Orientador: Mônica Barbosa de Melo / Dissertação (mestrado - Universidade Estadual de Campinas, Intituo de Biologia / Made available in DSpace on 2018-08-20T18:09:19Z (GMT). No. of bitstreams: 1 Braghini_CarolinaAyumi_M.pdf: 2316733 bytes, checksum: 1031a8585bbf2541b2b39db621d9f45d (MD5) Previous issue date: 2012 / Resumo: O glaucoma compreende um grupo heterogêneo de neuropatias ópticas, caracterizadas pela escavação do disco óptico e perda progressiva do campo visual, representando uma das maiores causas mundiais de perda irreversível da visão. Em 1997, o gene Myocilin (MYOC) foi descoberto, e mutações neste gene foram envolvidas no desenvolvimento do glaucoma primário de ângulo aberto (GPAA) e do GPAA do tipo juvenil (GPAA-J). No Brasil, 35,7% e 3,85% dos pacientes com GPAA-J e GPAA, respectivamente, são portadores de mutações no gene MYOC. O gene citocromo P450, família 1, subfamília B, polipeptídeo 1 (CYP1B1), primeiramente associado ao glaucoma congênito primário, tem sido apontado como modulador do fenótipo do GPAA na presença de alterações no gene MYOC. Recentemente, observaram-se mutações no gene CYP1B1 associadas ao GPAA e GPAA-J, independentemente da presença de alterações estruturais no gene MYOC, em diferentes populações. Este projeto se propôs a avaliar mutações nos genes MYOC e CYP1B1, utilizando técnicas de PCR e sequenciamento direto, em 100 indivíduos pertencentes a famílias com GPAA ou GPAA-J e 43 pacientes não relacionados portadores de GPAA-J, bem como avaliar a possível modulação do gene CYP1B1 no fenótipo da doença. Uma nova mutação no gene MYOC, c.1187_1188insCCCAGA, foi identificada segregando com a doença em três gerações de uma família. De acordo com análises in silico, esta mutação pode alterar os contatos internos da proteína, além de comprometer um sítio de fosforilação de caseína quinase II. Nas demais três famílias foi detectada a mutação C433R, já descrita anteriormente. Como os membros das famílias apresentavam fenótipos bastante variados, foi realizada a análise da possível modulação do fenótipo da doença pelo gene CYP1B1. Contudo, as análises mostraram a não associação de variantes deste gene na modulação do fenótipo do glaucoma nestas famílias. Nos casos não relacionados de GPAA-J, foram observadas as mutações P370L, Q368X e C433R. Nenhuma mutação no gene CYP1B1 foi identificada nestes casos, mas somente polimorfismos: R48G, A119S, V243V, V432L, A443G, D449D e N453S. De acordo com este e outros estudos, é possível concluir que mutações no gene MYOC tem papel importante no desenvolvimento do GPAA e GPAA-J, sendo a mutação C433R a mais frequente na população brasileira. Além disso, o gene CYP1B1 parece ter menor contribuição no desenvolvimento destes tipos de glaucoma em nossa população / Abstract: Glaucoma comprises a group of heterogeneous optic neuropathies characterized by excavation of the optic disc and progressive loss of visual field, representing a major global cause of irreversible blindness. In 1997, the Myocilin gene (MYOC) was discovered, and mutations in this gene were involved in the development of primary open-angle glaucoma (POAG) and juvenile-onset of POAG (JOAG). In Brazil, 35.7% and 3.85% of patients with POAG and JOAG, respectively, are carriers of mutations in the MYOC gene. The gene cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1), primarily associated with primary congenital glaucoma, has been related to phenotypic modulation of POAG in the presence of MYOC gene mutations. Recently, mutations in the CYP1B1 gene associated with POAG and JOAG were observed, regardless the presence of structural alterations in the MYOC gene in different populations. This project proposed to evaluate mutations in the MYOC and CYP1B1 genes using PCR and direct sequencing, in 100 individuals belonging to families with JOAG or POAG and 43 unrelated JOAG patients, and assess the possible role of the CYP1B1 gene in modulating the disease phenotype. A novel mutation in the MYOC gene, c.1187_1188insCCCAGA, was detected, segregating with the disease in three generations of one family. According to in silico analysis, this mutation can change the internal contacts of the protein, and has altered a phosphorylation site of casein kinase II. In the other three families the C433R mutation was detected, as described earlier. As family members presented with very different phenotypes, the CYP1B1 gene was evaluated as a possible modulator of the disease. However, the analysis showed no association of variants in CYP1B1 gene in modulating the glaucoma phenotype in these families. In unrelated cases of JOAG, the mutations P370L, Q368X and C433R were detected. No mutation in the CYP1B1 gene was observed in these cases, but only polymorphisms: R48G, A119S, V243V, V432L, A443G, D449D, and N453S. According to the present and other studies, we conclude that mutations in the MYOC gene play an important role in the development of POAG and JOAG, and the C433R mutation is the most frequent MYOC alteration in the Brazilian population. Furthermore, the CYP1B1 gene seems to have less contribution to the development of these types of glaucoma in our population / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

