh8 vectors that encode for the two subunits (α and β) of HexA into the thalami and lateral ventricle. This strategy has also been shown to be safe and effective in treating the cat model of Sandhoff disease. We tested the feasibility and safety of this therapy in non-human primates, which unexpectedly lead to neurotoxicity in the thalami. We hypothesized that toxicity was due to high overexpression of HexA, which dose reduction of vector could not compensate for. In order to maintain AAV dose, and therefore widespread HexA distribution in the brain, six new vector designs were screened for toxicity in nude mice. The top three vectors that showed reduction of HexA expression with low toxicity were chosen and tested for safety in non-human primates. A final formulation was chosen from the primate screen that showed overexpression of HexA with minimal to no toxicity. Therapeutic efficacy studies were performed in Sandhoff disease mice to define the minimum effective dose.

GM2 gangliosidoses are a family of lysosomal storage disorders that include both Tay-Sachs and Sandhoff diseases. These disorders result from deficiencies in the lysosomal enzyme β-N-acetylhexosaminidase (HexA). Impairment of HexA leads to accumulation of its substrate, GM2 ganglioside, in cells resulting in cellular dysfunction and death. There is currently no treatment for GM2 gangliosidoses. Patients primarily present with neurological dysfunction and degeneration. Here we developed a central nervous system gene therapy through direct injection that leads to long-term survival in the Sandhoff disease mouse model. We deliver an equal mixture of AAVrh8 vectors that encode for the two subunits (α and β) of HexA into the thalami and lateral ventricle. This strategy has also been shown to be safe and effective in treating the cat model of Sandhoff disease. We tested the feasibility and safety of this therapy in non-human primates, which unexpectedly lead to neurotoxicity in the thalami. We hypothesized that toxicity was due to high overexpression of HexA, which dose reduction of vector could not compensate for. In order to maintain AAV dose, and therefore widespread HexA distribution in the brain, six new vector designs were screened for toxicity in nude mice. The top three vectors that showed reduction of HexA expression with low toxicity were chosen and tested for safety in non-human primates. A final formulation was chosen from the primate screen that showed overexpression of HexA with minimal to no toxicity. Therapeutic efficacy studies were performed in Sandhoff disease mice to define the minimum effective dose.

Orientador: Ricardo de Lima Zollner
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O camundongo não obeso diabético (NOD) é caracterizado por desenvolver naturalmente diabetes mellitus tipo 1 (DM-1) com similaridade ao diabetes mellitus tipo 1 em humanos. A manifestação espontânea do diabetes neste modelo animal é caracterizado por infiltração progressiva das ilhotas de Langerhans por células mononucleares linfócitos T (CD4+ e CD8+) e destruição das células ß pancreáticas produtoras de insulina. O fator de crescimento neural (NGF) e algumas citocinas estão associados a regeneração neural, além de atuarem sobre células do sistema imune. Em adição a estes efeitos, NGF age na liberação de insulina pelas células betas das ilhotas pancreáticas, tornando-se foco de interesse com relação as suas propriedades moduladoras no processo inflamatório na ilhota pancreática. O gangliosídeo GM1 liga-se ao receptor de alta afinidade (TrkA) do NGF-ß, mimetizando seus efeitos. No presente trabalho, avaliamos a ação modulatória de GM1 e NGF em cultura de ilhotas pancreáticas, provenientes de camundongos NOD. Foram avaliados por meio de RT-PCR a expressão gênica de NGF-ß, TrkA e insulina e, por ensaio imunoenzimático, a concentração de citocinas IL-1ß, IL-12, TNF-a, INF-y e insulina. Nossos resultados sugerem ação moduladora similar entre GM1 e NGF sobre as ilhotas de NOD não diabéticos e pré-diabéticos. NGF e GM1 aumentam a expressão gênica de NGF e TrkA e diminuem a expressão gênica de insulina em NOD não diabéticos e pré-diabéticos. Além disso, aumentam a liberação de insulina e diminui a de citocinas inflamatórias IL-1ß, IL-12, TNF-a, IFN-y que caracterizam a resposta Th1.
Abstract: The non-obese diabetic mice (NOD) lineage is characterized by developing type 1 diabetes mellitus (DM-1) naturally, bearing a similarity to DM-1 in human beings. The spontaneous manifestation of diabetes is characterized by gradual infiltration in pancreatic islets by mononuclear cells lymphocytes T (CD4+ and CD8+) and destruction of the ß-cells producers of insulin. One consequence of this effect, is the release of neurotrophins trying modulate the insulin release by the ß cells of pancreatic islets. Thus, the neurotrophins have been the focus of interest in the modulation of the inflammatory process in the pancreatic islets. The ganglioside GM1 binds to the high affinity receptor (TrkA) of the NGF-ß, enhancing its effect. In the present work, we evaluate the immune modulation properties of GM1 and NGF in culture of pancreatic islets from NOD mice. The gene expression of NGF-ß, TrkA and insulin for immune enzymatic assay, the concentration of cytokines IL 1ß, IL-12, TNF-a, IFN-y and insulin were evaluated by RT-PCR and ELISA. Our results suggest similar modulation action between GM1 and NGF on islets of NOD non-diabetic and pre-diabetic. GM1 and NGF action increases the gene expression of NGF and TrkA and the decrease of insulin in mice NOD non-diabetic and pre-diabetic. Moreover, GM1 and NGF increase the insulin release and decrease inflammatory cytokines that characterize the Th1 reply.
Doutorado
Ciencias Basicas
Doutor em Clínica Médica

