Fan Baojian. / "June 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 101-126). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

In the Kuehn lab, it has been shown that inducible pluripotent stems cells that have been induced to be trabecular meshwork cell-like (iPSC-TM) have a unique ability to regenerate dysfunctional trabecular meshwork (TM) cells by sharing specific unknown factors. In this thesis will discuss the novel means by which I isolate primary human Trabecular Meshwork (pTMs) and efficiently prepare cell cultures for experimentation, such as a sequencing experiment in which I studied expression changes that arose when the TM cell culture’s cell cycle control is manipulated. Previous research has shown that pTM grow atypical when 100% confluent compared to other epithelial cells creating an interesting time frame by which to observe their unique cell cycle control. Using newly isolated TM cell cultures I investigated expression of mRNA and miRNA to understand their roles in cell cycle control of these atypical cultures. With regards to the isolation of TM cell cultures were able to show that the “Crawling Out” methodology is an effective way to establish a pure TM cell line with both a low contamination rate and less passages/time. With these cultures we were able to establish 50 mRNAs and 19 miRNAs that were differential expressed in the TM cell cultures that were atypically grown. When reviewing the literature many of these expression changes were linked to carcinogenics, and the progression/prognosis of various cancer types.

Myocilin, expressed in the trabecular meshwork of the eye, has been linked to inherited primary open-angle glaucoma (POAG). The biological function of myocilin is unknown, but mutant myocilin exhibits a gain-of-function mechanism, aggregating within the endoplasmic reticulum of human trabecular meshwork cells, causing cell stress and eventually apoptosis. After apoptosis occurs, the trabecular meshwork is compromised, leading to an increase in intraocular pressure, a symptom of glaucoma. In this thesis, I have expressed and purified the wild-type olfactomedin (OLF) domain and 24 reported disease-causing variants. I developed a facile thermal stability assay using differential scanning fluorimetry, which follows the unfolding of a protein through the fluorescence of a dye sensitive to hydrophobic regions of a protein. Also in this thesis I have determined melting temperatures for the wild-type and for each of the disease-causing mutants. I have tested the stability of the mutants in the presence of seven osmolytes, with sarcosine and trimethylamine-N-oxide restoring the melting temperature closest to wild-type. Additionally, I expressed and purified three reported single nucleotide polymorphisms (SNPs) (E352Q, E396D, K398R), which are considered benign variants. Variants were also compared by circular dichroism, revealing high b-sheet content and wild-type structure. When compared to previous studies, there is a positive correlation between the melting temperature, and previously reported qualitative assays, which measure the mutant myocilin solubility in detergent, secretion from mammalian cells, and aggregation propensity. Taken together, these data give insight into the relationship between glaucoma genotypes and phenotypes.

