Contrairement aux mammifères adultes, la rétine des amphibiens possède la particularité de croître durant toute la vie de l'animal grâce à l'activité continue d'une population de cellules souches localisée au sein d'une niche bien délimitée, la zone marginale ciliaire (ZMC). Ce modèle offre ainsi la possibilité d'étudier in vivo les mécanismes moléculaires à l'origine du maintien et de la prolifération des cellules souches neurales à des stades post-embryonnaires. Dans ce but, l'identification et la caractérisation des différentes voies de signalisation présentes au sein de la niche biologique des cellules souches rétiniennes est une première étape indispensable. Mon projet de thèse a été divisé en deux objectifs principaux: l'étude des interactions entre les voies Wnt et Hedgehog au sein de la ZMC chez le xénope et la réalisation de l'étude fonctionnelle de Yap, l'effecteur principal de la voie de signalisation Hippo dans ce modèle. Par des approches génétiques et pharmacologiques, la première partie de ce projet a permis de mettre en évidence un antagonisme inattendu entre les signaux Wnt et Hedgehog au sein de la ZMC qui régule l'activité proliférative des cellules souches et des progéniteurs rétiniens. Ce travail nous a conduit à proposer un modèle dans lequel ces deux voies réguleraient la balance prolifération/différenciation dans la rétine post-embryonnaire. Dans un deuxième temps, les expériences de gain et de perte de fonction du gène Yap ont montré que ce dernier joue un rôle essentiel dans la régulation du programme temporel de la phase de réplication de l'ADN des cellules souches rétiniennes. En effet, l'inhibition de Yap entraîne une importante réduction de la durée de la phase S du cycle cellulaire associée à une instabilité génomique. Une surexpression de c-Myc et de la voie p53-p21 semble impliquée dans ce phénotype. Nos travaux nous ont également permis d'identifier un nouveau partenaire de YAP, le facteur de transcription PKNOX1. L'ensemble de ces données nous a ainsi conduit à proposer un modèle selon lequel le complexe YAP/PKNOX1 pourrait être nécessaire au bon déroulement de la phase de réplication des cellules souches, indispensable à la maintenance de l'intégrité du génome de ces cellules et de leur descendance. / In contrast to the adult mammals, the retina of amphibians shows continuous growth during adulthood through active neural stem cells localized in the defined niche called ciliary marginal zone (CMZ). This model offers an exceptional tool to study in vivo the molecular mechanisms involved in the maintenance and proliferation of neural stem cells during post-embryonic stages. In this order, the identification and the characterization of the signaling pathways acting in biological retinal stem cell niche is an essential step.My PhD research was divided in two main parts: the study of the interaction between the Wnt and Hedgehog pathways within the CMZ and the functional study of Yap, the downstream effector of the Hippo pathway in this model. By using genetic and pharmacological tools, the first part of this project demonstrated an unexpected antagonism between the Wnt and the Hedgehog signaling in the CMZ that regulates proliferative activity of retinal stem and progenitor cells. In this article, we propose a model in which an antagonistic interplay of Wnt and Hedgehog pathways may regulate the balance proliferation/differentiation in the post-embryonic retina. Second, gain and loss of function experiments of Yap have shown that this factor plays a key role in the regulation of temporal replication of DNA retinal stem cells. Indeed, inhibition of Yap leads to strong reduction of the S-phase length during the cell cycle associated with genomic instability. c-Myc and p53-p21 overactivation seems to be involved in this phenotype. This work also allowed us to identify a novel YAP partner, the transcriptional factor PKNOX1. We indeed propose a model in which the YAP/PKNOX1 complex may be required for the successful convening of the replication phase on stem cells, essential for the maintenance of genome integrity on the cells and their progeny.