Tesis - Doctor en Ciencias de la Salud - Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Secretaría de Graduados en Ciencias de la Salud, 2018
163 h. : gráf, tabl., 29 cm
During the period from 1970-2017, 151 patients were diagnosed with cystic fibrosis and 110 patients with Sandhoff disease in CEMECO, making these diseases the two most diagnosed according to the local registry of patients. Objective 1: To contribute to the molecular charaterization of CFTR and HEXB genes and to integrate this knowledge into the multidisciplinary study of cystic fibrosis and Sandhoff disease, including a perspective from the genome to phenome level.
Durante el período 1970-2017, en CEMECO se han diagnosticado 151 pacientes con fibrosis quística y 110 pacientes con enfermedad de Snadhoff, convirtiéndose éstas en las enfermedades metabólicas con mayor registro local de pacientes. Objetivo general 1: Contribuir a la caracterización molecular de los genes CFTR Y HEXB e integrarlo al estudio multidisciplinar de las enfermedades fibrosis quística y de Sandhoff, dede una perspectiva que abarque desde el genoma al fenoma.
2020-11-06
Fil: Mugnaini, Julia. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas.; Argentina.
Fil: Mugnaini, Julia. Provincia de Córdoba. Hospital de Niños de Córdoba; Argentina.

Thesis advisor: Thomas N. Seyfried
Gangliosides are glycosphingolipids (GSLs) containing sialic acids that play numerous roles in neuronal maturation, apoptotic signaling, angiogenesis, and cell surface receptor activity. The GM1 and GM2 gangliosidoses are a series of autosomal recessive lysosomal storage disorders (LSDs) characterized by an inability to degrade these lipid molecules. GM1 gangliosidosis is caused by a mutation in the lysosomal hydrolase β-galactosidase, resulting in neuronal storage of ganglioside GM1 and asialo GA1. Tay-Sachs (TS) and Sandhoff Disease (SD) are GM2 gangliosidoses caused by mutations in either the α or β subunits, respectively, of the heterodimeric protein β- hexosaminidase A, resulting in the storage of ganglioside GM2 and asialo GA2. The accumulation of excess ganglioside in the central nervous system leads to abnormal intracellular vacuoles, neuronal loss, demyelination, ataxia, dementia, and premature death. In my studies, I have shown that accumulation of GM1 ganglioside may not coincide with secondary storage of cholesterol, by providing evidence that cholesterol-binding fluorescent molecule filipin reacted to GM1 ganglioside in the absence of cholesterol. In an effort to combat the early-onset gangliosidoses, I have explored the effects of combining Neural Stem Cells (NSCs) with Substrate Reduction Therapy (SRT) in juvenile Sandhoff mice. The analysis showed that SRT was more effective than NSCs in reducing stored GM2 and GA2 in young mice, and no synergy was observed. In adult GM1 gangliosidosis, Tay- Sachs, and Sandhoff mice, Adeno-Associated Viral (AAV) vector gene therapy was used to restore therapeutic levels of wild-type enzyme to the CNS. AAV therapy corrected ganglioside storage and ameliorated myelin-associated lipid loss in all tissues assayed, increasing motor performance and life in effected animals. Lastly, AAV therapy was also successful in a feline model of Sandhoff disease. These results in juvenile and adult model systems point the way towards multiple effective clinical therapies in the near future
Thesis (PhD) — Boston College, 2011
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

The infantile GM2 gangliosidoses are severe neurodegenerative disorders, caused by a defect in the β-hexosaminidase system. They are characterized by lysosomal accumulation of the substrate, GM2 ganglioside, which results in severe neuronal damage and death in the early years of life. Sandhoff mice deficient in both major hexosaminidase isozymes, Hex A and Hex B, mimic the disease severity in the human condition including the motor deterioration, histopathological findings, and premature death. To investigate the utility of systemic adeno-associated virus 9 (AAV9)-based gene delivery in treating GM2 gangliosidoses, we evaluated the therapeutic outcome of a single intravenous injection of recombinant AAV9 encoding the complementing Hexb gene in a Sandhoff mouse model. We showed prolonged survival, preserved motor function, and reduced GM2 ganglioside accumulation as well as inflammation when systemic AAV9 therapy was administered to 1-2 days old mice. However, the formation of liver or lung tumours accompanied the positive therapeutic effect.