The aim of this study was to compare the efficacy of trabeculectomy (TE), single XEN microstent implantation (solo XEN) or combined XEN implantation and cataract surgery (combined XEN) in primary open-angle glaucoma cases, naïve to prior surgical treatment, using a monocentric retrospective comparative cohort study. Intraocular pressure (IOP) and the number of IOP-lowering drugs (Meds) were monitored during the first 24 months after surgery. Further disease progression was monitored using peripapillary retinal nerve fiber layer (RNFL) thickness examinations using spectral domain optical coherence tomography (OCT) as well as visual acuity (VA) and visual field (VF) tests. In the TE group (52 eyes), the mean IOP decreased from 24.9 ± 5.9 to 13.9 ± 4.2 mmHg (p < 0.001) and Meds decreased from 3.2 ± 1.2 to 0.5 ± 1.1 (p < 0.001). In the solo XEN (38 eyes) and the combined XEN groups, the mean IOP decreased from 24.1 ± 4.7 to 15.7 ± 3.0 mmHg (p < 0.001) and 25.4 ± 5.6 to 14.7 ± 3.2 mmHg (p < 0.001), while Meds decreased from 3.3 ± 0.8 to 0.8 ± 1.2 (p < 0.001) and 2.7 ± 1.2 to 0.4 ± 1.0 (p < 0.001), respectively. The VF and VA indices showed no sign of further deterioration, the RNFL thickness further decreased in all treatment groups after surgery. TE and XEN led to comparable reductions in IOP and Meds. Although the VA and VF indices remained unaltered, the RNFL thickness continuously decreased in all treatment groups during the 24-month follow-up.

A busca visual é uma habilidade crítica para várias tarefas da vida diária e pode estar prejudicada em pacientes com deficiência visual. O objetivo deste estudo foi comparar a busca visual exploratória entre pacientes com glaucoma primário de ângulo aberto (GPAA) e controles saudáveis, avaliando a correlação espacial entre áreas com perdas localizadas na busca visual exploratória e os defeitos perimétricos, em pacientes com GPAA e com visão normal. Cinquenta e sete indivíduos com visão normal (acuidade visual corrigida melhor que 0.2 logMAR) diagnosticados (grupo GPAA, n = 29) ou não (grupo CONTROL, n = 28) com GPAA, realizaram um exame oftalmológico completo, incluindo perimetria visual (Humphrey -Fast 24.2) e uma tarefa exploratória de busca visual baseada em uma tela com dígitos. Um software personalizado quantificou o (s) tempo (s) gasto (s) até o encontro do número \"4\" em uma matriz aleatória de dígitos distribuídos em cinco áreas, em nove telas sequenciais do programa. Cinco áreas da tela de busca visual foram espacialmente correlacionadas com cinco setores do mapa total deviation (TD) da perimetria visual, após ajustes de ângulo e distância. A análise de covariância (ANCOVA) e testes de correlação foram utilizados para correlacionar parâmetros perimétricos e da busca visual exploratória, por meio de avaliação do tempo individual (gasto para encontrar cada dígito) e tempo total (gasto para completar a tarefa). Os pacientes com GPAA apresentaram pior sensibilidade perimétrica (MD) e de busca visual exploratória do que os controles (MD: -8,02 ± 7,88 dB vs -1,43 ± 1,50 dB; p <0,0001 e tempo total: 106,42 ± 59,64 s vs 52,75 ± 19,07 s; p < 0.0001). A sensibilidade do MD de ambos os grupos correlacionou-se significativamente com o tempo total (GPAA: r = -0.45; p = 0,01 e CONTROL: r = 0,37; p = 0,049). Os testes de ANCOVA mostraram uma correlação significativa entre a busca visual exploratória (tempo individual) e a acuidade visual (P = 0,006) e o diagnóstico de glaucoma (p = 0,005). A sensibilidade média das áreas perimétricas periféricas do grupo GPAA mostrou correlação significativa com o tempo de busca individual nas áreas espaciais correspondentes, exceto na área periférica temporal superior (r = -0,35, p = 0,06). Os controles não mostraram correlação significativa para nenhuma dessas áreas perimétricas, exceto a área periférica temporal superior (r = 0,43, p = 0,02). Com base em nossos resultados, regiões com pior desempenho na busca visual exploratória puderam ser correlacionadas às perdas periféricas localizadas dos pacientes com GPAA. Uma vez que foram estudados pacientes com acuidade visual normal, estes achados destacam a importância do uso de ferramentas de busca visual na avaliação do impacto das perdas perimétricas periféricas