In patients with Parkinson’s disease (PD), the molecularly misfolded form of α-synuclein was recently identified in cutaneous autonomic nerve fibers which displayed increased accumulation even in early disease stages. However, the underlying mechanisms of synucleinopathic nerve damage and its implication for brain pathology in later life remain to be elucidated. To date, specific diagnostic tools to evaluate small fiber pathology and to discriminate neurodegenerative proteinopathies are rare. Recently, research has indicated that deposition of α-synuclein in cutaneous nerve fibers quantified via immunohistochemistry in superficial skin biopsies might be a valid marker of PD which could facilitate early diagnosis and monitoring of disease progression. However, lack of standardization of techniques to quantify neural α-synuclein deposition limits their utility in clinical practice. Additional challenges include the identification of potential distinct morphological patterns of intraneural α-synuclein deposition among synucleinopathies to facilitate diagnostic discrimination and determining the degree to which structural damage relates to dysfunction of nerve fibers targeted by α-synuclein. Answering these questions might improve our understanding of the pathophysiological role of small fiber neuropathy in Parkinson’s disease, help identify new treatment targets, and facilitate assessment of response to neuroprotective treatment.

Submitted by Maria Medeiros (maria.dilva1@ufcg.edu.br) on 2018-08-16T11:47:56Z No. of bitstreams: 1 FABIANA MEDEIROS DO NASCIMENTO SILVA - TESE (PPGEQ) 2017.pdf: 6622967 bytes, checksum: bfed827814b744a5e9e9e565d28f0682 (MD5) / Made available in DSpace on 2018-08-16T11:47:56Z (GMT). No. of bitstreams: 1 FABIANA MEDEIROS DO NASCIMENTO SILVA - TESE (PPGEQ) 2017.pdf: 6622967 bytes, checksum: bfed827814b744a5e9e9e565d28f0682 (MD5) Previous issue date: 2017 / Capes / O presente trabalho tem como objetivo geral sintetizar as membranas zeolíticas (Mordenita/α-alumina), utilizando os métodos de síntese hidrotérmica, crescimento secundário: dip-coating e transporte em fase vapor, para serem avaliadas no processo de separação emulsão óleo/água. Dentro deste contexto foram avaliados alguns parâmetros, destacando-se, a influência do tempo de cristalização na síntese da zeólita mordenita, a influência do precursor (sulfato de alumínio e gibsita) na síntese da membrana zeolítica, influência dos métodos de síntese das membranas zeolíticas e os testes de permeação de água e o processo de separação emulsão óleo/água. A síntese da zeólita mordenita foi realizada utilizando o método hidrotérmico a 170°C, com tempos de cristalização de 24, 36, 48, 72, 96 e 120 horas, a fim de avaliar a cristalinidade da zeólita, e selecionar o melhor tempo para a síntese das membranas zeolíticas. Os suportes cerâmicos α-alumina foram preparados a partir da decomposição dos precursores sulfato de alumínio a 1000°C e gibsita a 1200ºC por 2 horas, e conformados, compactados e sinterizados a 1300ºC por 2h, e então submetidos às técnicas de caracterização: Difratometria de raios X (DRX), Adsorção Física de Nitrogênio, Microscopia Eletrônica de Varredura (MEV), Espectroscopia de Fluorescência de raios X por Energia Dispersiva (FRX-ED) e Termogravimétrica/Térmica Diferencial (TG/DTA). As membranas zeolíticas foram sintetizadas pelos métodos de síntese hidrotérmica, crescimento secundário: dip-coating e transporte em fase vapor a 170°C por 72h e caracterizadas por DRX e MEV. Os suportes cerâmicos e as membranas zeolíticas foram avaliadas em testes de permeação de água e no sistema de separação emulsão óleo/água de um efluente sintético, utilizando um processo de separação por membrana (PSM). Os ensaios foram realizados nas condições de concentração inicial da emulsão 100 mg.L-1, temperatura de 25 °C e pressão de 2,5 bar, permitindo avaliar a permeabilidade e a seletividade a partir da variação da concentração do permeado em (mg.L-1) e da percentagem de rejeição ao óleo (%R). A partir dos resultados obtidos para a síntese dos materiais, pode-se observar a efetiva formação da zeólita mordenita em fase pura e cristalina. Os precursores foram decompostos de maneira satisfatória obtendo a fase α-alumina. A manutenção da fase α-alumina pós-produção dos suportes cerâmicos foi confirmada após caracterização. De acordo com os resultados exibidos pelas análises de DRX e MEV as membranas zeolíticas MZMOR/α-alumina apresentaram uma distribuição homogênea e uniforme dos cristais zeolíticos correspondentes à fase mordenita, sem a presença de impurezas, livres de defeitos e sem fissuras, confirmando a formação da estrutura da membrana zeolítica pelos três métodos de síntese, utilizados neste trabalho. A zeólita mordenita se mostrou excelente em relação à adesão e formação da camada zeolítica sobre o suporte cerâmico α-alumina. A partir da avaliação da permeabilidade e seletividade nos testes de separação da emulsão óleo/água, pode-se concluir que a inserção da zeólita mordenita aos suportes cerâmicos melhorou o processo de separação da emulsão óleo/água. Em termos de eficiência no processo de separação, considera-se que a membrana zeolítica MZMOR/α – Al2O3 (SHGB) foi a que apresentou melhor relação entre fluxo e capacidade seletiva, mostrando a eficácia da utilização das membranas zeolíticas. Todas as membranas zeolíticas sintetizadas e avaliadas mostraram-se promissoras. / The objective of the present work is to synthesize zeolite membranes (Mordenite/α-alumina) using hydrothermal synthesis, secondary growth: dip-coating and vapor-phase transport, to be evaluated in the oil/water emulsion separation process. The influence of the crystallization time on the synthesis of the mordenite zeolite, the influence of the precursor (aluminum sulphate and gibsite) on the synthesis of the zeolite membrane, influence of the synthesis methods of the zeolite membranes and the water permeation tests and the oil/water emulsion separation process. The synthesis of the mordenite zeolite was performed using the hydrothermal method at 170°C, with crystallization times of 24, 36, 48, 72, 96 and 120 hours, in order to evaluate the crystallinity of the zeolite, and to select the best time for the synthesis of zeolite membranes. The α-alumina ceramic supports were prepared from the decomposition of the aluminum sulfate precursors at 1000°C and gibsite at 1200°C for 2 hours, and conformed, compacted and sintered at 1300°C for 2h, and then submitted to the characterization techniques: (XRD), Nitrogen Physical