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Congenital disorders of glycosylation (CDG) represent a heterogeneous group of mul- tisystemic metabolic disorders which are caused by defects in biosynthetic pathways of glycoproteins. The screening test for N-glycosylation disorders is the analyses of sialylated isoforms of serum transferrin (Tf) by means of isoelectric focusing (IEF). Two distinct pathological IEF patterns of Tf are observed. A type I pattern is cha- racterized by a decrease of tetra- and an increase of di- and asialotransferrin, whereas a type II pattern shows in addition an increase of tri- and monosialotransferrin. The aims of diploma thesis were: 1) to evaluate reference range for spectrum of sialylated forms of Tf separated by IEF and 2) to perform biochemical and molecular analyses in three patients (P1-P3) with clinical suspicion for CDG. Serum and genomic DNA from three patients with clinical suspicion for CDG and family members of P1 were analysed. Sera from 99 healthy volunteers within the age range of 2-42 years served as a control group. Tf was analysed by IEF with direct immunofixation, SDS-PAGE and Western blot using specific antibody against human Tf (Dako). Profiles of Tf were quantified by AlphaEaseFC software (Alpha Innotech). Data were analysed by software STATISTICA 9.0 (StatSoft). TF a PMM2 genes were analysed...

Submitted by Leonardo Nunes (assisnunes@gmail.com) on 2015-11-27T22:25:42Z No. of bitstreams: 1 DISSERTACAO_LeonardoAssisNunes-UFBA.pdf: 3102159 bytes, checksum: 56b93c54bc9a734ac341b8aa28e2c922 (MD5) / Made available in DSpace on 2015-11-27T22:25:42Z (GMT). No. of bitstreams: 1 DISSERTACAO_LeonardoAssisNunes-UFBA.pdf: 3102159 bytes, checksum: 56b93c54bc9a734ac341b8aa28e2c922 (MD5) / Capes / Este trabalho propõe uma discussão sobre o confronto entre limites que cerceiam e limites que libertam, no ensino de Música na modalidade a distância mediada por Tecnologias da Informação e Comunicação (TIC). Seu objeto de estudo é formado pelo conjunto de ações educativas pertinentes a um processo específico para formação de professores de Música para a Escola Básica brasileira. Trata-se, esse, do ensino para composição de Microcanções CDG (Cante e Dance com a Gente), conforme ocorrido no curso pioneiro no país na área de Licenciatura em Música, em modalidade a distância mediada pela internet, da UFRGS e Universidades Parceiras (PROLICENMUS), ao longo dos semestres 2008/01 a 2011/02, no âmbito de cinco interdisciplinas de seu currículo. Constatou-se, por um lado, que à Educação a Distância (EAD), processo predominantemente assíncrono, é inerente a necessidade de antecipar respostas, antes mesmo que os alunos tenham enunciado suas dúvidas, o que tende a tornar a comunicação estruturada, consequente e previsível; por outro, que o desejável num processo de criação, como o que conduz à composição musical e se realiza tradicionalmente de modo síncrono, é que ele seja aberto, flexível e surpreendente. Espera-se, desse modo, ampliar a discussão sobre a oferta de formação profissional para professores de Música nessa modalidade de ensino, mundo esse globalizado e informatizado que, para além do aproveitamento tecnicista e instrumental dos sempre novos recursos tecnológicos colocados à disposição das pessoas, busca perceber também o que eles produzem de novos sentidos para elas, e essas, por meio deles. / This paper discusses the confrontation between limits that restrain and limits that set free in Music Teaching in Distance Education modality, mediated by Information and Communication Technologies (ICT). Its object of study is composed by the set of educational activities which are relevant to a specific training process of Brazilian Primary School music teachers. This process is the training for Microsongs CDG (Cante e Dance com a Gente) composition as taught in the pioneer Music Teaching course in Brazil in Distance Education modality through the internet by UFRGS and Partner Universities (PROLICENMUS). This training took place in five of the curriculum's interdisciplinary courses taught over the 2008/01 to 2011/02 semesters. It was observed that, in one hand, the Distance Education, predominantly an asynchronous process is inherent to the need to anticipate answers, even before students have stated their doubts. This tends to make the communication structured, consequent, and predictable. On the other hand, the desirable in a process of creation, as it leads to musical composition and traditionally takes place synchronously, is that it is open, flexible, and surprising. It is expected, thus, that this could broaden the discussion on professional training offering for Music teachers in this kind of education in a globalized and computerized world which, beyond the technical and instrumental use of the ever-new technological resources available to people, also seeks to realize what they produce in terms of new meanings to them, and those through them.

