Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-10-20T16:27:57Z No. of bitstreams: 1 DIS_ELISAMAR_SANTOS_MARIN_WAGNER_COMPLETO.pdf: 909420 bytes, checksum: 855d83fc62f091978eab516fd6b1df35 (MD5) / Made available in DSpace on 2016-10-20T16:27:57Z (GMT). No. of bitstreams: 1 DIS_ELISAMAR_SANTOS_MARIN_WAGNER_COMPLETO.pdf: 909420 bytes, checksum: 855d83fc62f091978eab516fd6b1df35 (MD5) Previous issue date: 2016-03-01 / Gaucher disease, first described in 1882, is a rare autosomal recessive genetic disorder characterized by a deficiency of glucocerebrosidase, an enzyme that catalyses the hydrolysis of lysosomal glucocerebroside into glucose and ceramide. The presence of two mutant alleles located on chromosome 1 q21 region confirms the diagnosis of Gaucher disease, with N370S and L444P being the most common mutations. Clinical characteristics commonly associated with Gaucher disease include hepatosplenomegaly, anemia, thrombocytopenia and bone involvement, hematological and neurological impairment in type II and III. The accumulation of mutant enzyme and subsequent depletion of normal enzyme can be treated with enzyme replacement therapy using recombinant glucocerebrosidase. Enzyme replacement therapy is quite effective in the treatment of this disease, often reversing the cascade of biochemical events that lead to clinical manifestations and improving many of the patient's signs and symptoms. There are three recombinant enzymes for enzyme replacement therapy in Gaucher disease: Velaglucerase alfa, Alfataliglucerase and Imiglucerase. These enzymes differ from each other mainly in relation to the form of production, the amino acid sequence, and the glycosylation pattern. Currently, enzyme replacement therapy is administered by a drug called Cerezyme? (imiglucerase), produced by recombinant DNA technology using cell cultures obtained from Chinese hamster ovary. The imiglucerase differs from natural glucocerebrosidase, from placental origin, by an amino acid at position 495, where histidine is replaced with arginine. However, the cost of Cerezyme? is very high, hindering access to therapy. In Brazil, there are nearly 600 carriers of the disease. Although the treatment is guaranteed by law to all patients, the expenses on the purchase of the medicine are very high. The cost could be considerably reduced if the enzyme could be produced in an alternative way. Studies have shown that advanced reproductive techniques allow transgenic animals to be used as bioreactors for production of recombinant proteins of high potential biological and pharmaceutical interest. The Molecular Biology Laboratory and the development of University of Fortaleza in partnership with Quatro G developed the first Latin America transgenic goat clone, whose mammary gland is used as a bioreactor for producing recombinant protein human glucocerebrosidase. This study presented an alternative of glucocerebrosidase enzyme purification. The alternative method was achieved by using milk obtained through an induced lactation of the transgenic and cloned goat. According to the results, one can create a positive expectation for glucocerebrosidase through the procedure cited throughout this study. The confirmation of the presence of glucocerebrosidase enzyme activity was carried out by fluorimetry test. Cerezyme? and goat?s milk control (not genetically modified) were used with positive and negative control, respectively. The objective of this study was to establish protocols for purification of glucocerebrosidase enzyme from the cloned and transgenic goat's milk and confirm the enzyme activity, to evaluate the expression of the enzyme by electrophoresis, and to characterize the recombinant protein by mass spectrometry. / A doen?a de Gaucher, descrita pela primeira vez em 1882, ? uma rara desordem gen?tica autoss?mica recessiva caracterizada pela defici?ncia da atividade da glucocerebrosidase, uma enzima lisossomal que catalisa a hidr?lise de glucocerebros?deo em glicose e