Adsorption, Scanning Electron Microscopy (SEM), X-ray Fluorescence Spectroscopy (FRX-ED) and Thermogravimetric/Differential Thermal (TG/DTA). The zeolite membranes were synthesized by hydrothermal synthesis, secondary growth: dip-coating and vapor-phase transport at 170°C for 72 hours and characterized by XRD and SEM. Ceramic supports and zeolite membranes were evaluated in water permeation tests and in the oil/water emulsion separation system of a synthetic effluent using a membrane separation process. The tests were carried out under the conditions of initial concentration of the emulsion 100 mg.L-1, temperature of 25°C and pressure of 2,5 bar, allowing to evaluate the permeability and the selectivity from the variation of the permeate concentration in (mg. L-1) and the percentage of oil rejection (% R). From the results obtained for the synthesis of the materials, it is possible to observe the effective formation of zeolite mordenite in pure and crystalline phase. The precursors were satisfactorily decomposed to give the α-alumina phase. The maintenance of the post-production α-alumina phase of the ceramic supports was confirmed after characterization. The MZMOR/α-alumina zeolite membranes presented a homogeneous and uniform distribution of the zeolite crystals corresponding to the mordenite phase, without the presence of impurities, free of defects and without cracks, confirming the formation of the structure of the zeolite membrane by the three methods of synthesis, used in this work. The mordenite zeolite showed excellent adhesion and formation of the zeolitic layer on the ceramic support α-alumina. From the evaluation of the permeability and selectivity in the oil/water emulsion separation tests, it can be concluded that the insertion of the mordenite zeolite to the ceramic supports improved the separation process of the oil/water emulsion. In terms of efficiency in the separation process, the zeolite membrane MZMOR/α-Al2O3(SHGB) was considered to have the best relationship between flow and selectivity, showing the efficacy of zeolite membranes. All zeolite membranes synthesized and evaluated were promising.

Les alliages de titane β métastables sont des matériaux de structure essentiels pour les applications aéronautiques de part leurs très bonnes propriétés mécaniques. En effet, ils présentent une résistance spécifique élevée, une bonne ductilité et forgeabilité et une excellente réponse aux traitements thermiques. Toutefois, il existe encore aujourd'hui à leur sujet des controverses et des questions ouvertes et ce, malgré les efforts pour comprendre les mécanismes d'évolution microstructurale au cours de traitements thermo-mécaniques et pour déterminer les phases en présence et leur contribution aux les propriétés mécaniques résultantes. Ce travail de thèse a pour objectif de déterminer la nature de la phase β et de caractériser la transformation β→α à haute et basse températures par des caractérisations fines en microscopie électronique à balayage et à transmission couplées à des mesures d'orientations cristallographiques et de composition chimique. L'alliage étudié est un Ti-5553 avec une microstructure initiale 100% β obtenue par mise en solution et trempe. Il a été démontré expérimentalement que la structure de la phase β métastable n'est pas purement cubique centrée. Les points de la phase β dans les clichés de diffraction présentent un allongement (streaking) et des points supplémentaires sont visibles aux positions de diffraction 1/2, 1/3 et 2/3. Par ailleurs, les images MET ont un aspect en moiré. A partir de ces résultats et de calculs crystallographiques, il a été prouvé que des déplacements atomiques sur les plans {110}β et {112}β forment une structure intermédiaire entre celle de la phase β parente et celles des phases α et ω, prouvant que la phase β a intrinsèquement initié une transformation. L'étude de la précipitation au cours du procédé thermomécanique dans le domaine α+β a révélé que des précipités α discontinus, équiaxes ou légèrement allongés (1~2μm) se forment aux joints β de forte et de faible désorientation mais rarement au coeur des grains β produisant ainsi une microstructure en "collier". La relation d'orientation de Burgers (ROB) entre les phases α et β est progressivement détruite par la déformation. L'écart à la ROB est plus marqué pour les précipités α qui se forment au joint de grains qu'à l'intérieur des grains. L'écart à la ROB augmente aussi avec la déformation, mais diminue avec la vitesse de déformation. Au cours des déformations en bas du domaine α+β, les précipités α ont une morphologie qui dépend de leur position. Au coeur des bandes de glissement, les grains α/β sont équiaxes et ne respectent pas la ROB. Entre les bandes de glissement, la microstructure est lamellaire où les phases α/β alternent et respectent la ROB. Dans ce dernier cas, une forte sélection de variantes a été observée: Seuls les deux ou trois variants favorisant l'accommodation de la déformation se sont formés. A titre de comparaison, dans l'état non déformé, les 12 précipités sont présents. La transformation β→α est retardée en cours de compression à haute température. Ceci est attribué à une compétition entre adoucissement et transformation de phase. Au contraire, celle-ci est favorisée au cours de la compression à plus basse température du fait que les défauts cristallins induits par la déformation jouent le role de sites de germination et que la croissance des précipités soit accéléré alors que l'adoucissement soit ralenti. Dans le Ti-5553, le mécanisme de déformation dominant est le glissement des dislocations. Dans les déformations en bas du domaine α+β, du glissement simple ou multiple avec deux ou trois systèmes de glissement activés. L'identification de ces systèmes a pu être effectuée par des analyses de traces. Cette thèse a résolu la nature de la phase β métastable et constitue un travail de référence pour l'étude de la transformation β→α au cours de traitement thermomécanique / Metastable β titanium alloys are important structural materials for aeronautical applications due to their high strength to density ratio, good ductility and workability and excellent hardenability. Despite the efforts in resolving the complex microstructural evolution related to thermomechanical processes and in gaining knowledge on the produced phases and their contribution to the resultant mechanical properties, there are still some controversial and unresolved issues. The aim of the present PhD work is to determine precisely the metastable nature of β phase and