CDG-Ia ist eine genetisch bedingte Stoffwechselstörung, die eine fehlerhafte Synthese von Glykoproteinen zur Folge hat. Ursache sind Mutationen in der genetischen Information für das Enzym Phosphomannomutase II (PMM2). Inhalt dieser Arbeit ist die Generierung von zwei Gene-Targeting-Vektoren (GTV) und den entsprechenden transgenen embryonalen Maus-Stammzellen, welche jeweils die beiden häufigsten Mutationen R141H und F119L (R137H und F115L im Maus-Genom) tragen. Ausgangsprodukt ist ein Teil des murinen Pmm2-Gens, welcher in einen pBlueScript-Vektor subkloniert wird. Mit gezielter in-vitro-Mutagenese erfolgt das Einbringen der jeweiligen Mutation. Als Selektionsmarker für beide GTV dient ein von zwei loxP- Sequenzen flankiertes Neomycin-Resistenz-Gen (Neo). Die beiden derart konstruierten GTV, welche jeweils die Mutation F115L bzw. R137H beinhalten, werden als linearisiertes Konstrukt durch Elektroporation in embryonale Mausstammzellen transferiert. Nach Überprüfung der erfolgreichen homologen Rekombination wird je ein Stammzell-Klon mit dem Genotyp F115L/WT und R137H/WT mit einem Cre-Rekombinase-Gen transfiziert und die erfolgreiche Entfernung des Neo-Gens überprüft. Mit diesen transgenen Stammzellen sollen hypomorphe Mausmodelle für CDG-Ia generiert werden.

Deficiency of antithrombin caused by mutations affecting the gene encoding this key anticoagulant (SERPINC1) significantly increases the risk of thrombosis. We aim to identify new mechanisms involved in antithrombin deficiency. Using molecular, cellular and biochemical methods, we studied 29 patients with antithrombin deficiency without SERPINC1 mutation, a family study, three case-control studies including 2,980 patients and 3,996 controls, and two patients with congenital disorder of glycosylation (CDG). We identified the first mutation affecting the SERPINC1 promoter causing antithrombin deficiency. We confirmed the low genetic variability of SERPINC1 and its minor role on the heritability of antithrombin. Genome wide association studies and silencing experiments identified the first modulating gene of antithrombin, LARGE. We diagnosed a patient with CDG based on his antithrombin deficiency. Finally, we described a new disorder with identical biochemical features than CDGs, but only thrombosis, which is caused by a single mutation in PMM2 and concomitant alcohol consumption. / La deficiencia de antitrombina causada por mutaciones en el gen SERPINC1 incrementa el riesgo trombótico. Nuestro objetivo fue identificar nuevos mecanismos implicados en la deficiencia de este anticoagulante. Empleando metodología molecular, celular y bioquímica estudiamos 29 pacientes con deficiencia de antitrombina sin mutaciones en SERPINC1, un estudio familiar, tres estudios caso-control (2,980 pacientes/3,996 controles) y dos pacientes con trastornos congénitos de glicosilación (CDG). Identificamos la primera mutación en el promotor de SERPINC1 que causa deficiencia de antitrombina. Confirmamos la baja variabilidad genética en SERPINC1 y su escasa influencia en la heredabilidad de antitrombina. Un GWAS y experimentos de silenciamiento mostraron que LARGE es el primer gen modulador de antitrombina. Diagnosticamos un CDG por la deficiencia de antitrombina de un paciente con trombosis recurrente y descubrimos nuevo desorden con patrón bioquímico similar al CDG pero solo con trombosis que es causado por una sola mutación en PMM2 y consumo de alcohol.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This dissertation questioned if a particular proposal for music education, the Proposal Musicpedagogical CDG Sing and Dance with Us, could be considered a pedagogical poetics, of artistic creation in and for school. In order to achieve this aim, the research began characterizing CDG from documents produced by and about it. At the same time, a study about poetics from the thought of Paul Valéry e René Passeron was carried out. The authors cited served as theoretical and methodological lenses through which I meant to amplify CDG, in order to demonstrate how a pedagogical proposal can be understood as a poetics; this claim did not aim to restrict CDG to art teaching, but the actual production of art. The poetics, as presented here, is: poïein (VALÉRY , 2011), a do that leads to a process of creation; poiesis (PASSERON, 1997), the study of creative behavior; poiesis (CASTRO, 2004), when studied from outside the metaphysical field, essence of acting, inseparable from the concept of physis (incessant arise) and ethos (the act as a sense of being); the poíesis (AGAMBEN, 2012), while production in the presence, of something that cross the not going to be to the to be. Therefore, in order to demonstrate how the CDG constitutes a poetics, the conducts of creation in the proposal, through the analysis of an article written by the author of the CDG and a report of the author of the dissertation, both related to experiences of creation were studied. The conclusion was that the conduct of creation guided by CDG has always a collective development and is an open work of art; that in the process of creating the work, each person involved also creates a self, able to take responsibility for the success of them all and the work. The work in this context is the result of willingness and possibilities of each individual and the group. / A presente dissertação questionou a possibilidade de uma determinada proposta de ensino de música, qual seja, a Proposta Musicopedagógica CDG Cante e Dance com a Gente, ser considerada como uma poética pedagógica, de criação artística na e para a escola. Para tal, iniciou-se a pesquisa realizando uma caracterização do CDG a partir dos documentos produzidos pela e sobre a mesma. Concomitantemente, realizou-se o estudo da poética a partir do pensamento de Paul Valéry e René Passeron. Os autores citados serviram como lentes teóricas e metodológicas por intermédio das quais pretendi amplificar o CDG, a fim de demonstrar de que maneira uma proposta pedagógica pode ser entendida como uma poética; essa pretensão teve por objetivo não restringir o CDG à uma proposta de ensino de arte, mas à produção propriamente dita de arte. A poética, conforme apresentada neste trabalho, é: o poïein (VALÉRY, 2011), fazer conduzido por um processo de criação; a poiesis (PASSERON, 1997), estudo da conduta criadora; a poiesis (CASTRO, 2004) estudada fora do campo metafísico, essência do agir, inseparável da concepção de physis (surgir incessante) e ethos (agir como sentido do ser); a poíesis (AGAMBEN, 2012), enquanto produção na presença, do algo que passa do não ser ao ser. Portanto, a fim de demonstrar como o CDG se constitui em uma poética, estudou-se as condutas de criação presentes na proposta, por intermédio da análise de um artigo publicado pela autora do CDG e de um relato da autora da dissertação, ambos relacionados à experiências de criação no CDG. Concluiu-se que a conduta de criação no CDG é pautada pelo desenvolvimento coletivo de uma obra de arte que é sempre aberta; que no processo de criar a obra, cada pessoa envolvida cria, também um si mesmo, capaz de assumir responsabilidades pelo sucesso de todos e da obra. A obra, neste contexto, será o resultado dos quereres e das possibilidades de cada um e do grupo.

Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...

Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...

Compact Models are the physically based accurate mathematical description of the cir-cuit elements, which are computationally efficient enough to be incorporated in circuit simulators so that the outcome becomes useful for the circuit designers. As the multi-gate MOSFETs have appeared as replacements for bulk-MOSFETs in sub-32nm technology nodes, efficient compact models for these new transistors are required for their successful utilization in integrated circuits. Existing compact models for common double-gate (CDG) MOSFETs are based on the fundamental assumption of having symmetric gate oxide thickness. In this work we explore the possibility of developing models without this approximation, while preserving the computational efficiency at the same level. Such effort aims to generalize the compact model and also to capture the oxide thickness asymmetry effect, which might prevail in practical devices due to process uncertainties and thus affects the device performance significantly. However solution to this modeling problem is nontrivial due to the bias-dependent asym-metric nature of the electrostatic. Using the single implicit equation based Poisson so-lution and the unique quasi-linear relationship between the surface potentials, previous researchers of our laboratory have reported the core model for such asymmetric CDG MOSFET. In this work effort has been put to include Non-Quasistatic (NQS) effects, different small-geometry effects, and noise model to this core, so that the model becomes suitable for practical applications. It is demonstrated that the quasi-linear relationship between the surface potentials remains preserved under NQS condition, in the presence of all small geometry effects. This property of the device along with some other new techniques are used to develop the model while keeping the mathematical complexity at the same level of the models reported for the symmetric devices. Proposed model is verified against TCAD simulation for various device geometries and successfully imple-mented in professional circuit simulator. The model passes the source/drain symmetry test and good convergence is observed during standard circuit simulations.

CONTEXTE: Les " Type I Congenital disorders of glycosylation " (CDG-I) comportent des déficits de biosynthèse de l'oligosaccharide lié au dolichol (DLO) qui est nécessaire pour la N-glycosylation des protéines. Ces déficits induisent : 1) une hypoglycosylation des protéines qui serait à l'origine de la pathologie ; et 2) une accumulation de DLO tronqués à partir desquels, par un mécanisme encore inconnu, des structures oligosaccharidiques libres phosphorylées (OSP) sont générées dans le cytosol. Afin de comprendre le rôle de ce processus dans le CDG, il était donc nécessaire de caractériser l'activité qui est à l'origine des OSP.RESULTATS: J'ai caractérisé biochimiquement une DLO diphosphatase (DLODP) qui génère des OSP et du dolichol phosphate à partir de DLO. L'activité DLODP co-fractionne avec un marqueur de l'appareil de Golgi (AG) mais pas avec les enzymes réticulaires qui utilisent le dolichol phosphate. Cette localisation inattendue de DLODP m'a conduit à étudier la génération des OSP dans les cellules en utilisant la bréfeldine A (BFA) qui fusionne l'AG avec le RE. La BFA ne modifie pas les taux de DLO tronqués ni ceux des OSP cytoplasmiques dans un modèle cellulaire de CDG-I. Cependant, dans ces cellules et dans les cellules témoins, la BFA induit une forte augmentation des OSP dans le système endomembranaire à partir de DLO non-tronqués.CONCLUSION: L'identification de différents pools d'OSP, topologiquement distincts et pouvant être modulés de façon indépendante, révèle la multiplicité des mécanismes pour la génération d'OSP et suggère que la DLODP Golgienne n'est pas forcément l'enzyme responsable de la génération des OSP dans le contexte de CDG-I. / BACKGROUND: Type I congenital disorders of glycosylation (CDG-I) are caused by genetic defects in the biosynthetic pathway for the dolichol-linked oligosaccharide (DLO) that is required for protein N-glycosylation. These mutations result in the accumulation of truncated DLO and protein hypoglycosylation. Although protein hypoglycosylation is thought to be the main pathogenic factor in CDG-I, the role of truncated DLO intermediates in cellular homeostasis is not clear. Truncated DLO intermediates are known to give rise to cytoplasmic oligosaccharyl phosphates (OSP) by an uncharacterized mechanism. To understand this DLO editing process biochemical and molecular characterization of the activity that generate OSP is needed.RESULTS: I biochemically characterized a DLO diphosphatase (DLODP) that generates OSP and dolichol phosphate from DLO. Subcellular fractionation of mouse liver homogenates demonstrated a microsomal activity that co-distributes with a Golgi apparatus (GA) marker but not with endoplasmic reticulum (ER)-situated dolichol phosphate utilizing enzymes. This unexpected localization of DLODP prompted me to study OSP generation in cells using brefeldin A (BFA), which fuses the GA with the ER. BFA did not affect the levels of truncated DLO or cytoplasmic OSP, present in a cellular model of CDG-I. However, in these, and control cells, BFA caused striking increases of OSP within the endomembrane system. CONCLUSION: the identification of topologically distinct, independently modulated, OSP pools indicates multiple mechanisms for OSP generation and suggest that the GA-situated DLODP may not be the enzyme responsible for OSP generation in CDG-I.