ceramida. A presen?a de dois alelos mutantes localizado na regi?o q21 do cromossomo 1 confirma o diagn?stico, sendo as muta??es mais comuns s?o N370S e L444P. O ac?mulo desta enzima pode ser tratada com terapia de reposi??o enzim?tica, usando glucocerebrosidase recombinante, bastante eficaz no tratamento da doen?a, revertendo toda a cascata de eventos bioqu?micos que acabam por ocasionar as manifesta??es cl?nicas apresentadas pelos pacientes ou melhorando muitos dos seus sinais e sintomas. As caracter?sticas cl?nicas comumente associados com a doen?a de Gaucher incluem hepatoesplenomegalia, anemia, trombocitopenia e envolvimento ?sseo, hematol?gico e comprometimento neurol?gico no tipo II e III. H? tr?s enzimas recombinantes para a terapia de reposi??o enzim?tica na doen?a de Gaucher: Velaglucerase alfa, Alfataliglucerase e Imiglucerase que diferem entre si principalmente em rela??o ? forma de produ??o, ? sequ?ncia de amino?cidos e ao padr?o de glicosila??o. Atualmente, a terapia de reposi??o enzim?tica ? administrada por um medicamento chamado Cerezyme? (imiglucerase), produzido atrav?s da tecnologia do DNA recombinante utilizando culturas de c?lulas obtidas do ov?rio de hamster chin?s. A imiglucerase difere da glucocerebrosidase natural, de origem placent?ria, por um amino?cido na posi??o 495, onde a histidina ? substitu?da por arginina. Entretanto, o custo do medicamento dispon?vel ? muito alto, dificultando o acesso ? terapia. No Brasil, h? aproximadamente 600 portadores da doen?a e o tratamento ? garantido por lei a todos os pacientes, embora os gastos com a compra do medicamento sejam muito elevados. Este custo poderia ser consideravelmente reduzido se a enzima pudesse ser produzida de forma alternativa. Estudos demonstraram que t?cnicas reprodutivas avan?adas permitem que animais transg?nicos possam ser usados como biorreatores para a produ??o de prote?nas recombinantes de elevado potencial biol?gico e interesse farmac?utico. O Laborat?rio de Biologia Molecular e do Desenvolvimento da Universidade de Fortaleza em parceria com a Quatro G Pesquisa e Desenvolvimento desenvolveu o primeiro clone transg?nico de cabras da Am?rica Latina cuja gl?ndula mam?ria ? usada como um biorreator para a produ??o da prote?na glucocerebrosidase humana recombinante. Neste presente estudo, foi apresentado uma alternativa de purifica??o da enzima glucocerebrosidase a partir do leite obtido atrav?s da lacta??o induzida da cabra transg?nica e clonada. De acordo com os resultados, pode-se criar uma expectativa positiva para a obten??o da glucocerebrosidase atrav?s do procedimento citado ao longo deste estudo. A confirma??o da presen?a de atividade enzim?tica de glucocerebrosidase foi realizada pelo ensaio fluorim?trico, Cerezyme? e leite da cabra controle (n?o transg?nica) foram usados com controle positivo e negativo, respectivamente. O objetivo deste estudo foi estabelecer protocolos de purifica??o da enzima glucocerebrosidase a partir do leite da cabra clonada e transg?nica e confirmar a atividade enzim?tica, avaliar a express?o da enzima por eletroforese e caracterizar a prote?na recombinante atrav?s da espectrometria de massa.

Submitted by Helmut Patrocinio (hell.kenn@gmail.com) on 2017-11-24T03:31:01Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Cl?udio_Queiroz_TESE.pdf: 3430647 bytes, checksum: cd4fc13e7f5364c58653d2f2c6808b34 (MD5) / Approved for entry into archive by Ismael Pereira (ismael@neuro.ufrn.br) on 2017-11-27T16:14:10Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Cl?udio_Queiroz_TESE.pdf: 3430647 bytes, checksum: cd4fc13e7f5364c58653d2f2c6808b34 (MD5) / Made available in DSpace on 2017-11-27T16:15:20Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Cl?udio_Queiroz_TESE.pdf: 3430647 bytes, checksum: cd4fc13e7f5364c58653d2f2c6808b34 (MD5) Previous issue date: 2005 / As epilepsias do lobo temporal s?o as que apresentam maior refratariedade ao tratamento farmacol?gico e perfazem 2/3 das interven??es cir?rgicas de