em atividades diárias de pacientes com glaucoma. / Visual search is a critical skill for several daily tasks and may be declined in patients with impaired vision. The objectives of this study were to compare the exploratory visual search performance (EVSP) between patients with primary open-angle glaucoma (POAG) and healthy controls, and evaluate the spatial correlation between localized decreases in the EVSP and areas of visual field (VF) loss in normally-sighted patients POAG. Fifty-seven normal vision subjects (best corrected visual acuity better than 0.2 logMAR) diagnosed (POAG group; n= 29) or not (CONTROL group; n= 28) with POAG yielded a complete comprehensive ophthalmological examination, including Humphrey VF tests (SITA-Fast 24.2), and an exploratory visual search digit-based task. A custom software quantified the time (s) spent until patients found the number \"4\" on a random array of digits distributed in five areas on nine sequential screens. Each area was spatially matched with five sectors of the total deviation map from VF, after angle and distance adjustments. Covariance (ANCOVA) and correlation tests were used for correlating VF parameters and EVSP, evaluated through individual time (spent for finding each digit) and total time (spent for completing the task). POAG patients presented worse VF mean deviation (MD) sensitivity and EVSP than controls (MD: -8.02±7.88 dB vs -1.43±1.50 dB; p<0.0001, and total time: 106.42±59.64 s vs 52.75±19.07 s; p<0.0001). MD sensitivity of both groups significantly correlated with total time (POAG: r = -0.45; p = 0.01 and CONTROL: r = 0.37; p = 0.049). ANCOVA tests showed a significant correlation between EVSP (individual time) and both visual acuity (p = 0.006) and glaucoma diagnosis (p = 0.005). The mean sensitivity of the peripheral VF areas of the POAG group showed significant correlation with the individual search time in the corresponding spatial areas, except in the peripheral temporal superior area (r = -0.35, p =0.06). Controls did not show a significant correlation for any of those VF areas, except the peripheral temporal superior area (r =0.43, p =0.02). Based on our results, worse EVSP can be attributable to localized losses in the peripheral VF areas in patients with POAG. Since only normally sighted patients were studied, these findings highlight the importance of using visual search tools to evaluate the impact of peripheral VF loss in daily activities of glaucoma patients, such as driving.

Made available in DSpace on 2016-08-10T10:38:44Z (GMT). No. of bitstreams: 1 Leonardo Mariano Reis.pdf: 8263558 bytes, checksum: d4842ddc6615e7587ee43da1794b8d63 (MD5) Previous issue date: 2013-03-18 / Abstract of the First Chapter Objective: The objective was to make a wide review of the literature about the genetic aspects of glaucoma, with a highlight at the works dealing with mitochondrial DNA. Methods: Bibliographical research was carried out accessing the sites Scopus, Science Direct, PubMed, Scirus, Scielo and Google. Books, theses and dissertations were also used at the research. Results: The works brought to evidence that there is a relation between certain polymorphisms in mitochondrial DNA and a higher loss of retinal ganglion cells, which leads to the development of Glaucoma, especially Primary Open Angle Glaucoma. Conclusion: Higher incidence of Primary Open Angle Glaucoma (POAG) in afro-descendants is justified by the mitochondrial haplotypes, since it was observed a higher susceptibility of POAG in individuals of the L haplogroups, who are exactly the ones of African origin. It could also justify, based on mitochondrial inheritance, the major incidence of POAG in the maternal lineages, more frequent than paternal inheritance. Abstract of the Second Chapter Objective: To quantify the volume of the scientific production about Primary Open Angle Glaucoma and Mitocohndrial DNA and assess which are the main institutions of research; verify the contribution of the authors that most published about the subject; list the main journals with their Impact Factor; compare the countries and other bibliometric results. Methods: Bibliographical research about mitochondrial DNA and Primary Open Angle Glaucoma was carried out accessing the site Scopus. Then, search with the keywords mitochondrial DNA and glaucoma in all fields for publications until august of 2012 was performed. Finally, data was tabulated and the descriptive statistics was done, with the principal data, as: authors that published about mitochondrial DNA and Primary Open Angle Glaucoma; journals that had works about the subject; Research Centers and Universities that most published; countries where the researchs were performed. Results: The