to characterize finely the characteristics of the β→α transformation during high and low temperature thermomechanical treatments. Investigations were performed on a Ti-5553 alloy with the single β phase initial microstructure obtained by solution treatment followed by quenching using scanning and transmission electron microscopy (SEM/TEM) coupled to crystallographic orientation measurements and chemical analyses. It was demonstrated experimentally that the structure of the β phase in the metastable titanium alloy is not “pure” body centered cubic. Diffraction diagrams presents streaking of the β diffraction spots and additional spots at the 1/2, the 1/3 and 2/3 diffraction positions. Also, striations are observed in TEM images. From this experimental evidence and crystallographic calculations, it was proved that atomic displacements on the {110}β and {112}β planes formed a structure between that of the parent β phase and that of the α or ω phase, demonstrating pre-phase transformation tendency. The study of the precipitation during thermomechanical processing at higher temperature in the α+β region revealed that discontinuous equiaxed or short rod shaped α precipitates (1~2μm) mainly form on the high angle and low angle β grain boundaries but seldom in β grain interiors, forming the “necklace” microstructure. The Burgers orientation relationship (BOR) between the α and β phases is destroyed gradually by the deformation. The BOR deviation of grain boundary α is larger than that of intragranular α. The deviation from the BOR increases both with the increasing strain and decreasing strain rate. During the deformation at the lower temperature in the α+β region, the α precipitates exhibit different morphologies: such as lamellar α, equiaxed α and irregular α depending on their localization. Within the slip bands, equiaxed α/β grains which do not respect the BOR are present. However, between the bands, lamellar α and β phases maintaining the BOR are distributed alternately. In that last case a strong variant selection is observed as only the two or three variants that form are those which can accommodate the macroscopic deformation. Comparatively, in absence of compression all 12 variants are formed. The β→α phase transformation is retarded during the hot compression at higher temperature region, which is attributed to the competition between softening and phase transformation. On the contrary, it is promoted during compression at lower temperature region due to the more inducted deformation defects acting as α phase nucleation sites and due to accelerating growth of α precipitates and retarded softening. Dislocation slip is the leading deformation mechanism for the Ti-5553 alloy. Under the lower temperature deformation condition, single or multiple-slip bands with two or three different activated slip systems would form during the hot deformation process. Identification of these slip systems have been done by trace analysis. These results provide new insights into the structural nature of β metastable phase and valuable reference for β→α phase transformation during thermo-mechanical treatment in metastable β titanium alloys

Depuis quelques années, l’élaboration de molécules spiraniques connait un essor considérable avec, en particulier, comme but essentiel l’accroissement de la diversité moléculaire considérée à ce jour comme insuffisamment développée. L’objectif de cette thèse de doctorat a été la synthèse de nouvelles entités tricycliques arylaliphatiques possédant un carbone spiranique et ce, en complément des travaux antérieurement effectués au laboratoire. Dans un premier temps, nous avons choisi de générer des molécules regroupant des structures de type indane-1,2’-(azétidine, pyrrolidine et pipéridine). La possibilité de fixer par exemple un groupe fonctionnel tel qu’un amide ou un bras espaceur voire de substituer le noyau aromatique nous a permis d’exploiter les différentes directions de l’espace. Dans un second temps, nous avons développé une réaction d’arylation intramoléculaire en position α de groupements électroattracteurs. Cette arylation catalysée par des métaux (dans notre cas le cuivre) permet d’accéder à des composés aux motifs spiroindane- ou spirotétraline-1,3’-(azétidine, pyrrolidine et pipéridine). Dans un troisième temps, nous avons étudié les réactions d’addition nucléophiles intramoléculaires de pyridines N-activées pour accéder à des structures spirocycliques pyridiniques fonctionnalisées. Des essais préliminaires utilisant de l’anhydride acétique comme agent activant nous ont permis de générer les intermédiaires recherchés. Afin d’accroître la diversité moléculaire et découvrir de nouveaux fragments susceptibles de nous mener à des agents thérapeutiques, nous nous sommes intéressé, dans un dernier temps, au domaine des biotechnologies blanches en exploitant le potentiel des micro-organismes et de leurs enzymes pour fonctionnaliser des liaisons C-H non activées présentes au sein de charpentes spirocycliques préalablement préparées. / In recent years, the elaboration of spirocyclic molecules has arisen, particularly with an essential purpose to increase of molecular diversity which is not sufficiently developed to date. The objective of this work was the synthesis of new arylaliphatic tricyclic entities with spiranic carbon and it in addition to previous work in the laboratory. Firstly, we have chosen to generate molecules having indane-1,2’-(azetidine, pyrrolidine and piperidine) moiety. The possibility of incorporating a functional group such as an amide, a spacer group or even a substituent on the aromatic ring has allowed us to exploit all space directions. Secondly, we have developed an intramolecular arylation in α position of the electroattractive groups. This metal catalyzed arylation, (in this case copper) provides access to compounds with spiroindane or spirotetraline-1,3’-(azetidine, pyrrolidine and piperidine) patterns. Thirdly, we have studied the intramolecular nucleophilic addition of N-activated pyridine to accede to spirocyclic functionalized pyridine structures. Preliminary tests using acetic anhydride as the activating agent allowed us to generate some desired intermediates. Last but not least, in an effort to increase the molecular diversity and the discovery of new fragments that could lead us to therapeutic agents, we were interested in the field of white biotechnology by harnessing the potential of microorganisms and their enzymes to functionalize in activated C-H bonds in previously prepared spirocyclic scaffolds.