epilepsia, sendo portanto de grande custo social, econ?mico e psicol?gico. Assim, modelos animais de epilepsia do lobo temporal s?o de grande relev?ncia n?o s? para o entendimento das bases neurais dessa patologia, mas tamb?m para o desenvolvimento de abordagens terap?uticas capazes de evitar a instala??o da doen?a. Esses s?o os objetivos da presente disserta??o de doutorado. Ap?s um evento traum?tico (no caso deste trabalho, um estado de mal epil?ptico), diversas altera??es morfol?gicas e fisiol?gicas acontecem, caracterizando a g?nese da s?ndrome epil?ptico (epileptog?nese). Dentre as altera??es podemos destacar a intensa fosforila??o de prote?nas em res?duos de tirosina e a ativa??o de diferentes segundos mensageiros. Os dois primeiros cap?tulos desta tese descrevem a tentativa de bloquear os processos de epileptog?nese por meio da inibi??o da fosforila??o de res?duos de tirosina atrav?s do tratamento farmacol?gico com inibidores das tirosina cinases, a herbimicina A e o K-252a. O terceiro cap?tulo analisa eletrofisiologicamente o circuito neural do giro denteado em animais que apresentavam uma muta??o em um s?tio inibit?rio da prote?na c?lcio/calmodulina cinase do tipo II (CaMKII). No primeiro cap?tulo, mostramos que o tratamento agudo com herbimicina A (348?M, 5?L, icv), ? capaz de bloquear a potencia??o duradoura (LTP) induzida por um est?mulo tet?nico bem como de atenuar (~40%) a ativa??o neuronal (express?o de c-Fos) decorrente de um estado de mal epil?ptico induzido pela administra??o sist?mica de pilocarpina (SE). Apesar dos significativos efeitos agudos, este tratamento mostrou-se incapaz de atenuar a freq??ncia de crises espont?neas, bem como o padr?o de morte neuronal observado ap?s o estado de mal epil?ptico induzido pela pilocarpina. Entretanto, o tratamento com herbimicina A alterou o padr?o de marca??o de metais pesados (Zn+2) no hilo do giro denteado e na regi?o de CA3 do hipocampo, por?m n?o apresentou efeito sobre o padr?o de brotamento das fibras musgosas observado na camada molecular do giro denteado. No segundo cap?tulo, mostramos que a herbimicina e o K- 252a modificam a atividade epileptiforme induzida pela administra??o intra-hipocampal de ?cido ca?nico, sem alterar o padr?o de morte neuronal. Esses resultados sugerem que o tratamento com inibidores de prote?nas tirosina cinases ? capaz de modificar o padr?o de ativa??o agudo do hipocampo ap?s um est?mulo (i.e., o estado de mal epil?ptico ou a LTP), por?m sem qualquer efeito sobre o processo de epileptog?nese. No terceiro cap?tulo, estudamos a excitabilidade e a plasticidade do giro denteado ? estimula??o da via perfurante (principal afer?ncia da forma??o hipocampal) em animais que apresentam uma CaMKII geneticamente modificada. Essa prote?na uma vez ativada n?o pode ser inibida. A caracteriza??o eletrofisiol?gica demonstrou que esses animais apresentam potenciais de campo evocados no giro denteado aparentemente semelhante aos animais controle (wild-type), por?m sua responsividade a padr?es de estimula??o em salvas e sua plasticidade apresentaram clara altera??o. Essa modifica??o foi caracterizada por uma maior variabilidade nas respostas ? trens de estimula??o (freq??ncias de 1 e 2 Hz) e maior inibi??o do pulso pareado em trens de estimula??o (para pulsos pareados aplicados a freq??ncia de 5 Hz). Al?m disso, conforme j? descrito na literatura, mostramos que a susceptibilidade a atividade epileptiforme depende do padr?o de estimula??o utilizado para os diferentes animais (mutantes vs. wild-type). Assim, utilizando o modelo cl?ssico do abrasamento demonstramos que a muta??o n?o altera a evolu??o da epileptog?nese. Entretanto, ao utilizarmos duas variantes de um padr?o de estimula??o similar ? freq??ncia teta (5Hz, Intermittent vs. Continuous theta-burst stimulations), demonstramos a import?ncia da muta??o na manuten??o da excitabilidade do giro denteado. Esses resultados destacam a import?ncia da CaMKII na atividade epileptiforme al?m de sugerir novas abordagens