Ng, Tsz Kin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 151-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Hypothesis 1. MfERG is useful in the assessment of retinal dysfunction in patients following photodynamic therapy with verteporfin. / Hypothesis 2. MfERG is useful in the evaluation of retinal dysfunction in patients following safety-enhanced photodynamic therapy using half-dose verteporfin. / Hypothesis 3. MfERG is useful in the assessment of retinal dysfunction in patients receiving hydroxychloroquine therapy. / Hypothesis 4. MfERG findings correlate to certain extent with the visual field findings in patients receiving hydroxychloroquine therapy and mfERG might be more sensitive compared with 10-2 visual field testing in assessing retinal dysfunction associated with hydroxychloroquine therapy. / Hypothesis 5. MfERG is useful In the assessment of retinal dysfunction associated with intraoperative application of indocyaniue green for internal limiting membrane staining in epiretinal membrane surgery. / Multifocal electroretinography (mfERG) is an investigation which can provide objective assessment of retinal function, In contrast with full-field electroretinography which measures the mass electrical activity of the entire retina, mfERG allows simultaneous measurements of multiple retinal responses from the macula. / Several studies have demonstrated that mfERG might be useful in assessing retinal dysfunction caused by pharmacological agents and following laser therapy. This thesis aims to demonstrate the application of mfERG in the evaluation of retinal dysfunction associated with various ocular or systemic pharmacological agents. Three treatment modalities including photodynamic therapy with verteporfin, systemic use of hydroxychloroquine, and intraoperative application of indocyanine green dye were chosen for evaluation. These pharmacological agents were selected as they are associated with potential retinal dysfunction and are commonly encountered in the ophthalmic clinical practice. The thesis examines the following hypotheses: / Summary of studies arising from the thesis. Based on the findings from the above studies, it was demonstrated that mfERG can objectively evaluate the retinal dysfunction caused by a variety of ocular or systemic pharmacological agents. These included PDT with verteporfin, systemic therapy with hydroxychloroquine, as well as intraoperative application of ICG dye for ILM staining. The use of mfERG has enhanced the understanding of the underlying pathophysiology of drug-associated retinal toxicity. As mfERG becomes more widely available, its application will provide a valuable option for clinicians to assess toxic retinopathy objectively and enable safer administration of treatment to minimize potential retinal toxicity. (Abstract shortened by UMI.) / by Lai Yuk Yau, Timothy. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0951. / Thesis (M.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 161-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

Submitted by Irvana Coutinho (irvana@ufpa.br) on 2012-06-08T16:17:12Z No. of bitstreams: 2 Dissertacao_MELLINAValoresNormativosEletro.pdf: 4797106 bytes, checksum: b86a051a620a524491d2f888ddfea0be (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Irvana Coutinho(irvana@ufpa.br) on 2012-06-08T16:17:53Z (GMT) No. of bitstreams: 2 Dissertacao_MELLINAValoresNormativosEletro.pdf: 4797106 bytes, checksum: b86a051a620a524491d2f888ddfea0be (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2012-06-08T16:17:53Z (GMT). No. of bitstreams: 2 Dissertacao_MELLINAValoresNormativosEletro.pdf: 4797106 bytes, checksum: b86a051a620a524491d2f888ddfea0be (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2012 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / FINEP - Financiadora de Estudos e Projetos / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Muitos laboratórios de eletrofisiologia visual não possuem seus próprios valores de normalidade para o eletrorretinograma de campo total. Isto prejudica a confiabilidade dos diagnósticos de diversas doenças que afetam as vias visuais. Desta forma, o objetivo deste trabalho foi estabelecer os valores normativos para o teste Eletrorretinograma de Campo Total para o Laboratório de Neurologia Tropical (LNT) da Universidade Federal do Pará (UFPA). Realizaram o eletrorretinograma 68 indivíduos saudáveis e