No description available.

The present work aims to determine the physiological and sanitary quality of Sweet sorghum (Sorghum bicolor (L.) Moench) seeds, as well as to identify pathogens associated with seed, their transmission to seedlings and the subsequent pathogenicity of isolates obtained, In addition, molecularly identify the fungal species pathogenic to this crop. The experiments were carried out in the Teaching and Seed Research Laboratory (TSRL), of the Plant Engineering Department; In the Elocy Minussi Phytopathology Laboratory, Department of Plant Protection, and at the Biological Institute of São Paulo. Sweet sorghum seeds were used, all without chemical treatment. Sanitary quality was evaluated by sanity test, and physiological characteristics by germination and vigor tests (seedling length, dry mass, emergence, rate of emergence and accelerated aging). It was performed the test of transmission of the pathogens associated to the seeds and the subsequent pathogenicity of the obtained isolates, culminating with the molecular characterization of the identified pathogens, in which were sequenced the Internal Transcribed Spacer (ITS) genomic regions and the Elongation Factor 1 - alpha (TEF1-α) gene. The design used was the completely randomized design, with four cultivars of Sweet sorghum (BRS 506, F19, BRS 511 and BRS 509); For the evaluation of pathogenicity, the factorial scheme is represented by four cultivars and three isolates of Fusarium spp., besides the witness. The seeds of the BRS 509 cultivar were considered to have lower physiological quality than the other cultivars. The DNA sequencing allowed identifying the Fusarium thapsinum species as a pathogenic agent in the sweet sorghum crop, and proven its transmission via seeds. / O presente trabalho teve como objetivo determinar a qualidade fisiológica e sanitária de sementes de cultivares de sorgo sacarino (Sorghum bicolor (L.) Moench), bem como identificar os patógenos associados à semente, sua transmissão às plântulas e a posterior patogenicidade de isolados obtidos, além disso, identificar molecularmente as espécies fúngicas patogênicas a esta cultura. Os experimentos foram realizados no Laboratório Didático e de Pesquisas em Sementes (LDPS), do Departamento de Fitotecnia; no Laboratório de Fitopatologia Elocy Minussi, do Departamento de Defesa Fitossanitária e, no Instituto Biológico de São Paulo. Foram utilizadas sementes de sorgo sacarino, todas sem tratamento químico. A qualidade sanitária foi avaliada pelo teste de sanidade, e as características fisiológicas por meio dos testes de germinação e de vigor (comprimento de plântulas, massa seca, emergência, índice de velocidade de emergência e envelhecimento acelerado). Foi realizado o teste de transmissão dos patógenos associados à semente e a posterior patogenicidade dos isolados fúngicos obtidos, culminando com a caracterização molecular dos patógenos identificados, na qual foram sequenciadas as regiões genômicas Internal Transcribed Spacer (ITS) e o gene do fator de elongação 1-α (TEF1α). O delineamento utilizado foi o inteiramente casualizado com quatro cultivares de sorgo sacarino (BRS 506, Fepagro 19, BRS 511 e BRS 509); já para a avaliação da patogenicidade, o esquema fatorial foi representado pelas quatro cultivares e três isolados de Fusarium sp., além da testemunha. As sementes da cultivar BRS 509 foram consideradas de qualidade fisiológica inferior as demais cultivares. O sequenciamento de DNA permitiu identificar a espécie Fusarium thapsinum como agente patogênico na cultura do sorgo sacarino, sendo comprovada sua transmissão via sementes.

Made available in DSpace on 2015-05-14T13:00:16Z (GMT). No. of bitstreams: 1 parte1.pdf: 4104748 bytes, checksum: 578975146349baff07079d6c3f9756b6 (MD5) Previous issue date: 2008-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Snake venoms contain a complex arsenal of protein bio-active components, many of these being neurotoxins (NTXs). These snakes have high neurotoxic activity venom, corresponding to the Elapidae family, which includes coral snakes (Micrurus) whose venom contains circa 90-95% of low molecular mass protein components. Among these, several are postsynaptic neurotoxins or α- NTXs (MM = 6-9 kDa). The Micrurus ibiboboca (Merren, 1820) is a snake of the Elapidae family witch is quite common in the Northeast of Brazil. In spite of the great diversity of species of Micrurus, scarce works involving the nervous system with isolated and pure toxins of those serpents has been developed in level biochemical, pharmacological and electrophysiological. The aim of this study was to purify the toxin Mic6c7NTX of the Micrurus ibiboboca venom, characterize to biochemically and electrophysiologically the toxin Mic6c7NTX in the peripheral nervous system (PNS) of rats, evaluating alterations in the record of the Compound Action Potential (CAP) of the isolate nerve and the toxin activity on the voltage-dependent sodium channels (Nav) in the neurons of the dorsal root ganglion (DRG). The venom was extracted from the Micrurus ibiboboca collected in Paraiba State (Brazil). Initially, electrophysiological tests (current clamp method) using the single sucrose gap technique were accomplished with crude venom (100μg/mL). It was observed that in this concentration the crude venom caused reduction in the CAP amplitude (25%). This neurotoxity led into an intriguing question: what components of the venom would promote to reduction in the excitability of the nerve? Based upon this question, I decided to purify the venom throughout the Liquid Chromatography of the High Performance (HPLC) of the Cation Exchange Chromatography (CIEX) and the Reverse Phase Chromatography (RPC). The molecular mass (MM) of the raw toxin was determined by mass-spectrometry (MALDI-QTOF/ MS) and N-terminal sequence by means of Edman s Degradation. The search for similarity with other toxins was accomplished against proteomic data bank. The CIEX profile showed 19 fractions and the highest peak fraction was used for the second dimension. The toxin Mic6c7NTX obtained by RPC showed elution in 26.7%of the acetonitrile (ACN) and MM 7.047.56Da. The obtained partial N-terminal sequence showed 31 aminoacid residues. The search for similarity of structure and function showed great similarity (65%) with other short chain α-NTXs Australian elapids snakes. The electrophysiological studies (single sucrose gap technique) showed that the toxin Mic6c7NTX (1 μM) reduced the excitability of the isolate nerve similarly to the reduction observed in the crude venom about 21%. Other CAP parameters such as despolarization speed (DSCAP), repolarization time (τCAP) and peak of time (PTCAP) did not show alterations. This suggests that the toxin may be affecting the Nav channels. For the confirmation of that hypothesis experiments were accomplished with whole cell patch-clamp technique in DRG neurons. This results showed that the toxin Mic6c7NTX (1 WM) abolished completely the current of Nav channels sensitive the tetrodotoxin (TTX-S). Also the Nav channels TTX resistant (TTX-R) were investigated in the presence of the Mic6c7NTX toxin previously using TTX (100 nM). This results showed that the toxin Mic6c7NTX (100 nM) abolished completely the current of Nav channels TTX-R and IC50 = 30nM. However, reversion of this blocking was not observed. The present study biochemically and electrophysiologically characterized an α-NTX of the Micrurus ibiboboca elapid snake. Furthermore, it showed a potent toxin with affinity Nav channels TTX-S and TTX-R of the PNS. This is the first α-NTX isolated and identified of the venom from the Micrurus ibiboboca (Merrem, 1820) snake. / As serpentes da família Elapidae possuem uma peçonha com alta atividade neurotóxica e capacidade de letalidade. Fazem parte dessa família as serpentes corais americanas (gênero Micrurus) com suas peçonhas contendo cerca de 90-95% de componentes protéicos, sendo na sua maior parte neurotoxinas com baixa massa molecular (6-8 kDa), podendo ser destacadas as neurotoxinas com ação pós-sinápticas ou α-Neurotoxinas (α-NTX). A Micrurus ibiboboca (Merrem, 1820) é uma serpente da família Elapidae, comum na região Nordeste. Apesar da grande diversidade de espécies do gênero Micrurus sp., escassos trabalhos envolvendo atividade de toxinas isoladas e puras destas peçonhas e sistema nervoso têm sido desenvolvidos em nível bioquímico, farmacológico ou eletrofisiológico. O objetivo desse estudo foi purificar a toxina Mic6c7NTX da peçonha de M. ibiboboca, caracterizar bioquímicamente e investigar com ferramentas eletrofisiológicas a ação da toxina no Sistema Nervoso Periférico (SNP) de ratos avaliando alterações no Potencial de Ação Composto (PAC) do nervo isquiático isolado e a atividade da toxina nos canais para sódio dependentes de voltagem (Nav) em neurônios do gânglio da raiz dorsal (DRG). A peçonha da M. ibiboboca foi extraída de serpentes coletadas no Estado da Paraíba (Brasil). Inicialmente, ensaios eletrofisiológicos com o método de current clamp utilizando a técnica de single sucrose gap foram realizados com a peçonha bruta (100 Wg/mL). Os resultados mostraram que a peçonha bruta nessa concentração promoveu redução na amplitude do PAC (25%). Esse efeito da toxina na excitabilidade do nervo levantou o questionamento: Que componentes da peçonha estariam causando essa diminuição da excitabilidade? A peçonha foi purificada por meio de Cromatografia Líquida de Alta Performance (HPLC), de troca catiônica (CIEX) e fase reversa (RPC). Na sequência, os picos da CIEX foram submetidos à RPC e posteriormente analisados por espectrometria de massas (MALDI-TOF/MS) que detectou a massa molecular da toxina Mic6c7NTX de 7.047,56 Da. Em seguida, foi determinado o seu N-terminal por Degradação de Edman que apresentou 31 resíduos de aminoácidos e serviu de estudo para a bioinformática na busca por similaridade em banco de dados proteômicos com outras toxinas protéicas, demonstrando que a toxina Mic6c7NTX apresentou similaridade (65%) com α-NTXs de cadeia curta de serpentes elapídicas australianas. Posteriormente, foi investigado o efeito da toxina isolada no SNP. Os estudos eletrofisiológicos em single sucrose gap demonstraram que a toxina Mic6c7NTX (1 WM) reduziu a excitabilidade do nervo isolado de forma similar à observada pela peçonha bruta. Não foram observadas alterações significantes em outros parâmetros do PAC, como velocidade de despolarização (VDPAC), tempo de repolarização (τPAC) e tempo de pico (PTPAC), sugerindo que a toxina atuasse num sítio de ligação específico dos [Escreva uma citação do documento ou o 11 canais Nav no SNP. Para a confirmação dessa hipótese foram realizados experimentos de voltage clamp com a técnica de whole cell patch-clamp em cultura primária de neurônios DRG da medula espinhal de ratos. Os resultados mostraram que a toxina Mic6c7NTX (1 WM) aboliu completamente as correntes dos canais Nav sensíveis à tetrodotoxina (TTX-S). Também foi investigado o efeito da toxina sobre a população de canais Nav resistentes à TTX (TTX-R), utilizando previamente TTX (100 nM) para bloquear os canais Nav TTX-S. Os registros com a toxina Mic6c7NTX (100 nM) demonstraram um bloqueio total da corrente nos canais Nav TTX-R dos DRGs e uma IC50 da toxina em torno de 30 nM. Também foi observado que essa toxina se liga aos canais Nav de forma lenta e irreversível. O presente estudo caracterizou bioquímica e eletrofisiologicamente uma α-NTX da serpente elapídica Micrurus ibiboboca. Farmacologicamente, trata-se de uma potente toxina com afinidade aos canais Nav TTX-S e TTX-R do SNP. Essa é a primeira α-NTX isolada e caracterizada da peçonha da serpente Micrurus ibiboboca (Merrem, 1820).

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-14T12:30:34Z No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:05:05Z (GMT) No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Made available in DSpace on 2016-10-22T13:05:05Z (GMT). No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) Previous issue date: 2008-02-15 / Nos países tropicais, as perdas causadas pela infestação de carrapatos em bovinos acarretam um grande impacto no sistema de produção animal. Recentes estudos têm mostrado a importância de fatores genéticos ligados a resistência a carrapato em Bos taurus indicus e Bos taurus taurus e que as citocinas têm um papel crítico na prevenção ou progressão de doenças. O objetivo desse trabalho foi avaliar os níveis de expressão dos genes IL-10 e IL-4 relacionados ao perfil imunológico Th2 associado à susceptibilidade ao carrapato e os genes IL-2 e IFN- relacionados ao perfil imunológico Th1 associado à resistência ao parasito. Além destes genes, analisou-se o perfil de expressão do gene TLR-2, importante no processo de reconhecimento de patógenos e os genes IL-8 e TNF-α importantes no processo inflamatório inicial. Seis animais mais resistentes e seis animais mais susceptíveis de uma população F2 de 332 animais, originária do cruzamento de animais F1(½ Holandês: ½ Gir), foram selecionados baseado na contagem de carrapatos e valor genético. Amostras de tecido foram coletadas de pele no 5° e 12° dias após a infestação para extração de RNA total. As PCRs em tempo real foram realizadas usando o gene GAPDH como controle endógeno. Os animais resistentes e susceptíveis apresentaram aumento de expressão do gene IL-10 no 5° (p<0,01) e 12 ° dias após a infestação (p<0,05). O gene IL-2, nos animais resistentes e susceptíveis, no 5° dia após a infestação não apresentou alteração da expressão sendo que 12° dia, em ambos os grupos de animais, este gene passou a ser mais expresso em relação ao animal controle sugerindo um perfil de resposta imunológica do tipo de Th2 nos animais resistentes e susceptíveis nos primeiros dias após a infestação. O gene IL-4 apresentou uma tendência ao aumento de expressão nos animais resistentes e susceptíveis em relação ao controle, sendo o perfil Th2 sugerido atribuído a IL-10 produzida por linfócitos T regulatórios (p>0,05). O gene TNF- apresentou aumento de expressão nos animais susceptíveis no 5° dia após a infestação com posterior diminuição no 12° dia após a infestação (p<0,05). Nos animais resistentes não foi observada alteração da expressão deste gene, isto sugere que ele possa estar mais atuante no início do processo inflamatório, logo após a fixação do carrapato. A mesma observação estende-se para o gene IL-8, em que não foi verificada alteração de expressão nos animais resistentes, embora nos animais susceptíveis este gene apresentou diminuição da expressão no 12° dia após a infestação (p<0,05). Quanto ao gene IFN-, não houve diferença de expressão entre os animais resistentes e susceptíveis, sendo que este gene parece não estar relacionado ao mecanismo de resistência. O gene TLR-2 apresentou diminuição da expressão em ambos os grupos de animais. Estes resultados sugerem que a resposta imune adquirida avaliada neste trabalho não apresenta papel preponderante no mecanismo de resistência e que resposta imune inata poderia está envolvida no mecanismo de resistência ao carrapato. Portanto, avaliação da resposta imunológica horas após a fixação do carrapato poderia nos fornecer resultados mais conclusivos. / In tropical countries losses caused by tick infestation in cattle lead to a major impact on animal production systems. Recent studies have shown the importance of genetic factors linked to tick resistance in Bos indicus and Bos taurus as well as the critical role in the prevention or progression of diseases mediated by cytokines. The aim of this work was to evaluate gene expression of IL-10 and IL-4 in relation to tick susceptibility associated with the Th2 profile and gene expression of IL-2 and IFN- in relation to tick resistance associated with the Th1 profile. In addition, the expression of TLR-2, important in the process the recognition of pathogens, and TNF-α and IL-8 genes, important in the initial inflammatory process, were evaluated. Six tick-resistant and six tick-susceptible animals from a F2 population of 332 animals, originated from the cross of F1 animals (½ Holstein: ½ Gir), were selected based on tick count and breeding value for tick resistance. Skin biopsies were collected in the 5th and 12th days after tick infestation. The GAPDH was used as endogenous control to normalize the amount of starting cDNA target in the real-time PCR assay. Both resistant and susceptible animals showed increased gene expression of IL-10 in the 5th and 12th days after infestation in relation to control animal (p<0.05). The IL-2 gene showed no change of expression in the 5th day after infestation for the resistant and susceptible animals. In the 12th post infestation, both resistant and susceptible animals showed increased gene expression in relation to control animal. These results suggest an enhancement of Th2 profile through the increase of IL-10 mRNA levels and a possible inhibition of the Th1 pattern in both groups (resistant and susceptible) starting 5 days after infestation and return to normal by day 12. Despite our results suggest the occurrence of the Th2 profile, the susceptible and resistant animals did not show variation on gene expression for IL-4 in relation to control animal. The susceptible animals showed increased expression of TNF-α in the 5th day after infestation. However, in the 12th day post infestation it was noted a decrease in the gene expression level. The resistant animals showed no change in the expression of this gene in relation to control animals suggesting that TNF-α could be more actively expressed in the early steps of the inflammatory process. Similarly, the resistant animals showed no variation in the expression of IL-8 while the susceptible animals showed increased expression in the 12th day post infestation. There were no differences of expression between resistant and susceptible animals in relation to IFN-γ what suggests that this gene might not be involved in the resistance mechanism. The TLR-2 gene showed decreased expression in both resistant and susceptible animals (p<0.05). Finally, there was no difference in expression between susceptible and resistant animals in relation to all selected genes in the 5th and 12th days after infestation. These results suggest that the acquired immunity evaluated in this work might not have preponderant role in the resistance mechanism. The innate immunity might be playing a major role in the bovine tick resistance/susceptibility mechanism in early hours after infestation.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-10-05T15:16:05Z No. of bitstreams: 2 Dissertação - Iara Barreto Neves Oliveira - 2017.pdf: 2505199 bytes, checksum: 7432c019307e551f15f5fb4cd61ad45c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-10-05T15:17:21Z (GMT) No. of bitstreams: 2 Dissertação - Iara Barreto Neves Oliveira - 2017.pdf: 2505199 bytes, checksum: 7432c019307e551f15f5fb4cd61ad45c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-10-05T15:17:21Z (GMT). No. of bitstreams: 2 Dissertação - Iara Barreto Neves Oliveira - 2017.pdf: 2505199 bytes, checksum: 7432c019307e551f15f5fb4cd61ad45c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-09-29 / Introduction. Multiple Sclerosis (MS) is a Central Nervous System disease, mediated by the Immune System, whose symptoms occur in episodes of relapses. Interferon-beta (IFN-β) is considered a safe treatment for the reduction of relapses, but its mechanisms of action have not yet been clear. Studies have shown involvement of tumor necrosis factor alpha (TNF-α) and of Interleukin-10 (IL-10) in the immunopathogenesis of MS. The role of IL-32, a proinflammatory cytokine has role on several chronic inflammatory diseases, was not elucidated in MS. The effect of IFN-β on these cytokines and disease severity, as measured by the Expanded Disability Status Scale (EDSS), has not yet been established. Objective. The objective of the present study was to evaluate TNF-α, IL-10 and IL-32γ concentrations in the peripheral blood and gene expression of patients with IFN-β. Methods. The sample were patients of the Department of Neurology of the Clinics Hospital of the Federal University, and healthy individuals. Blood collection, blood culture with lipopolysaccharide (LPS), Toll 4 receptor agonist (TLR4), and PAM3Cys, TLR2 agonist, and the quantification of cytokines by real-time polymerase chain reaction were performed. Mann Whitney tests were used for statistical analysis of unpaired data, Wilcoxon for paired samples and Spearman's correlation test, adopting significance level p <0.05. Results. Of the 30 MS patients, 19 were treated with IFN-β and 11, untreated, with a mean age of 40.52 and 42 years, respectively, and female prevalence. TNF-α did not differ between groups but it was less produced after stimulation with Pam3Cys in treated patients compared to controls and untreated patients. IL- 10 concentrations were higher in cultures with LPS in patients treated compared to healthy controls. The mean EDSS of patients treated with IFN-β and untreated did not differ, and the correlation between and TNF-α and IL-10 concentrations produced in blood cultures and EDSS was not significant in the patients. There was a significant correlation between TNF-α concentrations and disease time in untreated patients in non-stimulated cultures and those with TLR2 agonist stimulus. Gene expression of IL-32γ was higher in IFN-β treated patients compared to controls. The gene expression of cytokines correlated positively and significantly in patients and controls and the IL-10 expression was correlated negative e significantly with the disease time in untreated patients. Conclusions. IFN-β reduced patients' response to Pam3Cys. IL-10 was higher in treated patients relative to controls. The correlations were not conclusive about the possible association between these cytokines and the clinic parameters of the disease. IL-32γ was higher in patients treated with IFN-β than in healthy subjects. / Introdução. A Esclerose Múltipla (EM) é uma doença do Sistema Nervoso Central, mediada pelo Sistema Imune,cujos sintomas ocorremem episódios de surtos.OInterferon-beta (IFN-β) é considerado um tratamento seguro para redução dos surtos, masseus mecanismos de ação ainda não foram bemesclarecidos. Estudos mostraram participaçãodo fator de necrose tumoral alfa (TNF-α) e da Interleucina-10 (IL-10) na imunopatogênese da EM. O papel da IL-32, citocina pró-inflamatória atuante emvárias doenças inflamatórias crônicas, não foi elucidado na EM. O efeito do IFN-β nestas citocinas e na gravidade da doença, medida pela Escala do Estado de Incapacidade Expandida (EDSS), ainda não foi estabelecido. Objetivo.O objetivo do presente estudo foi avaliar as concentrações de TNF-α, IL- 10 e IL-32γ no sangue periférico de pacientes com EM tratados com IFN-β e não tratados. Métodos.Foram recrutados portadores da doença, no Serviço de Neurologia do Hospital das Clínicas da Universidade Federal,e indivíduos sadios. Foi feita a coleta de sangue, hemoculturas com lipopolissacarídeo (LPS), agonista do receptor do tipo Toll 4 (TLR4), e PAM3Cys, agonista de TLR2, e a quantificação gênica das citocinas por reação em cadeia da polimerase em tempo real. Foram utilizados os testes Mann Whitney, para análise estatísticados dados não pareados, Wilcoxon para as amostras pareadas e o teste de correlação de Spearman, adotando nível de significância p<0,05. Resultados. Dos 30 pacientes com EM, 19 eram tratados com IFN-β e 11, não tratados, com idade média de 40,52 e 42 anos, anos, respectivamente, e prevalência do sexo feminino. As concentrações de IL-10 foram mais elevadasnas culturas dos pacientes tratados com IFN-β estimuladas com LPS comparado aos controles sadios.TNF-α não diferiu entre os grupos, mas foi menos produzido após estimulação com Pam3Cys nos pacientes tratados comparado aos controles e aos pacientes não tratados. O EDSS médio dos pacientes tratados com IFN-β e dos não tratados não diferiu, e a correlação entre e as concentrações de TNFα e IL-10 produzidas nas hemoculturas e o EDSS não foi significante nos pacientes. Houve correlação significante entre as concentrações de TNF-α e o tempo de doença nos pacientes não tratados nas culturas não estimuladas e naquelas com estímulo de agonista de TLR2. A expressão gênica de IL-32γ foi mais elevada nos pacientes tratados com IFN-β comparado aos controles. As expressões gênicas das citocinas se correlacionaram positiva e significantemente nos pacientes e controles e a expressão de IL-10 se correlacionou negativa e significantemente com o tempo de doença nos pacientes não tratados. Conclusões. O IFN-β reduziu a resposta dos pacientes ao Pam3Cys. IL-10 foi mais elevada nos pacientes tratados em relação aos controles. As correlações não foram conclusivas sobre a possível associação entre essas citocinas e os parâmetros clínicos da doença. IL-32γ foi mais elevada nos pacientes tratados com IFN-β em relação às pessoas sadias.