works brought to evidence the influences of certain polymorphisms in mitochondrial DNA at the development of Primary Open Angle Glaucoma. Statistics showed that theese articles have increased in the last few years, though yet confined, mostly, to some centers of research and concentrated in selected researchers in ophthalmology of developed countries. In Brazil, we do not have any article published about the issue, yet. Conclusion: 98 works were listed, until august of 2012, carried out in 30 countries, with higher concentration in the United States and England, but also with contributions of the Arabic world: Qatar and Saudi Arabia. There were more publications in the following journals: Molecular Vision, Investigative Ophthalmology and Visual Science, Archives of Ophthalmology, British Journal of Ophthalmology, Experimental Eye Research, Journal of Glaucoma and Progress in Retinal and Eye Research. More studies about the subject must be encouraged, for they are extremely important to the elucidation of the genetic causes of glaucoma and for the development of new therapies aiming not only the reduction of intraocular pressure. / RESUMO DO CAPÍTULO I Objetivo: O objetivo foi fazer uma ampla revisão de toda a literatura sobre os aspectos genéticos do glaucoma, com enfoque nos trabalhos que envolvem pesquisa com DNA mitocondrial. Métodos: A pesquisa bibliográfica foi realizada por meio das bases de dados: Scopus, Science Direct, PubMed, Scirus, Scielo e pelo sítio de busca Google. Foi feito também levantamento através de livros, dissertações e teses. Resultados: Os trabalhos evidenciaram que há alguma correlação de determinadas linhagens de DNA mitocondrial com maior perda de células ganglionares da retina, o que acaba levando ao desenvolvimento do glaucoma propriamente dito, sobretudo do glaucoma primário de ângulo aberto. Conclusão: Explica-se a maior incidência de glaucoma primário de ângulo aberto em indivíduos afrodescendentes a partir de haplótipos mitocondriais, uma vez que houve maior susceptibilidade ao glaucoma primário de ângulo aberto em haplótipos pertencentes aos grupos L, que são justamente os de origem africana. Também poder-se-ia justificar, a partir da herança mitocondrial, a maior incidência de glaucoma primário de ângulo aberto quando a linhagem materna apresenta a doença, bem mais frequente do que a herança paterna. RESUMO DO CAPÍTULO II Objetivo: Quantificar o volume da produção científica que relaciona o DNA mitocondrial com o Glaucoma Primário de Ângulo Aberto e avaliar quais são as principais instituições; verificar a contribuição dos autores que mais produziram publicações sobre o assunto; listar os principais veículos com os respectivos Fatores de Impacto; comparar os países que mais fizeram trabalhos sobre o tema e outros resultados bibliométricos. Métodos: A pesquisa bibliográfica de trabalhos envolvendo DNA mitocondrial e Glaucoma Primário de Ângulo Aberto foi realizada por meio da base de dados do sítio Scopus. Foi feito o levantamento a partir das palavras-chaves Mitochondrial DNA e Glaucoma em todos os campos para publicações até agosto de 2012. Finalmente, realizada a tabulação dos dados obtidos e a estatística descritiva, com o principais dados como: autores que publicaram sobre o assunto DNA mitocondrial e glaucoma; revistas e outras publicações que tiveram trabalhos relacionados com o tema; Centros de Pesquisa e Universidades que mais publicaram e países onde foram realizados os estudos sobre DNA mitocondrial e glaucoma. Resultados: Os trabalhos evidenciaram a influência de determinados polimorfismos de DNA mitocondrial no desenvolvimento do Glaucoma Primário de Ângulo Aberto. A estatística mostrou que esses estudos têm aumentado sobremaneira ao longo dos últimos anos, mas ainda se encontram confinados, na maior parte, a alguns centros de pesquisa e concentrados em seletos grupos de autores na área da oftalmologia em países desenvolvidos. No Brasil, ainda não temos pesquisas publicadas sobre o assunto até o momento. Conclusão: Foram listados 98 trabalhos até agosto de 2012, realizados em 30 países, com maior concentração nos Estados Unidos e Inglaterra; mas também com contribuições do mundo árabe: Arábia Saudita e Catar. Esses estudos foram mais publicados nas revistas: Molecular Vision, Investigative Ophthalmology and Visual Science, Archives of Ophthalmology, British Journal of Ophthalmology, Experimental Eye Research, Journal of Glaucoma e Progress in Retinal and Eye Research. Mais estudos sobre o assunto devem ser encorajados pois são de fundamental importância para a elucidação das causas genéticas do glaucoma e para o desenvolvimento de novas terapias que não visem tão somente a diminuição da pressão intraocular.