sem queixas visuais divididos em três grupos de acordo com a faixa etária: 36 indivíduos pertenceram ao grupo 1 (entre 17 e 30 anos), 21 indivíduos ao grupo 2 (entre 31 e 45 anos) e 11 indivíduos ao grupo 3 (entre 46 e 60 anos). O protocolo de realização do teste seguiu as recomendações da ISCEV, com a utilização de seis tipos de estimulação. Quatro após adaptação escotópica e estimulação com intensidades de: 0,01 cd.s/m2 (resposta de bastonetes), 3,0 cd.s/m2 (resposta mista de cones e bastonetes e potenciais oscilatórios) e 10,0 cd.s/m2 (resposta mista adicional). Dois após adaptação fotópica em fundo de 30 cd/m2: 3,0 cd.s/m2 (resposta de cones e Flicker 30Hz). Para a análise dos resultados foram calculados os valores de amplitude e tempo implícito das ondas a e b obtidas em resposta a cada um dos seis tipos de estimulação utilizados. Estes valores foram descritos estatisticamente através da mediana, intervalos de confiança, 1º e 3º quartis, coeficiente de variação, média, desvio padrão e valores mínimos e máximos. Os grupos de maior faixa etária apresentaram menores valores de amplitude e atraso no tempo implícito. A utilização da transformada wavelet permitiu a melhor visualização das ondas sem alteração de amplitude e tempo implícito. Portanto, os valores normativos obtidos podem servir como parâmetros de normalidade confiáveis para auxiliar o diagnóstico de doenças retinianas. / Many visual electrophysiology laboratories don’t have their own normal values for full-field electroretinogram. This impairs the reliability of the diagnosis of various diseases affecting the visual pathways. Thus, the purpose of this study was to establish normative values for the full-field electroretinogram to the Laboratory of Tropical Neurology (LNT) of the Federal University of Pará (UFPA). Were tested using the electroretinogram 68 healthy subjects without visual complaints, divided into three groups according to age group: 36 subjects belonged to group 1 (17 to 30 years), 21 subjects in group 2 (31 to 45 years) and 11 individuals in group 3 (46 to 60 years). Six types of stimulus that follow ISCEV standards were presented. Four dark-adapted: 0.01 cd.s/m2 (rod response), 3.0 cd.s/m2 (mixed response and oscillatory potentials) and 10.0 cd.s/m2 (mixed additional response). Two light-adapted, 3.0 cd.s/m2 (Cone response and Flicker 30Hz), with 30 cd/m2 background adaptation. For analysis, a-wave and b-wave amplitude and implicit times values were calculated. These values were statistically described using the following values: median, confidence interval, 1st and 3rd quartiles, coefficient of variation, mean, standard deviation and minimum and maximum values. The older age groups had lower amplitude and delayed implicit time. Wavelet transform allowed better visualization of waves without change of amplitude and implicit time. Therefore, the normative values obtained can serve as reliable parameters of normality to assist the diagnosis of retinal diseases.

Submitted by Ana Rosa Silva (arosa@ufpa.br) on 2012-07-23T15:40:16Z No. of bitstreams: 2 Dissertacao_CaracterizacaoCineticaCaptacao.pdf: 1215404 bytes, checksum: 148a3dc60f186dfc13a547ed24bfbf3e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-07-23T15:41:13Z (GMT) No. of bitstreams: 2 Dissertacao_CaracterizacaoCineticaCaptacao.pdf: 1215404 bytes, checksum: 148a3dc60f186dfc13a547ed24bfbf3e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2012-07-23T15:41:13Z (GMT). No. of bitstreams: 2 Dissertacao_CaracterizacaoCineticaCaptacao.pdf: 1215404 bytes, checksum: 148a3dc60f186dfc13a547ed24bfbf3e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2011-08 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente estudo descreve um método eficiente e simples utilizando cromatografia líquida de alta eficiência (CLAE) acoplada a detector de fluorescência para determinação dos parâmetros cinéticos da captação de glutamato (glu) no sistema nervoso central (SNC). O tecido retiniano embrionário de ave com sete dias de desenvolvimento foi incubado com concentrações conhecidas de glu (50-500 μM) por dez minutos. Os níveis do aminoácido derivado a partir de ortoftaldeído (OPA) no meio de incubação foram mensurados. Após avaliar a diferença entre a concentração de glu inicial e a final no meio, foi determinada a saturação do mecanismo de captação (Km = 8,2 e Vmax = 9,8 nmol/mg proteína/minuto). Estas determinações foram