INTRODUÇÃO: A trabeculectomia é a técnica de eleição para o tratamento cirúrgico do glaucoma. Entretanto, estudos recentes têm demonstrado perda da eficácia e menor redução da pressão intra-ocular dos pacientes submetidos à cirurgia ao longo dos anos. Esta diminuição decorre do contínuo processo de cicatrização e proliferação de fibroblastos na superfície epiescleral na região da bolha filtrante. Com o objetivo de diminuir esta proliferação fibroblástica e a conseqüente perda de função da trabeculectomia, introduziu-se o uso de antimetabólicos como o 5-fluorouracil e a mitomicina C. O uso dos antimetabólicos, no entanto, com freqüência é acompanhado dos indesejáveis efeitos da filtração excessiva e hipotonia. O uso da membrana amniótica em oftalmologia remonta aos anos de 1940, quando vários autores relataram seus efeitos benéficos no tratamento de doenças da superfície ocular. Além de promover a epitelização de superfícies, a membrana amniótica também é inibidora da fibrose. O efeito inibidor da fibrose pela membrana amniótica é altamente desejável na modulação da cicatrização após a trabeculectomia. O objetivo deste estudo é comparar a eficácia e a segurança do uso da membrana amniótica na trabeculectomia no tratamento cirúrgico do glaucoma primário de ângulo aberto. MATERIAL E MÉTODOS: Foi realizado um estudo prospectivo aberto, aleatório, com grupos paralelos de tratamento. Sessenta e três pacientes com indicação de cirurgia para glaucoma foram selecionados e aleatoriamente divididos em 2 RESUMO grupos. O primeiro grupo foi submetido a trabeculectomia com o uso peroperatório da membrana amniótica (grupo estudo) e o segundo grupo foi submetido a trabeculectomia sem o uso da membrana amniótica (grupo controle), na seção de glaucoma do Departamento de Oftalmologia da Santa Casa de São Paulo. Foram avaliados os efeitos redutores da pressão intraocular, número de medicações, aparência da bolha filtrante e complicações. Todos os pacientes foram acompanhados por 12 meses. RESULTADOS: A média das pressões pré-operatórias foi de 25,19 ± 7,34 mmHg no grupo da membrana amniótica e 25,42 ± 7,71 mmHg no grupo controle. A média das pressões pós-operatórias foi de 13,13 ± 2,50 mmHg no grupo da membrana amniótica e 15,47 ± 2,92 mmHg no grupo controle, diferença estatisticamente significante no seguimento de 1 ano. Não foi encontrada diferença estatisticamente significante entre os grupos controle e estudo em relação ao número de medicações pré e pós-operatória. No final de 12 meses de seguimento, no grupo de estudo, dois entre 31 olhos (6,45%) apresentaram bolha plana e vascularizada, 14 olhos (45,16%) bolha elevada e pouco vascularizada e 15 olhos (48,38 %) bolha fina e avascular. No grupo controle sete entre 32 olhos (21,87%) apresentaram bolha plana vascularizada, 22 olhos (68,75%) bolha elevada e pouco vascularizada e três olhos (9,37%) bolha fina e avascular. Foi encontrada diferença estatisticamente significante entre os grupos estudo e controle quanto a distribuição dos tipos de bolha encontradas. As complicações observadas no grupo de estudo foram um olho com câmara anterior rasa (3,22%) e dois olhos que apresentaram bolha encapsulada (6,45%). No grupo controle, dois olhos apresentaram câmara anterior rasa (6,25%), um olho descolamento de coróide (3,12%) e dois olhos RESUMO com bolha encapsulada (6,25%). CONCLUSÃO: O presente estudo demonstrou que a trabeculectomia com membrana amniótica causou maior redução da pressão intra-ocular e aparência menos vascularizada das bolhas filtrantes. Mostrou-se uma técnica segura e com baixo índice de complicações. / INTRODUCTION: Trabeculectomy is the procedure of choice for the surgical treatment of glaucoma until nowadays. However, recent studies have demonstrated a loss of efficacy and minor reduction of intraocular pressure in patients who underwent surgery over the years. This loss of efficacy of the trabeculectomy is related to the continuous process of healing and fibroblastic proliferation in the episcleral surface inside the filtering bleb. The use of 5-fluorouracil or mitomycin-C can improve the results of trabeculectomy, but they have been associated with an increased incidence of postoperative complications, especially in primary trabeculectomies. The use of amniotic membrane in ophthalmology retraces back to 1940, when some authors showed its beneficial effect in treatment of ocular surface disorders. Amniotic membrane can promote epitelization of ocular surface and act as an inhibitor of fibrosis. The purpose of this study was to compare the safety and efficacy of human preserved amniotic membrane in the trabeculectomy for treatment of primary open angle glaucoma. METHODS: The study was a prospective, randomized clinical trial comparing primary trabeculectomy with amniotic membrane (study group) and without amniotic membrane (control group) in the treatment of the glaucoma. Intraocular pressure (IOP), number of glaucoma medication, appearance of the bleb and complications were compared between the two groups. Sixty-three patients were divided in the study group of 31 patientes and control group of 32 patients and were followed for a period of 12 months in the glaucoma section of Santa Casa de Sao Paulo. RESULTS: The mean pre-operative IOP was 25.19 ± 7.34 mmHg in the amniotic membrane group and 25.42 ± 7.71 mmHg in the control group. The difference of the mean postoperative IOP between groups was statistically significant; in the control group it was 15.47 ± 2.92 mmHg and in the study group 13.13± 2.50 mmHg at one year follow up. Postoperative number of medication decreased in both groups. Analysis shows at the end of 12-month follow-up period in the study group two of 31 eyes (6.45%) exhibited flat vascularized bleb, 14 eyes (45.16%) had elevated but not avascular blebs and 9 eyes (48.38%) showed thin, avascular blebs. In the control group seven of 32 eyes (21.87%) exhibited flat vascularized bleb, 22 eyes (68.75%) had elevated but not avascular blebs and three eyes (9.37%) showed a thin avascular bleb. Complications were: one eye (3.22%) presented with shallow anterior chamber after surgery and two eyes (6.45%) had encapsulated bleb in the study group; in the control group, two eyes (6.25%) presented shallow anterior chamber after surgery, one eye (3.12%) had coroidal detachment and two eyes (6.25%) developed encapsulated bleb. CONCLUSIONS: Trabeculectomy with amniotic membrane and standard trabeculectomy promote lower postoperative IOP and the results showed statistically significant difference between groups in postoperative IOP after one year follow-up. The procedure is safe with low rate of complications.