En aquesta tesi s'ha realitzat una aproximació genètica i molecular a les malalties Gangliosidosi GM1 i Morquio B. Aquestes dues malalties són causades per mutacions en un únic gen, el gen GLB1. Aquest gen codifica per la proteïna beta-galactosidasa lisosòmica, de manera que mutacions al gen GLB1 alteren la funcionalitat d'aquesta proteïna. Això pot conduir a l'acumulació dels diferents substrats que normalment són degradats per la beta-galactosidasa: el gangliòsid GM1, el queratà sulfat i diferents oligasacàrids proteics. En aquesta tesi es presenten els resultats de la cerca de mutacions en 49 pacients amb Gangliosidosi GM1 i 5 amb Morquio B. Els pacients estudiats provenien bàsicament d'Espanya i d'Argentina. Es van poder identificar 52 mutacions diferents de les que 32 no havien estat descrites prèviament. A més a més, per a la majoria de mutacions identificades es va poder establir una correlació entre la mutació trobada i un fenotip determinat de la malaltia. La mutació R59H va ser la mutació més prevalent a les poblacions analitzades, essent especialment prevalent als pacients d'ètnia gitana, on aquesta va ser l'única mutació descrita. A més a més, en aquests pacients la mutació R59H sempre es va trobar associada a un mateix haplotip, cosa que indicaria un origen únic del canvi R59H dins de la població gitana.
Posteriorment, algunes de les mutacions identificades van ser expressades in vitro utilitzant com a sistema d'expressió les cèl.lules COS-7 transfectades amb Lipofectamina. D'aquesta manera es van expressar 15 variants mutades de la beta-galactosidasa. Les variants causants de la forma infantil (la forma més severa) van mostrar una activitat enzimàtica residual nul.la, mentre que 3 variants associades a formes més lleus (el tipus adult i la malaltia de Morquio B) van resultar en activitats residuals amb un rang del 7-15%. Per altra banda, 3 canvis que eren polimòrfics (no patogènics) van ser els que van presentar una activitat enzimàtica residual més elevada, al voltant del 30-60%. Així doncs, es va poder concloure que el sistema d'expressió emprat era un bon sistema per establir correlacions entre les activitats residuals de les variants mutades i el fenotip associat a cadascuna d'elles.
Finalment, es van dur a terme una sèrie d'experiments per entendre el mecanisme que regula el procés d'splicing alternatiu del gen GLB1. Aquest gen codifica dues proteïnes diferents (la beta-galactosidasa i l'EBP) gràcies a un mecanisme d'splicing alternatiu. Al transcrit de l'EBP, els exons 3, 4 i 6 són exclosos del transcrit madur. Diferents experiments van mostrar que el mecanisme d'NMD ("Nonsense-mediated decay") s'encarrega d'eliminar les combinacions d'exons errònies, fent que únicament els dos transcrits funcionals romanguin estables a la cèl.lula. Paral.lelament, mitjançant la cotransfecció de plasmidis que codificaven diferents proteïnes SR junt amb un minigèn construït amb els exons implicats en l'splicing alternatiu del gen GLB1, es va poder comprovar que les proteïnes SR tenien la capacitat de modificar la proporció en que es generaven les diferents combinacions d'exons en els transcrits obtinguts a partir del minigèn. Això indicaria que les proteïnes SR, que juguen un paper essencial en el procés d'splicing cel.lular, podrien tenir també una funció en la regulació de l'splicing alternatiu del gen GLB1.

The GM2 gangliosidoses are a group of recessive disorders, which lead to the accumulation of GM2 ganglioside in neuronal cells. The genes responsible for these disorders are HEXA (Tay-Sachs disease and variants), HEXB (Sandhoff disease and variants) and GM2A (AB variant of GM2 gangliosidosis). We have established three relational locus-specific databases recording allelic variation at the HEXA, HEXB and GM2A genes, and these can be accessed through the G M2 Gangliosidoses home page (http://data.mch.mcgill.ca/gm2-gangliosidoses/). The purpose of these databases is to collect and distribute information on mutations in the genes responsible for GM2 gangliosidosis. These databases are available online for users to search and retrieve information about specific mutations either by mutation, phenotype or author(s). In addition, submission forms are available for the addition of new mutations to the databases.
In order to provide information concerning the effects of mutations on the manifestations of disease, we proceeded to model on the theoretical model of the alpha subunit a few missense mutations. (Abstract shortened by UMI.)

A deficiência hereditária da enzima lisossômica G-galactosidase, codificada pelo gene GLB1, causa duas doenças humanas clinicamente distintas, a Gangliosidose GM1 e Morquio B. Clinicamente, pacientes com Gangliosidose GM1 mostram graus variados de neurodegeneração e anormalidades esqueléticas, enquanto que os com Mórquio B apresentam displasia esquelética e opacidade de córnea, sem envolvimento do sistema nervoso central. Neste trabalho foi realizada a análise da freqüência populacional das mutações mais comuns no Brasil para Gangliosidose GM1, e a tentativa de comprovação da hipótese de efeito fundador destas mutações. Também foi realizada a pesquisa clínica e molecular de pacientes com Gangliosidose GM1 na tentativa de caracterizar os pacientes brasileiros, identificando novas mutações e traçando um panorama clínico e genético dessa população. Com a intenção de compreender os efeitos das novas mutações encontradas entre os pacientes brasileiros sobre a estrutura da proteína codificada por GLB1, modelos tridimensionais foram gerados através de ferramentas de bioinformática. Neste estudo foi possível predizer as conseqüências biológicas destas mutações, correlacionando às mesmas com os achados fenotípicos dos pacientes. Um esforço na busca de novas terapias para a doença também foi realizado, visto que não existe tratamento eficaz contra a Gangliosidose GM1. Para tanto, três terapias foram testadas em fibroblastos de paciente com a mutação mais comum encontrada na população brasileira: a terapia de tradução alternativa com o uso de geneticina e cloranfenicol, e a terapia de chaperonas farmacológicas, através do uso de galactose. Nenhuma das terapias foi eficaz no aumento da atividade enzimática da G-galactosidase, mas um aumento da expressão gênica pode ser observado para o gene GLB1.
The inherited deficiency of the lysosomal enzyme G-galactosidase, encoded by the gene GLB1, causes two clinically distinct human diseases: GM1Gangliosidosis and Morquio B. Clinically, patients with GM1 Gangliosidosis show varying degrees of neurodegeneration and skeletal abnormalities, while Morquio B shows skeletal dysplasia and corneal opacity, without involving the central nervous system. This work was performed to analyze the population frequency of the most common mutations in Brazil for Gangliosidosis GM1, and the attempt to prove the hypothesis of a founder effect for these mutations. A clinical and molecular research in patients with Gangliosidosis GM1 was also performed, in an attempt to characterize Brazilian patients, identifying new mutations and drawing a picture of the clinical and genetic aspects of this population. Trying to understand the effects of new mutations found among Brazilian patients on the structure of the protein encoded by GLB1, threedimensional models were generated using bioinformatics tools. In this study it was possible to predict the biological consequences of these mutations, correlating them with the phenotypic findings of the patients. An effort in finding new therapies for the disease was also performed, since there is no effective treatment for GM1 Gangliosidosis. Therefore, three treatments were tested in fibroblasts from patients with the most common mutation found in Brazil: the alternative translation therapy using geneticin and chloramphenicol, and the pharmacological chaperone therapy, using galactose. None of these therapies was effective in increasing the enzyme activity of G-galactosidase, but an increase in gene expression could be observed in the GLB1 gene.