dependentes e independentes de sódio e temperatura, indicando que o mecanismo que regula a diminuição dos níveis de glu no SNC, é a captação via transportadores de alta afinidade. Além disso, o cloreto de zinco (ZnCl) (um inibidor do transportador glu/aspartato) foi utilizado em diversas concentrações e evocou diminuição da captação de glu. Com isto, destaca-se a elevada aplicabilidade desta metodologia. Além deste trabalho caracterizar metodologia alternativa para avaliar captação de glu no SNC usando CLAE, também pode ser importante ferramenta para estudos relacionados à caracterização do transporte do neurotransmissor durante injúrias no SNC. / The present study describes a simple and efficient method utilizing high performance liquid chromatography (HPLC) coupled to fluorescence detection for the determination of kinetic parameters of glutamate uptake in nervous tissue. Retinal tissue obtained from 7-day-old chicks was incubated to known concentrations of glutamate (50-2000 μM) for 10 minutes, and the levels of the o-phtaldehyde (OPA)-derivatized neurotransmitter in the incubation medium were measured. By assessing the difference between initial and final concentrations of glutamate in the medium, a saturable uptake mechanism was characterized (Km = 8,2 e Vmax = 9,8 nmol/mg protein/minute). This measure was largely sodium- and temperature-dependent, strongly supporting that the mechanism for concentration decrements is indeed uptake by high-affinity transporters. Added to this, our results also demonstrated that zinc chloride (an inhibitor of glutamate/aspartate transporters) evoked a concentration dependent decrease in the glutamate uptake, demonstrating an elevate specificity of our methodology. Overall, the present work characterizes an alternative methodology to evaluate glutamate uptake in nervous tissue using HPLC. This approach could be an important tool for studies associated to the characterization of glutamate transport related with central nervous system injury

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2014-01-22T18:12:10Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoMetodoFotooxidacao.pdf: 132738540 bytes, checksum: 24543aac4b55976eda3a952ad9d57a98 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-01-24T14:33:10Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoMetodoFotooxidacao.pdf: 132738540 bytes, checksum: 24543aac4b55976eda3a952ad9d57a98 (MD5) / Made available in DSpace on 2014-01-24T14:33:11Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoMetodoFotooxidacao.pdf: 132738540 bytes, checksum: 24543aac4b55976eda3a952ad9d57a98 (MD5) Previous issue date: 2013 / A análise da morfologia celular é um aspecto crucial da neurobiologia, pois a relação entre forma e função pode definir os processos fisiológicos na saúde e na doença. Um dos principais métodos para avaliar a morfologia celular é por meio da fotooxidação de marcadores fluorescentes intracelulares, dentre os quais se tem o percloreto de 1,1’-dioctadecil-3,3,3’,3’-tetrametil-indocarbocianina (DiI), uma carbocianina lipofílica que pode marcar células vivas ou fixadas. O DiI foi escolhido para este trabalho devido, dentre outros fatores, à sua importante utilização para estudos de morfologia celular. Como modelo para avaliar a qualidade da fotooxidação do aparelho construído para essa finalidade, elegeu-se as células horizontais da retina humana com o intuito de prosseguir com estudo morfométrico posterior dessas células, tendo em vista a escassez de trabalhos com essa abordagem em retina humana. Assim, este estudo teve como objetivo avaliar a qualidade do método de fotooxidação do DiI através de LED e utilizando como modelo células horizontais da retina humana. O material foi obtido do Banco de Olhos do Hospital Ophir Loyola e, em sequência dissecado, marcado com cristais de DiI e fotooxidado com o aparelho de LED. As imagens que resultaram do novo método de iluminação para fotooxidação de traçador fluorescente apresentou elevada qualidade de detalhes da morfologia neuronal, similares aos resultados obtidos em reações de fotoconversão convencional com microscópio, o que permite concluir que o aparelho mantém a eficiência da fotooxidação por revelar detalhes finos da morfologia celular, inclusive com as vantagens de processar áreas maiores de tecido e considerável redução de custo por dispensar o emprego de microscópio para o processo. / The