Background: The aim of this retrospective study was to compare the efficacy and safety profile of a single XEN-microstent in different types of primary and secondary open angle glaucoma. Methods: A single XEN microstent was implanted in patients with primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), pseudoexfoliation glaucoma (PEX) and secondary glaucoma (Sec.Gl). The intraocular pressure (IOP), the active substances of the applied IOP-lowering drugs, the best corrected visual acuity (BCVA) and the mean deviation (MD) of the perimetry were measured at baseline and at regular follow-ups, scheduled at 2 days and 1, 3, 6 and 12 months after surgery. Results: 153 eyes were included in this analysis. 113 eyes were affected by POAG (74%), 5 eyes by NTG (3%), 22 eyes by PEX (14%) and 13 eyes by Sec. Gl (9%). Mean IOP decreased in all treatment groups during the 12 months of follow-up (complete group: 23.9 ± 7.4 to 15.4 ± 5.1 mmHg (p < 0.01); POAG: 22.8 ± 6.5 to 15.1 ± 4.6 mmHg (p < 0.01); NTG: 16.6 ± 3.4 to 11.6 ± 2.2 mmHg (p < 0.05); PEX: 28.0 ± 7.9 to 17.1 ± 6.6 mmHg (p < 0.01); Sec.Gl: 28.9 ± 13.9 to 15.5 ± 6.9 mmHg (p < 0.05)). In the 153 eyes the average number of IOP-lowering drugs applied decreased from 2.6 ± 1.2 to 0.8 ± 1.3 12 months after surgery (p < 0.01). BCVA and mean deviation of automated standard perimetry remained stable in all groups during follow-up. Conclusion: As in eyes suffering from POAG, IOP and number of IOP-lowering drugs applied can be effectively reduced by XEN implantation in eyes suffering from NTG, PEX and secondary glaucoma while leaving BCVA and visual field unchanged.

Primary open-angle glaucoma (POAG) is one of the leading causes of blindness worldwide. The only modifiable risk factor for POAG is elevated intraocular pressure, resulting from increased aqueous humor production or decreased drainage. Resistance to drainage in the aqueous outflow pathway is believed to reside in the juxtacanalicular connective tissue (JCT) and to be modulated by the inner wall (IW) endothelium of Schlemm’s canal (SC); however, the mechanisms that increase resistance in POAG remain unclear. To cross the IW, aqueous humor passes through I-pores on giant vacuoles (GVs) or B-pores between adjacent endothelial cells. Additionally, outflow around the circumference of the eye is segmental, or non-uniform, and fluorescent tracers can be used to label areas of high-flow and non-flow. The morphological differences in the endothelial cells of SC and their GVs in high- vs. non-flow areas have not been fully elucidated. In this project, we investigated the role of GVs and pores in the IW endothelial cells of SC in regulating segmental outflow in human eyes. We used serial block-face scanning electron microscopy to generate thousands of serial images and visualize these structures in 3D at the ultrastructural level. First, we 3D-reconstructed 45 individual IW cells and their GVs and quantified the number of connections each cell makes with the underlying JCT matrix/cells. We found that cells in high-flow areas made significantly fewer connections to JCT matrix/cells compared to cells in non-flow areas. Secondly, we analyzed 3,302 GVs for I-pores and basal openings and found a significantly greater percentage of GVs with both basal openings and I-pores in high-flow area compared to non-flow area, suggesting this type of GVs form a channel through which aqueous humor passes from JCT to SC. We also found that GVs with I-pores were significantly larger than those without I-pores. Our results suggest that decreasing number of cellular connections and increasing number of GVs with pores may be potential strategies to increase the amount of high-flow area and aqueous outflow for glaucoma treatment. Together, these studies add to our understanding of the role of GVs and pores in regulating segmental flow around the eye.

No / Purpose To document the magnitude of any learning effect for short-wavelength automated perimetry (SWAP) in patients with either ocular hypertension (OHT) or open-angle glaucoma (OAG) who are experienced in standard automated perimetry (SAP). Participants Thirty-five patients (22 with OHT and 13 with OAG) who had previously undergone at least 3 threshold SAP visual field examinations with the Humphrey Field Analyzer (HFA; Carl Zeiss Meditech Inc., Dublin, CA), and 9 patients with OHT who had not previously undertaken any form of perimetry. Methods Each patient attended for SWAP on 5 occasions, each separated by 1 week. At each visit, both eyes were examined using Program 24-2 of the HFA; the right eye was always examined before the left eye. Main Outcome Measures (1) Change over the 5 examinations, in each eye, of the visual field indices Mean Deviation (MD), Short-term Fluctuation (SF), Pattern Standard Deviation (PSD), and Corrected Pattern Standard Deviation. (2) Change in each eye between Visits 1 and 5 in proportionate Mean Sensitivity (pMS) for the central annulus of stimulus locations compared with that for the peripheral annulus thereby determining the influence of stimulus eccentricity on any alteration in sensitivity. (3) Change between Visits 1 and 5 in the number and magnitude of the Pattern Deviation (PD) probability levels associated with any alteration in sensitivity. Results The MD, SF, and PSD each improved over the 5 examinations (each at P<0.001). The improvement in pMS between Visits 1 and 5 was greater for the peripheral annulus than for the central annulus by approximately twofold for the patients with OAG. Considerable variation was present between patients, within and between groups, in the number of locations exhibiting an improving sensitivity between Visits 1 and 5 by 1 or more PD probability levels. Conclusions Care should be taken to ensure that, during the initial examinations, apparent field loss with SWAP in patients exhibiting a normal field by SAP is not the result of inexperience in SWAP. Apparently deeper or wider field loss in the initial examinations with SWAP compared with that exhibited by SAP in OAG also may arise from inexperience in SWAP.

<p>Two different principles were studied. 1st - statistical analysis techniques were used to obtain medical results from a patient population. 2nd - the patient population was used to study the statistical analysis techniques. </p><p>Medical conclusions: latanoprost and timolol treatment showed a statistically significant and clinically useful mean IOP-reduction in a typical worldwide clinical trial population. Latanoprost reduced the IOP 1.6 mm Hg more than timolol. The IOP-reduction was maintained with timolol and slightly enforced with latanoprost up to 6 months of treatment. The mean IOP-reduction was maintained during 2 years of latanoprost treatment. The overall risk of withdrawal due to insufficient IOP-reduction with latanoprost was 8%. </p><p>The statistical methodological issues are of a general and reoccurring character in trial design of the IOP-reduction: should the statistical hypothesis testing be based on the mean intraocular pressure (IOP) or the proportion of patients who reach a specific IOP level, should the estimate of the IOP or IOP-reduction be based on single eyes, mean of bilaterally eligible and identically treated eyes or the difference between an eye with active treatment and a placebo treated contralateral eye, and is mean of replicated recordings useful? Statistical methodological conclusions: the most effective response variable varies with the selected patient population. Therefore, the trial design process should include a comparison of the variability, test power and required sample size for the possible response variables in a sample of the target population. At minimum a statistical consideration should be done.</p>

Two different principles were studied. 1st - statistical analysis techniques were used to obtain medical results from a patient population. 2nd - the patient population was used to study the statistical analysis techniques. Medical conclusions: latanoprost and timolol treatment showed a statistically significant and clinically useful mean IOP-reduction in a typical worldwide clinical trial population. Latanoprost reduced the IOP 1.6 mm Hg more than timolol. The IOP-reduction was maintained with timolol and slightly enforced with latanoprost up to 6 months of treatment. The mean IOP-reduction was maintained during 2 years of latanoprost treatment. The overall risk of withdrawal due to insufficient IOP-reduction with latanoprost was 8%. The statistical methodological issues are of a general and reoccurring character in trial design of the IOP-reduction: should the statistical hypothesis testing be based on the mean intraocular pressure (IOP) or the proportion of patients who reach a specific IOP level, should the estimate of the IOP or IOP-reduction be based on single eyes, mean of bilaterally eligible and identically treated eyes or the difference between an eye with active treatment and a placebo treated contralateral eye, and is mean of replicated recordings useful? Statistical methodological conclusions: the most effective response variable varies with the selected patient population. Therefore, the trial design process should include a comparison of the variability, test power and required sample size for the possible response variables in a sample of the target population. At minimum a statistical consideration should be done.

Orientadores: José Paulo Cabral de Vasconcellos, Mônica Barbosa de Melo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T22:51:05Z (GMT). No. of bitstreams: 1 Santos_BibianaAmeliaCosimdos_M.pdf: 2658658 bytes, checksum: ff5d14370ccba56de1fdd33e8d363ac9 (MD5) Previous issue date: 2012 / Resumo: O glaucoma primário de ângulo aberto (GPAA) é uma doença neurodegenerativa. Suas características clínicas incluem lesão progressiva do disco óptico com perda de campo visual correspondente. Vários fatores de risco estão associados para sua instalação e desenvolvimento¸ o principal é o aumento da pressão intra-ocular (PIO). Apesar de ser uma doença de herança complexa, foi possível, através da biologia molecular, identificar quatro genes associados a esta forma de glaucoma por meio de estudos de ligação em famílias com esta afecção. O primeiro foi o gene MYOC, descrito em 1997, seguido pelos genes OPTN, WDR36 e NTF4, identificados em 2002, 2005 e 2009, respectivamente. Existem outros 14 loci já descritos em famílias com GPAA com padrão de herança autossômico dominante, cujos genes associados ao glaucoma não foram ainda identificados. Desta forma, através de marcadores do tipo microssatélites - escolhidos a partir do mapa genético humano Généthon serão avaliados os loci candidatos (GLC1A e GLC1J - GLC1N) associados ao glaucoma primário de ângulo aberto em duas famílias informativas portadoras de GPAA. Simultaneamente, será realizada a análise de ligação nestas mesmas famílias por meio de lâminas ou microarrrays de single nucleotide polymorphisms (SNPs) utilizando-se o GeneChip® Mapping 10K 2.0. Na primeira abordagem por meio de microssatélites foram avaliados 33 indivíduos da família 1 e 18 indivíduos da família 2 enquanto que na análise por meio das lâminas de SNPs foram investigados 19 membros da família 1 e 11 membros da família 2. Não houve ligação dos loci investigados: GLC1A, GLC1J - GLC1N em ambas as famílias com o GPAA. Entretanto, na análise de ligação por meio de lâminas de SNPs sugeriu possíveis regiões candidatas associadas ao GPAA (lod score maior do que 2) nos cromossomos 5q31.2 - 31.3 e 2p22.3 - 23.1 / Abstract: Primary open-angle glaucoma is a neurodegenerative disease. Clinical features include progressive damage of the optic disc with corresponding visual field loss. Several risk factors are associated with installation and development, the main is a increased of intraocular pressure (IOP). Although a disease of complex inheritance, it was possible, through molecular biology, identify four genes associated with this form of glaucoma through linkage studies in families with this disease. The first was the MYOC gene, described in 1997, followed by the OPTN genes, WDR36 and NTF4, identified in 2002, 2005 and 2009, respectively. There are 14 other loci already described in POAG families with autosomal dominant, whose genes associated with glaucoma have not yet been identified. Thus, through microsatellite markers - chosen from the human genetic map Généthon will evaluate candidate loci (GLC1A and GLC1J - GLC1N) associated with primary open-angle glaucoma in two informative families carriers from POAG. Simultaneously, will be performed a linkage analysis in these same families through "microarrrays" of "single nucleotide polymorphisms" (SNPs) using the GeneChip Mapping 10K 2.0 ®. In the first approach through microsatellites were evaluated 33 individuals of Family 1 and 18 individuals of Family 2 while the analysis through SNPs were investigated in 19 member of family 1 and 11 members of family 2. There was no linkage of investigated loci: GLC1A, GLC1J - GLC1N in both families with POAG. However, in linkage analysis through SNPs suggested possible candidate regions associated with POAG (lod score greater than two) in the chromosome 5q31.2 - 2p22.3 and 31.3 - 23.1 / Mestrado / Ciencias Basicas / Mestra em Clínica Médica