A Gangliosidose GM1 é um Erro Inato do Metabolismo (EIM) causado pela deficiência da enzima B-galactosidase ácida. Essa doença é caracterizada pelo acúmulo de metabólitos não degradados, principalmente gangliosídeo GM1, nos lisossomos de vários tipos celulares. Baseado na idade de início e na atividade residual da enzima, a Gangliosidose GM1 é classificada em três diferentes tipos: infantil, juvenil e adulto. O gene da B-galactosidase ácida (GLB1, GeneBank M27507) está situado no cromossomo 3 e possui mais de 60 kb, contendo 16 exons. Cerca de 50 mutações associadas à doença estão descritas na literatura. No sul do Brasil, há uma alta freqüência dessa doença (1:17.000 nascidos vivos). Neste trabalho, vinte pacientes diagnosticados no Hospital de Clínicas de Porto Alegre (Brasil) tiveram o gene GLB1 investigado por SSCP (Single Strand Conformational Polymorphism) usando DNA extraído de sangue periférico. Através desta triagem foram encontradas 52 alterações de mobilidade do DNA, indicando a presença de mutações. As amostras relativas aos exons 2 e 15 foram submetidas a sequenciamento direto com seqüenciador ABI31O(Applied Biosystens) utilizado kit BigDye 3.1. Cinco novas mutações no gene GLB1 (F63Y, R38G, Y36S, Y64F e R59C) e duas mutações já descritas (R59H e 1622-1627insG) foram encontradas. Este trabalho possibilitou a genotipagem completa de 6 pacientes e parcial de 5, e direcionou a investigação de mutações, contribuindo diretamente no diagnóstico da enfermidade e permitindo a realização de estudos de correlação genótipo/fenótipo destes pacientes.
GM1 Gangliosidosis is a Iysosomal storage disease caused by r3- galactosidase deficiency. As a result of this defect there is a huge accumulation of GM1 ganglioside in tissues of affected patients. Gangliosides are glycosphingolipids present in high concentration in neural tissues. The localization of these lipids explain in part the neurodegeneration present in patients. The r3-galactosidasegene (GLB1-Gene Bank M27507) is located on chromosome 3 and spands more than 60 kb and it is formed by 16 exons. Over than 45 mutations were found in this gene up to now. In southern Brazil the high frequency of GM1 Gangliosidosis (1:17.000 live born) justify the aim of this work, that is to genotype those patients with GM1 Gangliosidosis, by, fist screening they DNA by SSCP (Single Strand Conformational Polimorphism) and than, sequencing in automated apparatus. The screening of 16 exons from 20 patiens gave us the information that 52 mobility changes of DNA Single Strand were found, suggesting possibility of mutations. Samples from exons 2 and 15 were submitted to direct sequencing in ABI310 (Applied Biosystems) using Big Dye3.1 terminator Kit. Five new mutations were found in GLB1 gene (F63Y, R38G, Y36S, Y64F and R59C) and two mutations described previously (R59H and 1627insG). This investigation gave support to complete genotype 6 patients with GM1 Gangliosidosis, partial genotype 5 patients and gave a good support for future analysis in GLB1 gene to correlate genotype and phenotype easily.

Orientador: Ricardo de Lima Zollner
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A linhagem de camundongos NOD (non obese diabetic) desenvolve espontaneamente diabetes mellitus tipo 1 (DM-1) com marcante similaridade ao observado em humanos, que se estabelece entre 12ª e 24ª semana de vida. Os gangliosideos são glicoesfingolipídeos de membrana que contém ácido siálico em sua composição e estão presentes na maioria das células dos vertebrados sendo particularmente abundantes no sistema nervoso. Gangliosídeos exógenos são capazes de acelerar a regeneração de nervos periféricos danificados, porém tem sido relacionados com síndromes neuropáticas periféricas como a síndrome de Guilláin Barret onde os pacientes apresentam anticorpos anti-gangliosídeos especificamente contra o gangliosídeo GM1. Entretanto os mecanismos ainda permanecem controversos. Nossos resultados sugerem que administração de GM1 na dose de 100mg/kg/dia em camundongos NOD e Balb/C fêmeas a partir da 4ª semana de vida não é capaz de provocar neuropatia clínica e que animais diabéticos apresentaram maior imunoreatividade para GM1 nos nervos periféricos com presença de marcação para NGF somente em camundongos Balb/C. Os animais diabéticos tratados com GM1 demonstraram queda na atividade nervosa, em contraste os camundongos Balb/C tratados com GM1 apresentaram aumento significativo na atividade nervosa
Abstract: The strain of NOD mice (non obese diabetic) spontaneously develops diabetes mellitus type 1 (DM-1) similarity to the observed in humans. In this model, the diabetes manifestation occurs between 12th and 24th weeks of life, with presence of pancreas-specific autoantibodies. The gangliosides are glycosphingolipids of membrane that contains sialic acid in their composition and are present in the majority of cells from vertebrates and are particularly abundant in the nervous system. Exogenous gangliosides are capable to increase regeneration in damaged peripheral nerves. However, the gangliosides are related with peripheral neuropathics syndromes as the syndrome of Guilláin Barret in which the patients specifically present antibodies against gangliosides GM1, however these mechanisms still remain controversial. Our results suggest that administration of GM1 in the dose of 100mg/kg/day in female NOD and Balb/C mice at the 4th week of life is not capable to provoke clinical peripheral neuropathy and that diabetic animals present major immunoreactivity for GM1 in peripheral nerves with the presence of immunoreactivity to NGF only in Balb/C mice. Diabetic animals treated with GM1 showed lower nervous activity when compared to Balb/C mice, which presented significant increase
Mestrado
Ciencia Basica
Mestre em Clinica Medica