A busca visual é uma habilidade crítica para várias tarefas da vida diária e pode estar prejudicada em pacientes com deficiência visual. O objetivo deste estudo foi comparar a busca visual exploratória entre pacientes com glaucoma primário de ângulo aberto (GPAA) e controles saudáveis, avaliando a correlação espacial entre áreas com perdas localizadas na busca visual exploratória e os defeitos perimétricos, em pacientes com GPAA e com visão normal. Cinquenta e sete indivíduos com visão normal (acuidade visual corrigida melhor que 0.2 logMAR) diagnosticados (grupo GPAA, n = 29) ou não (grupo CONTROL, n = 28) com GPAA, realizaram um exame oftalmológico completo, incluindo perimetria visual (Humphrey -Fast 24.2) e uma tarefa exploratória de busca visual baseada em uma tela com dígitos. Um software personalizado quantificou o (s) tempo (s) gasto (s) até o encontro do número \"4\" em uma matriz aleatória de dígitos distribuídos em cinco áreas, em nove telas sequenciais do programa. Cinco áreas da tela de busca visual foram espacialmente correlacionadas com cinco setores do mapa total deviation (TD) da perimetria visual, após ajustes de ângulo e distância. A análise de covariância (ANCOVA) e testes de correlação foram utilizados para correlacionar parâmetros perimétricos e da busca visual exploratória, por meio de avaliação do tempo individual (gasto para encontrar cada dígito) e tempo total (gasto para completar a tarefa). Os pacientes com GPAA apresentaram pior sensibilidade perimétrica (MD) e de busca visual exploratória do que os controles (MD: -8,02 ± 7,88 dB vs -1,43 ± 1,50 dB; p <0,0001 e tempo total: 106,42 ± 59,64 s vs 52,75 ± 19,07 s; p < 0.0001). A sensibilidade do MD de ambos os grupos correlacionou-se significativamente com o tempo total (GPAA: r = -0.45; p = 0,01 e CONTROL: r = 0,37; p = 0,049). Os testes de ANCOVA mostraram uma correlação significativa entre a busca visual exploratória (tempo individual) e a acuidade visual (P = 0,006) e o diagnóstico de glaucoma (p = 0,005). A sensibilidade média das áreas perimétricas periféricas do grupo GPAA mostrou correlação significativa com o tempo de busca individual nas áreas espaciais correspondentes, exceto na área periférica temporal superior (r = -0,35, p = 0,06). Os controles não mostraram correlação significativa para nenhuma dessas áreas perimétricas, exceto a área periférica temporal superior (r = 0,43, p = 0,02). Com base em nossos resultados, regiões com pior desempenho na busca visual exploratória puderam ser correlacionadas às perdas periféricas localizadas dos pacientes com GPAA. Uma vez que foram estudados pacientes com acuidade visual normal, estes achados destacam a importância do uso de ferramentas de busca visual na avaliação do impacto das perdas perimétricas periféricas em atividades diárias de pacientes com glaucoma. / Visual search is a critical skill for several daily tasks and may be declined in patients with impaired vision. The objectives of this study were to compare the exploratory visual search performance (EVSP) between patients with primary open-angle glaucoma (POAG) and healthy controls, and evaluate the spatial correlation between localized decreases in the EVSP and areas of visual field (VF) loss in normally-sighted patients POAG. Fifty-seven normal vision subjects (best corrected visual acuity better than 0.2 logMAR) diagnosed (POAG group; n= 29) or not (CONTROL group; n= 28) with POAG yielded a complete comprehensive ophthalmological examination, including Humphrey VF tests (SITA-Fast 24.2), and an exploratory visual search digit-based task. A custom software quantified the time (s) spent until patients found the number \"4\" on a random array of digits distributed in five areas on nine sequential screens. Each area was spatially matched with five sectors of the total deviation map from VF, after angle and distance adjustments. Covariance (ANCOVA) and correlation tests were used for correlating VF parameters and EVSP, evaluated through individual time (spent for finding each digit) and total time (spent for completing the task). POAG patients presented worse VF mean deviation (MD) sensitivity and EVSP than controls (MD: -8.02±7.88 dB vs -1.43±1.50 dB; p<0.0001, and total time: 106.42±59.64 s vs 52.75±19.07 s; p<0.0001). MD sensitivity of both groups significantly correlated with total time (POAG: r = -0.45; p = 0.01 and CONTROL: r = 0.37; p = 0.049). ANCOVA tests showed a significant correlation between EVSP (individual time) and both visual acuity (p = 0.006) and glaucoma diagnosis (p = 0.005). The mean sensitivity of the peripheral VF areas of the POAG group showed significant correlation with the individual search time in the corresponding spatial areas, except in the peripheral temporal superior area (r = -0.35, p =0.06). Controls did not show a significant correlation for any of those VF areas, except the peripheral temporal superior area (r =0.43, p =0.02). Based on our results, worse EVSP can be attributable to localized losses in the peripheral VF areas in patients with POAG. Since only normally sighted patients were studied, these findings highlight the importance of using visual search tools to evaluate the impact of peripheral VF loss in daily activities of glaucoma patients, such as driving.