analysis of cell morphology is a crucial aspect of neurobiology, since the relationship between form and function can define the physiological processes in health and disease. One of the main methods to evaluate cell morphology is through photooxidation of intracellular fluorescent markers, among which is the perchlorate of 1,1 '-dioctadecyl-3, 3,3', 3'-tetramethyl-indocarbocianina (DiI) a lipophilic carbocyanine that can make living or fixed cells. The DiI was chosen for this work because, among other factors, it is used for important studies of cell morphology. As a model for evaluating the quality of photooxidation of the apparatus constructed for this purpose, horizontal cells of human retina were elected in order to proceed with morphometric study these cells further, in view of the lack of studies with this approach in human retina. This study evaluated the quality of the DiI method photooxidation via LED and used the horizontal cells of the human retina as a model. The material was obtained from the Eye Bank of Ophir Loyola Hospital and sequence dissected, labeled with DiI crystals and photooxided with the LED device. The images resulting from the new method of lighting fluorescent tracer showed photooxidation of high-quality detailed neuronal morphology, similar to the results obtained in reactions of convencional photoconversion with a microscope, which indicates that the device maintains the efficiency of the photooxidation for revealing fine details of cellular morphology, including the advantages of larger areas of tissue processing and a considerable cost reduction for the use of microscopy dispensed into the process.

Submitted by DIOGO SOUSA ZANONI (zanoni_19@hotmail.com) on 2019-01-31T11:46:22Z No. of bitstreams: 1 Defesa Doutorado - entrega Pós.pdf: 2160080 bytes, checksum: a016ec7f20ec85dc0433f4452821780e (MD5) / Approved for entry into archive by Ana Lucia de Grava Kempinas (algkempinas@fca.unesp.br) on 2019-01-31T19:07:54Z (GMT) No. of bitstreams: 1 zanoni_ds_dr_botfmvz.pdf: 2160080 bytes, checksum: a016ec7f20ec85dc0433f4452821780e (MD5) / Made available in DSpace on 2019-01-31T19:07:54Z (GMT). No. of bitstreams: 1 zanoni_ds_dr_botfmvz.pdf: 2160080 bytes, checksum: a016ec7f20ec85dc0433f4452821780e (MD5) Previous issue date: 2018-12-11 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O glaucoma é a principal causa global de cegueira irreversível e o número de pessoas com glaucoma em todo o mundo aumentará para 111,8 milhões em 2040, mesmo com os tratamentos vigentes. Deste modo, enseja uma necessidade de desenvolver terapias neuroprotetoras que possam ser usadas para reduzir os efeitos perniciosos do glaucoma, como a morte de células ganglionares da retina (CGR). Avanços na compreensão da fisiopatologia do glaucoma é um fator chave na compreensão da patogênese da neuropatia glaucomatosa. Neste contexto a isquemia retiniana desempenha um papel central em várias doenças da retina. A patogênese da isquemia retiniana envolve alterações temporais da morfologia e morfometria da retina assim como mudanças na expressão gênica e protéica. O Citrato de Sildenafila (SC) mostrou efeito protetor nos modelos de isquemia/reperfusão (I/R) com efeitos neuroprotetores. Contudo, a administração oral de CS, em humanos, encontra inúmeros efeitos colaterais, tais como redução da pressão arterial, dores de cabeça, rubor e congestão nasal são concomitantes, assim como pacientes com moderada a grave doença cardiovascular ou aqueles submetidos a terapia a base de nitrato apresentam riscos secundários aumentados para efeitos cardiovasculares adversos. Deste modo, propomos que a administração tópica de CS pode ser uma alternativa à via oral e também ser igualmente neuroprotetor no glaucoma e podem minimizar os riscos indesejados no uso sistêmico desse fármaco, além de oferecer uma nova abordagem para a intervenção terapêutica na patogênese da neuropatia glaucomatosa. Caso nossos resultados sejam positivos, em um estudo tão abrangente, pode levar a um ensaio clínico deste fármaco seguro e promissor em pacientes com glaucoma. / Glaucoma is the leading global cause of irreversible blindness and the number of people with glaucoma worldwide will increase to 111.8 million by 2040, even with current treatments. Thus, there is a need to develop neuroprotective therapies that can be used to reduce the deleterious effects of glaucoma, such as retinal ganglion