Juvenile GM2 gangliosidosis (jGM2) is a group of inherited neurodegenerative diseases caused by deficiency of lysosomal β-hexosaminidase A (Hex A) resulting in GM2 ganglioside accumulation in brain. Like many other lysosomal storage diseases (LSDs), no specific treatment currently exists. In order to establish clinical outcomes for the investigation of potential therapies for jGM2, I collected comprehensive information on the natural history of the condition by studying retrospective and prospectively a cohort of 21 patients with the disease, and reviewing previously published reports of 134 patients. Several symptoms at disease onset, symptom latencies, and the survival curve were described. Genotype-phenotype correlations and neuroradiological findings were also studied. Based on pre-clinical results in animal models, we studied substrate reduction therapy (SRT), with miglustat, in a phase I/II clinical trial to assess its pharmacokinetics (PK), safety, tolerability in infantile and jGM2. Miglustat showed a PK profile similar to the one found in adult patients. The drug was found to be safe and well-tolerated in patients with jGM2, with diarrhea and weight loss being the most common drug-related adverse events. The analysis of efficacy showed that SRT was unable to arrest the full neurological progression of the condition; however, relative stabilization of cognitive function was noted, which was consistent with brain MRI findings. Because of the limited efficacy obtained with SRT, enzyme-enhancement therapy was considered to be an attractive alternative therapy for the late onset forms of GM2 gangliosidosis. Screening of a FDA-approved library of approved therapeutic compounds resulted in the identification of pyrimethamine, as a potential pharmacological chaperone for mutant forms of Hex A. Relative enhancements of enzyme activity and protein levels were observed in patient cells treated with therapeutic concentrations of drug. Applying the same principles, ambroxol was identified as a potential PC for mutant glucocerebrosidase (GCC), the lysosomal enzyme that when deficient causes Gaucher disease (GD). Significant increases of residual mutant GCC were observed in cultured patients cells with type 1 GD. In conclusion, principles developed in the course of studies on jGM2 were shown to be useful for the investigation of novel small-molecule therapies for LSDs, associated with significant neurodegeneration.

碩士
長庚大學
光電工程研究所
101
Alzheimer’s disease (AD) is a progressive, neurodegenerative disease. It is characterized by the functional impairment and loss of neurons that results in a progressive decline in memory and other cognitive functions, leading to dementia. Currently without a cure, AD and related disorders will inevitably reach epidemic proportions in a few years. Recent studies have shown that the oligomeric forms of Aβ correlate more strongly with the actual diseased state than either fibril (plaque) or monomers. Thus there must be a mechanism(s) for Aβ oligomers to form, and unfortunately resulting in neurotoxicity. Our working hypothesis is that a highly structured, functional membrane construct, the lipid raft, plays an important role in: a) accumulating Aβ molecules, either in small aggregate forms or influences oligomerization of monomers, and b) membrane integrity is compromised to facilitate osmotic imbalances and/or transport of Aβ oligomers across the membrane.We utilized an ultrasensitive,label-free detection method, the dual-channel paired, surface plasmon wave detector system with ~pM sensitivity, to characterize the interaction between Aβ protein and the supported membrane constructs, starting on lipid bilayer constructs.

Orientação interna: Margarida Alves ; orientação externa: Rui Pedro Louro
A Gangliosidose GM1 é uma doença autossómica recessiva associada à diminuição da atividade da enzima beta-galactosidase 1 (GLB1), devido a mutações no gene GLB1, que levam à acumulação de gangliosídeos GM1 nos lisossomas. Esta acumulação resulta em manifestações clínicas neurológicas. Na raça Cão de Água Português os sinais clínicos têm início a partir dos quatro meses e envolvem, principalmente, sinais de disfunção cerebelar, tais como, tremores da cabeça, dismetria, ataxia e nistagmo. É uma doença fatal e de rápida evolução para a qual não existe qualquer tipo de tratamento. O conhecimento da existência da doença e a realização de testes genéticos, por parte dos criadores é o primeiro passo para a sua prevenção. O presente estudo teve como objetivo obter informação sobre o conhecimento da doença por parte de criadores e proprietários de cães da raça Cão de Água Português e também sobre a realização do teste genético para detecção da presença da mutação c.200G>A no gene GLB aos seus exemplares. O trabalho foi realizado em 70 cães, todos eles registados no Clube Português de Canicultura. Todos os criadores tinham conhecimento da doença e sabiam da existência do teste genético, sendo que, apenas, dois de três o realizavam. Nenhum dos proprietários conhecia a doença. Dos 70 cães em estudo, apenas 28 tinham sido submetidos ao teste genético, revelando serem homozigóticos para a ausência da mutação. Os resultados obtidos evidenciam a falta de conhecimento sobre a doença por parte dos proprietários de cães da raça Cão de Água Português e a necessidade de sensibilizar os criadores para a realização dos testes genéticos e para a importância de informar os novos proprietários sobre a doença.
GM1 gangliosidosis is an autosomal recessive disorder which involves a decrease of beta-galactosidase 1 enzyme (GLB1) activity, due to a mutation in the GLB1 gene, leading to accumulation of GM1 gangliosides in lysosomes. This accumulation results in clinical neurological manifestations. In the Portuguese Water Dog breed, the clinical signs begin at four months of age and involve, mainly, signs of cerebellar dysfunction: head tremors, dysmetria, ataxia, and nystagmus. It is a fatal and fast evolving disease for which there is no treatment. The knowledge about the existence of the disease and the genetic testing by breeders, is the first step to its prevention. This study aimed to obtain information about the knowledge of breeders and owners of dogs from the Portuguese Water Dog breed about the diasease and to evaluate if the breeders test their dogs for the presence of the c.200G>A mutation in the GLB1 gene. The study envolved 70 dogs, all of them registered in the Clube Português de Canicultura (Portuguese Kennel Club). All the tree breeders were all aware about the disease and the existence of the genetic test, but, only two out of three of them performed it on a regular basis. None of the owners knew the disease. Only 28 dogs, out of the 70 in study, were tested for this disease, being homozygous for the absence of the mutation. The results obtained showed the lack of knowledge about the disease by the owners of the Portuguese Water Dog breed and the need for greater education of breeders so that they understand the importance of genetic testing and to inform new owners about the disease.