<p>Glaucoma is a common disease in the elderly population. Open-angle glaucoma (OAG) is the predominant form of glaucoma. Chronic simple glaucoma and capsular glaucoma, characterized by the occurrence of pseudoexfoliation in the anterior eye segment, are the most frequent types of OAG. The purpose of the present thesis was to study the epidemiology of OAG in the municipality of Tierp, whose population has a high exposure to pseudoexfoliation.</p><p>In a case-finding study, the prevalence of known cases of OAG by December 31, 1983 was estimated to 1.4% in people ≥45 years of age. Sixty-three percent of all cases had capsular glaucoma. Patients with advanced glaucoma were older, had had the disease for longer, had higher mean initial intraocular pressure, and had more extensive visual field defects at the time of diagnosis.</p><p>A population survey of people 65–74 years of age was conducted in 1984–86. The prevalence of OAG was 5.3%. Pseudoexfoliation was found in 17%, being more common in females. Pseudoexfoliation was associated with OAG only in people previously diagnosed with the disease (odds ratio = 16). In cases detected at the survey, an intraocular pressure ≥20 mmHg was a serious risk factor of having OAG (odds ratio = 9.7).</p><p>In a 5-year follow-up study of participants in the population survey, increased intraocular pressure and pseudoexfoliation were recognized as independent risk factors for the development of OAG (standardized risk ratios = 3.4 and 9.8, respectively). Interaction between increased intraocular pressure and pseudoexfoliation was indicated. By May 2006, the incidence of OAG was estimated to 7.1 per 1,000 person-years. The incidence of capsular glaucoma was more than twice that of chronic simple glaucoma.</p><p>The prevalence and incidence of OAG was higher than that reported from other studies conducted on Caucasian populations. The probable explanation for this finding is exposure to pseudoexfoliation.</p>

Glaucoma is a common disease in the elderly population. Open-angle glaucoma (OAG) is the predominant form of glaucoma. Chronic simple glaucoma and capsular glaucoma, characterized by the occurrence of pseudoexfoliation in the anterior eye segment, are the most frequent types of OAG. The purpose of the present thesis was to study the epidemiology of OAG in the municipality of Tierp, whose population has a high exposure to pseudoexfoliation. In a case-finding study, the prevalence of known cases of OAG by December 31, 1983 was estimated to 1.4% in people ≥45 years of age. Sixty-three percent of all cases had capsular glaucoma. Patients with advanced glaucoma were older, had had the disease for longer, had higher mean initial intraocular pressure, and had more extensive visual field defects at the time of diagnosis. A population survey of people 65–74 years of age was conducted in 1984–86. The prevalence of OAG was 5.3%. Pseudoexfoliation was found in 17%, being more common in females. Pseudoexfoliation was associated with OAG only in people previously diagnosed with the disease (odds ratio = 16). In cases detected at the survey, an intraocular pressure ≥20 mmHg was a serious risk factor of having OAG (odds ratio = 9.7). In a 5-year follow-up study of participants in the population survey, increased intraocular pressure and pseudoexfoliation were recognized as independent risk factors for the development of OAG (standardized risk ratios = 3.4 and 9.8, respectively). Interaction between increased intraocular pressure and pseudoexfoliation was indicated. By May 2006, the incidence of OAG was estimated to 7.1 per 1,000 person-years. The incidence of capsular glaucoma was more than twice that of chronic simple glaucoma. The prevalence and incidence of OAG was higher than that reported from other studies conducted on Caucasian populations. The probable explanation for this finding is exposure to pseudoexfoliation.