cell death (RBC). Advances in understanding the pathophysiology of glaucoma are a key factor in understanding the pathogenesis of glaucomatous neuropathy. In this context, retinal ischemia plays a central role in various diseases of the retina. The pathogenesis of retinal ischemia involves temporal changes in retinal morphology and morphometry as well as changes in gene and protein expression. Sildenafil Citrate (SC) showed protective effect in the ischemia / reperfusion (I / R) models with neuroprotective effects. However, oral administration of CS in humans finds numerous side effects such as reduced blood pressure, headaches, flushing and nasal congestion are concomitant, as well as patients with moderate to severe cardiovascular disease or those undergoing nitrate therapy have increased secondary risks for adverse cardiovascular effects. Thus, we propose that the eye drops administration of CS may be an alternative to the oral route and also be equally neuroprotective in glaucoma and may minimize the undesirable risks in the systemic use of this drug, as well as offer a new approach for the therapeutic intervention in the pathogenesis of neuropathy glaucomatous. If our results are positive, in such a comprehensive study, it may lead to a clinical trial of this safe and promising drug in patients with glaucoma. / 142471\2015-1

Submitted by DIOGO SOUSA ZANONI (zanoni_19@hotmail.com) on 2019-02-01T17:03:15Z No. of bitstreams: 1 Defesa Doutorado - entrega Pós.pdf: 2159159 bytes, checksum: c32db30aea42543516cc0f930e17164d (MD5) / Approved for entry into archive by Ana Lucia de Grava Kempinas (algkempinas@fca.unesp.br) on 2019-02-01T18:10:48Z (GMT) No. of bitstreams: 1 zanoni_ds_dr_botfmvz.pdf: 2159159 bytes, checksum: c32db30aea42543516cc0f930e17164d (MD5) / Made available in DSpace on 2019-02-01T18:10:48Z (GMT). No. of bitstreams: 1 zanoni_ds_dr_botfmvz.pdf: 2159159 bytes, checksum: c32db30aea42543516cc0f930e17164d (MD5) Previous issue date: 2018-12-11 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O glaucoma é a principal causa global de cegueira irreversível e o número de pessoas com glaucoma em todo o mundo aumentará para 111,8 milhões em 2040, mesmo com os tratamentos vigentes. Deste modo, enseja uma necessidade de desenvolver terapias neuroprotetoras que possam ser usadas para reduzir os efeitos perniciosos do glaucoma, como a morte de células ganglionares da retina (CGR). Avanços na compreensão da fisiopatologia do glaucoma é um fator chave na compreensão da patogênese da neuropatia glaucomatosa. Neste contexto a isquemia retiniana desempenha um papel central em várias doenças da retina. A patogênese da isquemia retiniana envolve alterações temporais da morfologia e morfometria da retina assim como mudanças na expressão gênica e protéica. O Citrato de Sildenafila (SC) mostrou efeito protetor nos modelos de isquemia/reperfusão (I/R) com efeitos neuroprotetores. Contudo, a administração oral de CS, em humanos, encontra inúmeros efeitos colaterais, tais como redução da pressão arterial, dores de cabeça, rubor e congestão nasal são concomitantes, / Glaucoma is the leading global cause of irreversible blindness and the number of people with glaucoma worldwide will increase to 111.8 million by 2040, even with current treatments. Thus, there is a need to develop neuroprotective therapies that can be used to reduce the deleterious effects of glaucoma, such as retinal ganglion cell death (RBC). Advances in understanding the pathophysiology of glaucoma are a key factor in understanding the pathogenesis of glaucomatous neuropathy. In this context, retinal ischemia plays a central role in various diseases of the retina. The pathogenesis of retinal ischemia involves temporal changes in retinal morphology and morphometry as well as changes in gene and protein expression. Sildenafil Citrate (SC) showed protective effect in the ischemia / reperfusion (I / R) models with neuroprotective effects. However, oral administration of CS in humans finds numerous side effects such as reduced blood pressure, headaches, flushing and nasal congestion are concomitant, as well as patients with moderate to severe cardiovascular disease or those undergoing nitrate therapy have increased secondary risks for adverse cardiovascular effects. Thus, we propose that the eye drops administration of CS may be an alternative to the oral route and also be equally neuroprotective in glaucoma and may minimize the undesirable risks in the systemic use of this drug, as well as offer a new approach for the therapeutic intervention in the pathogenesis of neuropathy glaucomatous. If our results are positive, in such a comprehensive study, it may lead to a clinical trial of this safe and promising drug in patients with glaucoma. / 142471\2015-1

Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-09-21T17:57:32Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EfeitosFatorCrescimento.pdf: 20205989 bytes, checksum: 44c1e41ad424a7d873f2cf7feb0e9dad (MD5) / Rejected by Edisangela Bastos (edisangela@ufpa.br), reason: on 2017-10-10T17:00:41Z (GMT) / Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-10-17T18:31:48Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EfeitosFatorCrescimento.pdf: 20205989 bytes, checksum: 44c1e41ad424a7d873f2cf7feb0e9dad (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-11-24T14:54:19Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EfeitosFatorCrescimento.pdf: 20205989 bytes, checksum: 44c1e41ad424a7d873f2cf7feb0e9dad (MD5) / Made available in DSpace on 2017-11-24T14:54:19Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EfeitosFatorCrescimento.pdf: 20205989 bytes, checksum: 44c1e41ad424a7d873f2cf7feb0e9dad (MD5) Previous issue date: 2011-04-20 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / O fator de crescimento do nervo (NGF) e o glutamato têm papéis bem definidos durante o desenvolvimento do sistema nervoso e podem atuar de maneira sinérgica para induzir a sobrevivência neuronal. O NGF promove a liberação de glutamato em diferentes áreas corticais, mas pouco é conhecido sobre a regulação da liberação de glutamato por NGF na retina. Por este motivo, investigamos se NGF poderia modular a liberação de glutamato no tecido retiniano durante o seu pico de atividade neurotrófica (E10-E12). Além disso, estudamos os mecanismos de liberação de glutamato com relação a sua dependência de Ca2+ extracelular e a participação de transportadores dependentes e independentes de Na+. Uma vez que, níveis elevados de glutamato estão implicados na ocorrência de estresse oxidativo, investigamos também os efeitos de NGF sobre a liberação de compostos tióis. Para isto, tecidos retinianos íntegros de embrião de galinha (E11) foram incubados com NGF (10, 50, 100 ng/ml) por diferentes períodos de incubação (15, 30, 120). Os níveis extracelulares de glutamato e tióis foram medidos por cromatografia líquida de alta eficácia (CLAE) e ensaio colorimétrico, respectivamente. Observamos que NGF aumenta rapidamente a liberação basal de glutamato e também pode induzir a liberação de tióis em um tempo maior de incubação. De maneira interessante, o aumento dos níveis extracelulares de glutamato induzido por NGF foi revertido em meio sem Ca2+ somente em retinas tratadas por 15 min. Retinas que foram incubadas com NGF por 30 min apresentaram liberação de glutamato independente de Ca2+. Dado que, a liberação de glutamato e tióis induzida por NGF não foi bloqueada por Zn2+ e ocorreu na ausência de Na+, sugerimos o possível envolvimento do sistema independente de Na+, o trocador Xc- em ambos os processos. O aumento de tióis extracelulares induzido por NGF poderia representar um importante mecanismo protetor, possibilitando que os neurônios mantenham seu estado redox durante o desenvolvimento. / Nerve growth factor (NGF) belongs to the neurotrophin family and induces its effects through activation of two distinct receptor types. NGF was first described by Rita Levi-Montalcini and collaborators as an important factor involved in nerve differentiation and survival. Another role for NGF has been established in neurotransmitter release in the hippocampus, developing visual cortex and cerebellar neuron. However, this phenomenon has not been demonstrated in retina to date. We therefore investigated whether NGF can modulate the glutamate release in the retinal tissue at its peak of the neurotrophic activity (E10-E12). In addition this, we aimed to study the mechanisms of this effect about its dependence on extracellular Ca2+ and participation of Na+-dependent and Na+-independent glutamate transporters. Since high levels of glutamate signalization have been implicated in the oxidative stress, we also investigated the effects of NGF on the thiols compounds. We used intact retinal tissue from chicken embryos (E11) incubated with NGF (10, 50, 100 ng/ml) for different periods (15, 30, 45, 60, 120 min). Extracellular glutamate and thiols content was measured by HPLC methods and colorimetric assay, respectively. We found that NGF rapidly enhances the release of basal glutamate and it can induce thiol release in a more prolonged time of incubation, as well. Interestingly, the NGF-induced increase in the extracellular levels of glutamate was blocked by Ca2+-free medium only in retina treated for 15 min. Retina incubated for 30 min showed a non-vesicular NGF-induced glutamate release. Since glutamate and thiol release was not blocked by Zn2+, we suggested the possible involvement of system Xc- in both processes.NGF-induced increase in the extracellular thiol could be an important protective mechanism enabling retinal neurons to maintain their redox status during development.

The central nervous system is a complex, yet well-organised, often laminated, tissue. This robust organisation is evident in the architecture of the retina: consisting of 5 different neuronal types organised into distinct layers: Retinal Ganglion Cell (RGC), Amacrine Cell (AC), Bipolar Cell (BP), Horizontal Cell (HC) and Photoreceptor cell (PR) layers. This remarkable organisation is evolutionarily conserved in vertebrates, yet little is known about the mechanisms by which these cells form the correct layers. Live imaging has revealed overlapping periods of birth and extensive inter-digitation followed by cells sorting out into their appropriate positions, suggesting cell-cell interactions are important. To investigate possible cellular and molecular mechanisms responsible for the establishment of the tissue architecture I developed an organoid culture system for zebrafish retinal cells. To identify the cells in culture I used a Spectrum of Fates fish line which is a multiply transgenic line in which each retinal cell type can be identified based on expression of a combination of fluorescently tagged cell fate markers. The development of the protocol by which I cultured the cells and observed their cell-cell interactions involved establishing the best methods to dissociate and culture zebrafish retinal cells in a non-adhesive environment, then imaging the resulting reaggregates to examine the position of the different retinal cell types. By doing this I observed their inherent self-organising properties, in the absence of extrinsic cues or scaffolds. These cells appeared to be arranged in an inside-out layering, although all cell types are layered in the same relative order as they are in vivo. To analyse the organization in these aggregates I developed a Matlab script in collaboration with Leila Muresan which analyses the relative positioning of cells in concentric rings from the periphery to the centre of the aggregates according to the cell fate-tagged fluorescent markers. The script then fits this data as an empirical cumulative distribution function for different groups of cells to determine how spatially distinct populations of cells are. This gave me my measure of organisation. I then investigated the cell-cell interactions involved in this self-organisation by genetically or pharmacologically removing individual cell types and assaying the resulting organisation of the reaggregated, cell-type deficient, retinal organoids. I revealed that Müller Glia are important for retinal cell self-organisation. I also investigated the role of Retinal Pigment Epithelial (RPE) cells and Retinal Ganglion Cells and found they had no impact on the ability of the remaining cell types to organize. I began to investigate the role of Amacrine Cells but found that retinas void of ACs were susceptible to disaggregating in our dissection setup, preventing me from collecting the material needed for culture. I also investigated the role of candidate molecules in this system and revealed that R-Cognin is critical for retinal cells to reaggregate. Not only can I remove cells or molecules from the system, but I show how it can also be manipulated to replace molecules of interest such as laminin, by coating beads with the substance of choice and placing it amongst the cells to see if their organisational behaviour is affected. In summary, I have developed a system which provides a simple and easy platform to manipulate in various ways to help us potentially reveal some of the important players in neuronal patterning.