Tesis (Doctor en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2002.
Tanto la expresión endógena de gangliósidos así como la administración exógena de los mismos han sido implicadas en la regulación de eventos relacionados al crecimiento, maduración y diferenciación celular. En el presente trabajo hemos estudiado el efecto de la expresión de diferentes gangliósidos sobre el comportamiento del receptor del factor de crecimiento epidermal (EGFr). Para encarar este objetivo hemos desarrollado en nuestro laboratorio un modelo experimental consistente en la generación de clones estables de células CHOK1 con expresión diferencial de gangliósidos. Los diferentes clones fueron generados mediante transfección estable con vectores de expresión que contienen los cDNAs que codifican para las glicosíltransferasas involucradas en la biosíntesis de los gangliósidos. Además, se utilizó una condición de depleción de gangliósidos totales consistente en la incubación de células CHO-K1 con un inhibidor específico de la enzima UDP-glucosa:ceramida-glucosiltransferasa, mediante el cual fue posible eliminar en un 90 % los gangliósidos totales expresados en la membrana plasmática de estas células. Sobre la base de este modelo, se analizó el comportamiento del receptor para el factor de crecimiento epidermal de origen humano (EGFr), el cual fue expresado de manera heteróloga mediante la transfección transiente del cDNA que codifica para el mismo. Los estudios realizados sobre el comportamiento de EGFr expresado en los diferentes clones de células CHO-K1 con expresión diferencial de gangliósidos y depleción de gangliósidos totales revelaron que en condiciones donde se expresa el disialo gangliósido GD3 (clones ST18 e IST2A), y luego de estimular los mismos con factor de crecimiento epidermal (EGF), existe una marcada disminución en los niveles de actividad de EGFr. No se encontraron diferencias significativas con respecto a células CHO-K1 salvajes (GM3+), células CHO-K1 con expresión reducida de gangliósidos, ni en condiciones donde se expresan gangliósidos complejos de la serie "a" tales como GM2, GM1 y GD1a (clon 7), indicando que el efecto sobre EGFr no era causado por la aparición de otros tipos de gangliósidos complejos, ni por la disminución en los niveles del gangliósido GM3 en su membrana plasmática. Estudios desarrollados en el clon ST18 e IST2A revelaron que la disminución en la actividad de EGFr no sería atribuida a alteraciones en la cinética de activación de EGFr, ni en la afinidad de este por su agonista. Ensayos posteriores revelaron además que el comportamiento del receptor de insulina no se ve afectado bajo las mismas condiciones de expresión de gangliósidos que afectan a EGFr. Finalmente, analizamos la distribución de EGFr en membrana plasmática de células CHO-K1, así como su posible interacción con el gangliósido GD3. Estos estudios, revelaron que a diferencia de lo encontrado en otros sistemas celulares, EGFr localiza en dominios de membrana solubles al tratamiento con el detergente no ionico Tritón X-100 a 4°C, en cada una de las condiciones de expresión diferencial de gangliósidos estudiadas, y que dicha localización no es afectada por la presencia de gangliósido GD3 endógeno. Asimismo, no fue posible detectar la existencia de algún tipo de interacción estable entre EGFr y el gangliósido GD3.