experimentais (i.e., sensibilidade ? padr?es de estimula??o eletrofisiol?gica) no estudo da epileptog?nese. Em resumo, os resultados apresentados nessa tese contribuem para um melhor entendimento dos fen?menos subjacentes aos processos de plasticidade neuronal e da contribui??o destes para o fen?meno de epileptog?nese, al?m de sugerir / Temporal lobe epilepsies are highly refractory to pharmacological treatment. Up to 70% of these patients undergo chirurgical resection of temporal region, procedure with important consequences for the social, economic and psychological spheres. Experimental animal models that mimic temporal lobe epilepsy provide an insightful approach to study the neural basis of epilepsy as well as create opportunities to test promising therapeutic drugs. The present thesis tests the antiepileptogenic activity of two protein tyrosine kinase inhibitors and the relevance of Ca+2/calmodulin kinase type II (CaMKII) mutants. Multiples morphological and physiological alterations take place after a traumatic brain injury (in this thesis, the status epilepticus) leading the animal to an epileptic conditions. During this period, the epileptogenesis process, there is strong tyrsine phosphorylation with the activation of many second messengers. The first two chapters of the thesis describe experiments in which herbimycin A and K-252a, two protein tyrosine kinase inhibitors, were used to attenuate synaptic plasticity and epileptogenesis. The third chapter, the dentate gyrus network was studied after angular bundle stimulation in animals presenting one punctual mutation at the autoinhibitory phosphorylation site of the CaMKII. In the first chapter, we showed that one single herbimycin A injection (348?M, 5?L, icv) was able to attenuate long-term potentiation (LTP) in the commissural CA3 neurons and also, to decrease status epilepticus- (SE-) induced neuronal activation (c-Fos expression) in almost 40%. Although markedly acute effects, the present herbimycin A treatment was not able to diminish spontaneous seizure frequency, cell death or aberrant mossy fiber sprouting observed after the pilocarpine-induced SE. Curiously, herbimycin-treated animals presented decreased neo-Timm staining in the hilus and CA3 region despite the epileptic condition. In the second chapter, we confirmed the ability of protein tyrosine kinase inhibitors to decrease SE-induced neuronal activation. Herbimycin A icv treatment altered the kainic acid-induced epileptiform profile in EEG recordings. Cell death pattern was not altered by any pharmacological treatment. These results suggest that protein tyrosine kinase inhibitiors are able to modify the acute neuronal activation and plasticity (ictogenesis or LTP) but is ineffective in attenuating the epileptogenesis process. In the third chapter, we studied the dentate gyrus excitability and plasticity after angular bundle stimulation in CaMKII mutant animals. Once in its self-sustained mode, this mutation does not allow the reduction of the catalytic activity of the kinase. These animals present normal electrophysiological profiles (similar to wild-type animals) but with reduced amplitude. Shortterm plasticity was clearly altered. Mutant animals presented increased variability in the responses to trains of stimulation at 1 and 2 Hz, and at at 5Hz stronger paired-pulse inhibition. Accordingly to the literature, we also showed that the epileptiform susceptibility depends on the stimulation pattern used in both animals (mutants vs. wild-type). Thus, although the mutation did not altered the behavior and the electrographic kindling evolution, we showed that mutant animals were prone to afterdischarges when stimulate by an intermittent theta-burst stimulation. On the other hand, the same animals needed more bursts to induce afterdischarges when the stimulation was set in the continuos mode. Taken together, the present results contribute to a better understanding of the protein tyrosine kinase and CaMKII function in neuronal plasticity underlying the epileptogenesis process and sum efforts in searching for a clinic antiepileptogenic drug.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-09T00:26:04Z No. of bitstreams: 1 StefanoPupeJohann_TESE.pdf: 36592987 bytes, checksum: f44357f3110776cf9f7cb628b2d65a95 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-09T00:36:11Z (GMT) No. of bitstreams: 1 StefanoPupeJohann_TESE.pdf: 36592987 bytes, checksum: f44357f3110776cf9f7cb628b2d65a95 (MD5) / Made available in DSpace on 2016-04-09T00:36:12Z (GMT). No. of bitstreams: 1 StefanoPupeJohann_TESE.pdf: 36592987 bytes, checksum: f44357f3110776cf9f7cb628b2d65a95 (MD5) Previous issue date: 2015-03-20 / Os neurocientistas tem uma diversidade de perspectivas com as quais podem classificar diferentes partes do c?rebro. Com o surgimento de t?cnicas baseadas na gen?tica, como a optogen?tica, se torna cada vez mais importante identificar se um grupo de c?lulas, definidas atrav?s de morfologia, fun??o ou posi??o anat?mica possui um padr?o caracter?stico de express?o de um ou mais promotores gen?ticos. Isso permitiria melhores formas de estudar essas popula??es de neur?nios definidas geneticamente. Neste trabalho, eu apresento uma discuss?o te?rica e tr?s estudos experimentais nos quais essa foi a principal quest?o sendo abordada. O Estudo I discute as quest?es envolvidas em selecionar um promotor para estudar estruturas e subpopula??es na ?rea Tegmental Ventral. O Estudo II caracteriza uma subpopula??o de c?lulas na ?rea Tegmental Ventral que compartilha a express?o de um promotor, que ? anatomicamente muito restrita, e que induz avers?o quando estimulada. O Estudo II utiliza uma estrat?gia similar para investigar a subpopula??o no n?cleo subtal?mico que expressa PITX2 e VGLUT2 que, quando inativada, causa hiperlocomo??o. O Estudo IV explora o fato de que um grupo de c?lulas previamente identificadas no Hipocampo Ventral expressa CHRNA2, e indica que essa subpopula??o pode ser necess?ria e suficiente para o estabelecimento do ritmo teta (2-8 Hz) no Hipocampo Ventral de camundongos anestesiados. Todos esses estudos foram guiados pela mesma estrat?gia de identificar um promotor gen?tico capaz de permitir o controle de uma popula??o de neur?nios identificada geneticamente, e eles demonstram as diferentes formas em que essa abordagem pode generar novas descobertas. / Neuroscientists have a variety of perspectives with which to classify different parts of the brain. With the rise of genetic-based techniques such as optogenetics, it is increasingly important to identify whether a group of cells, defined by morphology, function or anatomical location possesses a distinct pattern of expression of one or more genetic promoters. This would allow for better ways to study of these genetically defined subpopulations of neurons. In this work, I present a theoretical discussion and threeexperimental studies in which this was the main question being addressed. Paper I discusses the issues involved in selecting a promoter to study structures and subpopulations in the Ventral Tegmental Area. Paper II characterizes a subpopulation of cells in the Ventral Tegmental Area that shares the expression of a promoter and is anatomically very restricted, and induces aversion when stimulated. Paper III utilizes a similar strategy to investigate a subpopulation in the subthalamic nucleus that expresses PITX2 and VGLUT2 which, when inactivated, causes hyperlocomotion. Paper IV exploits the fact that a previously identified group of cells in the ventral hippocampus expresses CHRNA2, and indicates that this population may be necessary and sufficient for the establishment of the theta rhythm (2-8 Hz) in the Local Field Potential of anesthetized mice. All of these studies were guided by the same strategy of characterizing and studying the role of a genetically defined subpopulation of cells, and they demonstrate the different ways in which this approach can generate new discoveries.