Made available in DSpace on 2016-08-19T12:56:48Z (GMT). No. of bitstreams: 1 Dissertacao LAISE NAYRA DOS SANTOS PEREIRA.pdf: 1329578 bytes, checksum: bb0923e1ddc2d57bb73b2390b4ceb375 (MD5) Previous issue date: 2014-07-18 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / This work describes the development of electroanalytical procedures for α-lipoic acid (ALA) determination in dietary supplement using a pyrolitic graphite electrode (PG) modified with cobalt phthalocyanine (CoPc) from electrochemical techniques and by Batch Injection Analysis (BIA). Cyclic voltammograms of ALA oxidation on the PG/ CoPc electrode presented an irreversible peak ~+0.85 V vs Ag/ AgCl, a value about 100 mV to less positive potential, well as a significant increase in the magnitude of peak current compared to the PG electrode unmodified. The analytical curves obtained with cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperometry showed good linearity and sensitivity. The DPV technique showed the best values of limit of detection (LOD) and quantification (LOQ) 3.4 x 10ˉ⁹ mol Lˉ¹ and 1.2 x 10ˉ⁸ mol Lˉ¹, respectively. Therefore was chosen for determination of ALA on technique. Voltammetric response of ALA was also evaluated in PG electrode unimodified and modified in hydrodynamic conditions. The modification of the PG/ CoPc electrode produced an increase of 2.33 times compared to the PG electrode. The analytical method was then proposed using the PG/ CoPc electrode for detection of ALA by BIA. The method showed a good linear range between (1.0 x 10ˉ⁶ to 1.0 x 10ˉ⁴ mol Lˉ¹), values of LOD and LOQ of 1.5 × 10ˉ⁸ mol Lˉ¹ and 5.0 × 10ˉ⁸ mol L-1, respectively, with analytical frequency of 208 injections/ hour. The quantification of ALA in dietary supplement showed concordant results with those obtained by chromatographic method, with a confidence level of 95%. Recovery tests were performed added a concentration of ALA in a sample of synthetic urine, where the recovery values were in the range 98 - 107%. / O presente trabalho descreve o desenvolvimento de procedimentos eletroanalíticos para determinação de ácido α-lipóico (ALA) em suplemento dietético utilizando eletrodo de grafite pirolítico (GP) modificado com ftalocianina de cobalto (CoPc) a partir de técnicas eletroquímicas e pela Análise por Injeção em Batelada (BIA). Os voltamogramas cíclicos da oxidação de ALA sobre o eletrodo GP/ CoPc apresentaram um pico irreversível com potencial ~ +0,85 V vs. Ag/ AgCl, mostrando um deslocamento do início da corrente de oxidação em cerca de 100 mV para potenciais menos positivos, bem como um aumento significativo na magnitude da corrente de pico, quando comparados ao eletrodo não modificado GP. As curvas analíticas obtidas com as técnicas de voltametria cíclica (VC), voltametria de pulso diferencial (VPD) e cronoamperometria apresentaram boa linearidade e sensibilidade. A técnica de VPD apresentou os melhores valores de limite de detecção (LD) e de quantificação (LQ), 3,4 x 10ˉ⁹ mol Lˉ¹ e 1,2 x 10ˉ⁸ mol Lˉ¹, respectivamente. Portanto foi a técnica escolhida para determinação de ALA. Resposta voltamétrica do ALA também foi avaliada no eletrodo GP não modificado e modificado em condições hidrodinâmicas. A modificação do eletrodo GP/ CoPc produziu um aumento de 2,33 vezes comparado com o eletrodo GP. O método analítico foi então proposto usando o eletrodo GP/ CoPc para detecção de ALA por BIA. O método apresentou uma boa faixa linear compreendida entre (1 x 10ˉ⁶ a 1 x 10ˉ⁴ mol Lˉ¹); valores de LD e LQ de 1, 5 x 10ˉ⁸ mol Lˉ¹ e 5, 0 x 10ˉ⁸ mol Lˉ¹, respectivamente, com frequência analítica de 208 injeções/ hora. A quantificação de ALA em suplemento dietético apresentou resultados concordantes com os obtidos pelo método cromatográfico, apresentando um nível de confiança de 95%. Testes de recuperação foram feitos adicionados uma concentração de ALA em amostra de urina sintética, onde os valores de recuperação foram na faixa de 98 a 107%.