Tesis (Doctor en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2017
Los gangliósidos, una gran familia de glicoesfingolípidos ácidos, se componen de un segmento hidrofóbico, la ceramida, y un oligosacárido mono o polisialilado de longitud y composición variable. En las células, los gangliósidos se encuentran principalmente en la hemicapa externa de la membrana plasmática a la cual se insertan a través del residuo ceramida exponiendo el oligosacárido hacia el medioambiente extracelular. Estos glicolípidos han sido implicados en numerosos procesos fisiológicos que incluyen, crecimiento, diferenciación, migración y apoptosis entre otros, a través de la modulación de la actividad de receptores de membrana y de interacciones célula-célula y célula-matriz extracelular. Además de su rol fisiológico, los gangliósidos también han sido asociados a un amplio espectro de procesos patológicos siendo receptores de virus, toxinas, lectinas y anticuerpos. Durante el presente trabajo de tesis se abordó el estudio de la unión y comportamiento endocítico de diferentes anticuerpos anti-glicolípidos (AGAb), particularmente de inmunoglobulinas que reconocen a los gangliósidos GD1a y GM1 en varias líneas celulares en cultivo. En primera instancia se caracterizó la unión, endocitosis y destino intracelular de diferentes anticuerpos monoclonales de isotipo IgG que unen con alta afinidad al gangliósido GD1a en células epiteliales derivadas de ovario de hámster chino (CHO-K1) y en células derivadas de neuroblastoma murino (Neuro-2a). Luego de unirse a membrana plasmática, una fracción minoritaria de un anticuerpo anti-GD1a (Ab1-GD1a) fue rápidamente endocitada a 37°C en células epiteliales mediante un mecanismo independiente de dinamina-2 y dependiente de Arf-6 colocalizando en una región yuxtanuclear principalmente con marcadores de endosomas de reciclado. Mientras que la cantidad asociada a la fracción celular fue mínima (20-25%) una fracción mayoritaria del anticuerpo inicialmente unida a membrana plasmática, fue recuperada en el medio de cultivo. Este anticuerpo permaneció principalmente localizado en la superficie celular en experimentos de endocitosis a 16°C, contrastando con la eficiente endocitosis de la proteína transferrina y de otro complejo gangliósido-anticuerpo (GD3-R24) a esta temperatura. Esta evidencia experimental sugiere la presencia de mecanismos selectivos de internalización celular de complejos gangliósidos-anticuerpos. Para determinar si la unión a membrana, endocitosis y destino intracelular es una característica compartida por los anticuerpos anti-GD1a, se incluyó en el estudio otro anticuerpo monoclonal anti-GD1a de diferente isotipo (Ab2-GD1a). Los resultados obtenidos muestran una mínima endocitosis de este anticuerpo en células epiteliales que involucra un mecanismo dependiente de Arf-6, junto con una drástica y rápida reducción de los niveles totales de Ab2-GD1a y localización del anticuerpo endocitado en una región yuxtanuclear. Esto indica un comportamiento comparable de ambos anticuerpos que reconocen GD1a sugiriendo que comparten procesos similares de unión y endocitosis en células epiteliales. Ab2-GD1a se une eficientemente a la superficie de células derivadas de neuroblastoma y se registra un moderado descenso de los niveles totales del anticuerpo comparado con la drástica reducción observada en las células epiteliales. Esto sugiere que el procesamiento endocítico de los anticuerpos anti-GD1a analizados también depende del tipo celular. Los estudios de unión y endocitosis fueron también investigados para un anticuerpo monoclonal contra el gangliósido GM1 (Ab2-GM1). Este anticuerpo se unió eficientemente a la membrana plasmática de células CHO-K1 y una fracción mayoritaria (60-70%) fue rápidamente endocitada a 37°C y acumulada en un compartimiento yuxtanuclear contrastando con la mínima endocitosis descripta para los anticuerpos que reconocen GD1a. Además, los niveles de Ab2-GM1 permanecieron inalterados y principalmente asociados a la membrana plasmática cuando los experimentos de endocitosis se realizaron a 37°C en células Neuro-2a, contrastando con lo observado para los anticuerpos anti-GD1a en esta línea celular. Por lo tanto, se observó que bajo idénticas condiciones experimentales, el tiempo de residencia en membrana plasmática y la fracción endocitada de anticuerpos anti-GD1a y anti-GM1 fueron notoriamente diferentes. En su conjunto, los datos experimentales sugieren que el comportamiento endocítico y procesamiento celular de cada anticuerpo anti-gangliósido puede variar en función del tipo de anticuerpo y del tipo celular, lo cual representa un aspecto clave a considerar en el estudio de sus roles patogénicos en neuropatías así como también en su uso como herramientas terapéuticas. Los gangliósidos son sintetizados por un conjunto de enzimas glicosiltransferasas y sialiltransferasas residentes de retículo endoplásmico (RE) y del complejo de Golgi. Éstas enzimas, en muchos casos organizadas como complejos multienzimáticos, utilizan como sustrato de partida ceramida (Cer) e incorporan monosacáridos de forma secuencial de manera que el producto de una enzima es sustrato de la siguiente en la vía biosintética (Fig. I-1). Como parte de este trabajo de tesis se caracterizó por primera vez la expresión, localización subcelular y modificaciones postraduccionales de la sialiltransferasa humana ST3Gal-II. Ésta es la principal enzima responsable de la biosíntesis in vivo del gangliósido GD1a a partir de GM1, ambos gangliósidos blanco de los anticuerpos anti-glicolípidos estudiados en la segunda parte de esta tesis. II es una proteína transmembrana tipo-II con un corto segmento citoplasmático N-terminal, un fragmento transmembrana y un gran dominio C-terminal orientado hacia el lumen del complejo de Golgi que contiene el sitio catalítico. La secuencia primaria de aminoácidos revela dos sitios potenciales de N-glicosilación, asparagina 92 y asparagina 211 (Asn92 y Asn211). Mediante ensayos bioquímicos, farmacológicos, de microscopía confocal y mutagénesis sitio dirigida se caracterizó la expresión, localización subcelular, ocupación y relevancia de los sitios de N-glicosilación en la localización y actividad in vitro de la enzima. ST3Gal-II se localiza en el complejo de Golgi de células CHO-K1 con predominio en compartimientos proximales y se expresa en similar proporción de monómero y dímero sensible al tratamiento con agentes reductores. La enzima se encuentra N-glicosilada principalmente en Asn211 y el glicano no es del tipo complejo, sino que contiene una alta proporción de residuos de manosa. La presencia del N-glicano en dicha posición es necesaria para la salida de la enzima del RE y su transporte y localización en el complejo de Golgi. La carencia del N-glicano en posición 211 influencia de manera negativa la actividad sialiltransferasa in vitro utilizando como aceptor un gangliósido (GM1) o una glicoproteína (asialofetuina), mientras que la falta del mismo en la posición 92 no afecta la actividad hacia la glicoproteína e influencia de manera positiva la actividad hacia el gangliósido. Una versión quimérica conteniendo el dominio N-terminal (NtD) de ST3Gal-II (aminoácidos 1-51) fusionada a la proteína fluorescente mCherry (ST3Gal-II-1-51-mCherry) se expresa en células CHO-K1 y se localiza en el complejo de Golgi. Esto sugiere que el NtD es necesario para el transporte desde el RE y retención de ST3Gal-II en el complejo de Golgi. Además, indicaría que el dominio C-terminal de ST3Gal-II depende de la N-glicosilación para alcanzar un estado conformacional óptimo de plegamiento que le permita salir del RE para localizarse adecuadamente en el aparato de Golgi, probablemente como un requerimiento del control de calidad de plegamiento de proteínas en RE, pero no sería un requerimiento excluyente para la retención de la enzima en el complejo de Golgi. Asimismo, el residuo cisteína presente en el NtD de ST3Gal-II tendría un rol en la formación y estabilización de la forma dimérica.
Ruggiero, Fernando Miguel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.
Daniotti, José Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.
Alvarez, Cecilia Inés. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.
Irazoqui, Fernando José. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.
Cuadra, Gabriel Ricardo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacología. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.
D´Alessio, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina.