The inherited form of primary open angle glaucoma, a disorder characterized by increased intraocular pressure and retina degeneration, is linked to mutations in the olfactomedin (OLF) domain of the myocilin gene. Disease-causing myocilin variants accumulate within trabecular meshwork cells instead of being secreted to the trabecular extracellular matrix thought to regulate aqueous humor flow and control intraocular pressure. Like other diseases of protein misfolding, we hypothesize myocilin toxicity originates from defects in protein biophysical properties. In this thesis, the first preparative recombinant high-yield expression and purification system for the C-terminal OLF domain of myocilin (myoc-OLF) is described. To determine the relative stability of wild-type (WT) and mutant OLF domains, a fluorescence thermal stability assay was adapted to provide the first direct evidence that mutated OLF is folded but less thermally stable than WT. In addition, mutant myocilin can be stabilized by chemical chaperones. Together, this work provides the first quantitative demonstration of compromised stability among identified OLF variants and placing myocilin glaucoma in the context of other complex diseases of protein misfolding. Subsequent investigations into the biophysical properties of WT myoc-OLF provide insight into its structure and function. In particular, myoc-OLF is stable in the presence of glycosaminoglycans (GAGs), as well as over a wide pH range in buffers with functional groups reminiscent of such GAGs. Myoc-OLF contains significant â-sheet and â-turn secondary structure as revealed by circular dichroism analysis. At neutral pH, thermal melts indicate a highly cooperative transition with a melting temperature of ~55°C. A compact core structural domain of OLF was identified by limited proteolysis and consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. This construct also is capable of generating 3D crystals for structure determination. This data, presented in Chapter 3, inform new testable hypotheses for interactions with specific trabecular extracellular matrix components. To gain further insight into the biological function of myoc-OLF, a facile fluorescence chemical stability assay was designed to identify possible ligands and drug candidates. In the assay described in Chapter 4, the target protein is initially destabilized with a chemical denaturant and is tested for re-stabilization upon the addition of small molecules. The assay requires no prior knowledge of the structure and/or function of the target protein, and it is amendable to high-throughput screening. Application of the assay using a library of 1,280 compounds revealed 14 possible ligands and drug candidates for myoc-OLF that may also generate insights into myoc-OLF function. Due to the high â-sheet content of monomeric myoc-OLF and presence of an aggregated species upon myoc-OLF purification, the ability of myoc-OLF to form amyloid fibrils was suspected and verified. The fibril forming region was confirmed to reside in the OLF domain of myocilin. Kinetic analyses of fibril formation reveal a self-propagating process common to amyloid. The presence of an aggregated species was confirmed in cells transfected with WT myocilin, but to a greater extent in cells transfected with P370L mutant myocilin. Both cell lines stained positive for amyloid. Taken together, these results provide further insights into the structure of myocilin and suggest a new hypothesis for glaucoma pathogenesis. Finally, in a related study, small molecule drug candidates were investigated to treat acid â-glucosidase (GCase), the deficient lysosomal enzyme in Gaucher disease, another protein conformational disorder. Three new GCase active-site directed 3,4,5,6-tetrahydroxylazepane inhibitors were synthesized that exhibit half inhibitory concentrations (IC50) in the low millimolar to low micromolar range. Although the compounds thermally stabilize GCase at pH 7.4, only one of the synthesized analogs exhibits chaperoning activity under typical assay conditions. This successful pharmacological chaperone is also one in which GCase is in its proposed active conformation as revealed by X-ray crystallography. Probing the plasticity of the active-site of GCase offers additional insight into possible molecular determinants for an effective small molecule therapy for GD.

Cette thèse constitue à la fois un apport de nature clinique et technologique dans l’approche diagnostique du glaucome. Plus précisément, nous nous proposons d’étudier une nouvelle façon de détecter le glaucome par la mesure de l’asymétrie du rapport de la surface de l’anneau neurorétinien et de la surface de la papille ou du disque optique ou rim to disc area asymmetry ratio (RADAAR). Pour atteindre cet objectif, nous avons recours à une base de données composée d’une population subdivisée en 4 différents groupes de diagnostic (normal, glaucome possible, glaucome probable et glaucome définitif). Les mesures du RADAAR sont calculées de différentes façons à partir des paramètres stéréométriques de la tête du nerf optique des sujets, produits par la microscopie confocale à balayage laser (Heidelberg Retina Tomograph (HRT) (Heidelberg Engineering, Germany)). Nous procédons à une analyse de données grâce au logiciel SPSS où nous mettons en exergue la distribution du RADAAR dans les différentes populations, sa validité et son utilité dans le dépistage du glaucome. Nous enrôlons donc 523 sujets dans cette étude avec 82 sujets atteints de glaucome définitif. La moyenne d’âge est de 62 ans. Il y a plus de femmes que d’hommes et plus de Caucasiens que d’Africains Caribéens. Nous trouvons que la distribution de la mesure du RADAAR est différente des sujets d’un groupe de diagnostic à l’autre. En termes de performance, la sensibilité de la mesure du RADAAR est très basse c'est-à-dire que sa capacité de détecter la maladie est basse. En revanche la mesure du RADAAR est plus spécifique c'est-à-dire que sa capacité d’identifier les sujets exempts de la maladie est plus grande. Elle tendrait à être aussi plus performante chez les Africains Caribéens que chez les Caucasiens. De même, elle serait plus sensible chez les hommes que chez les femmes. La mesure du RADAAR est utile si on l’associe à une autre méthode de diagnostic comme l’analyse de Régression de Moorfields (MRA) incluse dans le logiciel du HRT3 spécialement lors de la détection du glaucome dans la population à haut risque. En définitive, nous déterminons que la mesure du RADAAR se veut un outil d’aide au diagnostic. Elle est particulièrement intéressante dans le contexte de dépistage de glaucome. / This thesis describes a new clinical and technological approach to the diagnosis of glaucoma. Specifically, we intend to study a new way to detect glaucoma by measuring rim area to disc area asymmetry ratio (RADAAR). For this purpose, we use a database consisting of a population divided into 4 different diagnostic groups (normal, possible glaucoma, probable glaucoma and definitive glaucoma). The RADAAR measurements are calculated in different ways based on the stereometric parameters of the optic nerve head of subjects, produced by confocal scanning laser ophthalmoscopy (Heidelberg Retina Tomograph (HRT) (Heidelberg Engineering, Germany)). We conduct an analysis of data with SPSS or we put forward the RADAAR distribution in different populations, its validity in detecting open angle glaucoma and its usefulness in screening for glaucoma. We therefore enroll 523 subjects in this study with about 82 subjects with definitive glaucoma. The average age is 62 years. There are more females than males and more Caucasians than Africans Caraibeans. We find that the distribution of RADAAR measures is different in each diagnosis group. In terms of performance, the sensitivity of the RADAAR measurement is very low. So, its ability to detect the disease is low. However the RADAAR measure is much more specific so, its ability to identify subjects free of the disease is high. RADAAR measure would also be much more effective in African Caribbean’s than in Caucasians. Similarly, it would be much more sensitive in males than in females. The RADAAR measurement is useful if it is combined with another method of diagnosis like the Moorfields regression analysis (MRA) included in the HRT3 software especially in case of the detection of glaucoma in populations at high risk. Ultimately, we determine that the RADAAR is an interesting tool for the diagnosis of glaucoma particularly in the context of screening for glaucoma.

Le glaucome représente la première cause de cécité irréversible à l’échelle mondiale. C’est une maladie neuro-dégénérative caractérisée traditionnellement par une pression intraoculaire (PIO) élevée, un dommage du nerf optique et un défaut du champ visuel correspondant. En fait, la PIO élevée constitue le facteur de risque central associé au développement du glaucome. Cependant, en dépit d’un contrôle adéquat de la PIO, la maladie continue à progresser chez certains patients. Cela montre qu’il existe d’autres facteurs impliqués dans la pathogenèse du glaucome. Des études récentes indiquent qu’un dérèglement de l’oxygène est associé à son développement. En utilisant une nouvelle technologie multi-spectrale capable de mesurer la saturation en oxygène (SaO2) dans les structures capillaires de la rétine, cette étude tentera de déterminer si un état d’oxygénation anormal pourrait se retrouver à la papille optique des patients souffrant de glaucome. Une meilleure compréhension du rôle de l’oxygène pourrait aider à améliorer le pronostic du glaucome. Les résultats de l’étude indiquent que le facteur de position (supérieure, temporale et inférieure de la papille optique) n’a aucun effet sur la mesure SaO2 ainsi que sa variabilité chez les patients normaux. La comparaison de la SaO2 entre les sujets normaux et glaucomateux ne montre pas de différence statistiquement significative. En conclusion, la SaO2 «normale» mesurée dans les yeux glaucomateux n'exclut pas nécessairement que l'hypoxie ne soit pas impliquée dans la pathogenèse. Au moment de l’étude, la PIO était bien contrôlée par des médicaments topiques, ce qui pourrait influencer l’oxygénation à la papille optique. / Glaucoma is the leading cause of irreversible blindness worldwide. Traditionally, open-angle glaucoma was defined as a neurodegenerative disease characterized by high intraocular pressure (IOP), progressive retinal cell death with subsequent visual field loss. Elevated IOP has been identified as one of the major risk factors for glaucomatous optic nerve damage. However, adequate IOP control cannot prevent progression of the disease in all patients suggesting that there are other factors involved in the pathogenesis of glaucoma. Recent studies suggest that hypoxia may contribute to the development of glaucoma. Using a new multi-spectral detection system of oxygen saturation (O2Sa), this study determined whether an abnormal state of oxygenation at the optic disc could be found in glaucoma patients. Knowledge about the influence of the oxygen in glaucoma may help to improve the pronostic of the disease. The results of the study indicate that the position factor (superior, temporal and inferior of the optic nerve head) has no effect on the measurement of O2Sa and its variability in normal patients. Comparing the O2Sa between normal subjects and glaucoma subjects shows no statistically significant difference. In conclusion, the «normal» O2Sa measured in glaucomatous eyes does not necessarily exclude that hypoxia is not involved in the pathogenesis of glaucoma because glaucoma patients were under treatment with topical drops that lowered IOP. These medicines could affect the oxygenation of the optic disc.