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2016
Los gangliósidos, glicanos propios abundantes en tejido neural, han sido propuestos como los principales blancos antigénicos en el Síndrome de Guillain-Barré (SGB). Aunque la etiología de esta enfermedad de tipo autoinmune es desconocida, se cree que la respuesta inmune elaborada hacia un patógeno, podría reaccionar en forma cruzada con gangliósidos de los nervios periféricos. Conejos inmunizados con una mezcla de gangliósidos de cerebro bovino (BBG) y hemocianina de lapa californiana (KLH por su sigla en inglés) como proteína carrier, desarrollan una neuropatía clínicamente similar al SGB. Sin embargo, la enfermedad no se produce si en lugar de utilizar KLH se emplea albúmina de suero bovino metilada (BSAm). Con el objeto de caracterizar el desarrollo de la respuesta inmune humoral en este modelo animal, grupos de conejos fueron inmunizados con distintos inmunógenos. Un grupo fue inmunizado con KLH/BBG y otro con BSAm/BBG. Con el objetivo de evaluar la función carrier de KLH en el modelo experimental, otros dos grupos fueron inmunizados en base a un protocolo alternativo de inmunización (BSAm/BBG+KLH y KLH+BBG), en el que KLH se administra en emulsiones separadas. Además, se utilizaron dos grupos controles inmunizando sólo con BBG o sólo con KLH. Se evaluaron tanto los signos clínicos como la respuesta inmune generada (título, isotipo y especificidad de los anticuerpos anti-gangliósidos). Todos los animales inmunizados con KLH/BBG mostraron signos clínicos de neuropatía y se les detectaron inmunoglobulinas de isotipo G (IgG) que reconocen a gangliósidos de estructura simple (GA1) y compleja (GM1 y GD1b). Estos anticuerpos fueron parcialmente bloqueados por pre-incubación con KLH soluble indicando su reactividad cruzada. El porcentaje de reactividad cruzada con KLH se modificó a lo largo del tiempo (cambio de la especificidad de los anticuerpos). La reducción en el porcentaje de anticuerpos con reactividad cruzada coincide generalmente con el comienzo de los signos clínicos. Ningún animal de los grupos controles (Grupos BBG o KLH) se enfermó, y sólo en aquellos inmunizados con KLH se detectó una leve reactividad de anticuerpos de isotipo IgG que reconocen GA1 y que son totalmente bloqueados por preincubación con KLH soluble. Utilizando la lectina del maní (PNA) se corroboró en KLH la existencia de un determinante presente también en GA1/GM1/GD1b Galβ1-3GalNAc). Si bien el grupo BSAm/BBG no enfermó, al inmunizar por separado también con KLH (BSAm/BBG+KLH) se revirtió esta situación y se generó la neuropatía. Sin embargo, la inmunización con BBG y KLH por separado (KLH+BBG) no produce signos clínicos de enfermedad. Finalmente, entre los animales que desarrollaron la neuropatía, aquellos que enfermaron tempranamente presentaron un mayor porcentaje de anticuerpos anti-GA1 de alta afinidad en suero pre-inmune, comparado con aquellos que enferman de forma tardía. Estos resultados sugieren que, la presencia de KLH en la inmunización es requerida para generar la enfermedad. Los anticuerpos comenzarían reconociendo los azúcares terminales compartidos entre KLH y GA1 para luego expandir la región identificada. Como consecuencia, se produce un cambio de especificidad de los anticuerpos hacia los gangliósidos, acompañado del comienzo de la neuropatía. Si bien los gangliósidos no poseen la capacidad de generar una respuesta de isotipo IgG, podrían re-direccionar la respuesta anti-GA1 inducida por KLH hacia gangliósidos con estructuras más complejas. Tanto la especificidad fina, como la afinidad de los anticuerpos resultaría clave en el comienzo de la neuropatía, incluso un elevado porcentaje de anticuerpos anti-GA1 de alta afinidad en suero pre-inmune se asoció con el comienzo temprano de la misma. Se establecen dos conclusiones principales. En primer lugar, KLH estaría cumpliendo el papel tanto de carrier como de inmuno-estimulador específico. En segundo lugar, el factor de susceptibilidad a desarrollar la neuropatía podría estar relacionado a la afinidad de los NOAbs anti-GA1 presentes en el suero pre-inmune. Según nuestro conocimiento, este trabajo es el primero no sólo en describir en detalle la respuesta inmune generada durante el desarrollo de la neuropatía en un modelo experimental, sino también en establecer un posible factor de susceptibilidad del huésped.
Funes, Samanta. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.
Nores, Gustavo Alejandro. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.
Cuadra, Gabriel Ricardo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacología. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.
Irazoqui, Fernando José. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.
Pistoresi de Palencia, María Cristina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.
Cremaschi, Graciela Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina.