Development and regeneration of neuronal circuits in the vertebrate retina

Thesis (Ph.D.)--University of Washington, 2016-08

Variação sazonal e influência de fatores meteorológicos na incidência de descolamentos regmatógenos da retina na zona centro de Portugal e na Madeira

Objetivos: Analisar a variação sazonal e a influência de fatores meteorológicos na incidência a 5 anos de descolamento regmatógeno da retina (DRR), na região centro do país e região autónoma da Madeira (RAM). Métodos: Série de casos consecutiva, retrospetiva e multicêntrica. Incluíram-se doentes submetidos a cirurgia de DRR no Centro Hospitalar e Universitário de Coimbra e Hospital Doutor Nélio Mendonça entre Janeiro’10 e Dezembro’14. A seleção de casos foi feita através codificação ICD-9-CM e os critérios de inclusão foram: 1)idade ≥18 anos; 2)DRR de novo; 3)reparação cirúrgica do DRR. Consideraram-se critérios de exclusão: 1)DR tracional, exsudativo ou misto; 2)DR antigo ou re-descolamento; 3)soluções de continuidade ou lesões regmatógenas tratadas apenas com laser. Os dados meteorológicos diários das estações de Coimbra, Leiria, Aveiro, Viseu, Guarda, Castelo Branco e Funchal, foram utilizados na análise estatística. Resultados: Foram incluídos 1013 olhos (914 da região centro e 99 da RAM), com idade média 61,84±14,00 anos. Através de um modelo cronológico de regressão linear constatou-se a presença de sazonalidade com picos nos meses de Maio e Setembro. A temperatura média apresentou uma associação marginalmente significativa com a incidência de DRR num modelo biológico de regressão linear considerando os mais fortes preditores de DRR da amostra. Conclusões: Os nossos resultados demonstram que a incidência de DRR se associa a um padrão sazonal significativo que parece ser explicado pela temperatura. Este estudo alerta para a necessidade de um eficaz planeamento em saúde que deverá passar por uma gestão harmoniosa de recursos humanos em épocas de maior incidência.

Variação sazonal e influência de fatores meteorológicos na incidência de descolamentos regmatógenos da retina na zona centro de Portugal e na Madeira

Objetivos: Analisar a variação sazonal e a influência de fatores meteorológicos na incidência a 5 anos de descolamento regmatógeno da retina (DRR), na região centro do país e região autónoma da Madeira (RAM). Métodos: Série de casos consecutiva, retrospetiva e multicêntrica. Incluíram-se doentes submetidos a cirurgia de DRR no Centro Hospitalar e Universitário de Coimbra e Hospital Doutor Nélio Mendonça entre Janeiro’10 e Dezembro’14. A seleção de casos foi feita através codificação ICD-9-CM e os critérios de inclusão foram: 1)idade ≥18 anos; 2)DRR de novo; 3)reparação cirúrgica do DRR. Consideraram-se critérios de exclusão: 1)DR tracional, exsudativo ou misto; 2)DR antigo ou re-descolamento; 3)soluções de continuidade ou lesões regmatógenas tratadas apenas com laser. Os dados meteorológicos diários das estações de Coimbra, Leiria, Aveiro, Viseu, Guarda, Castelo Branco e Funchal, foram utilizados na análise estatística. Resultados: Foram incluídos 1013 olhos (914 da região centro e 99 da RAM), com idade média 61,84±14,00 anos. Através de um modelo cronológico de regressão linear constatou-se a presença de sazonalidade com picos nos meses de Maio e Setembro. A temperatura média apresentou uma associação marginalmente significativa com a incidência de DRR num modelo biológico de regressão linear considerando os mais fortes preditores de DRR da amostra. Conclusões: Os nossos resultados demonstram que a incidência de DRR se associa a um padrão sazonal significativo que parece ser explicado pela temperatura. Este estudo alerta para a necessidade de um eficaz planeamento em saúde que deverá passar por uma gestão harmoniosa de recursos humanos em épocas de maior incidência.

Dissecting signaling pathways that control glia migration in the developing Drosophila retina

The function of a complex nervous system relies on an intricate interaction between neurons and glial cells. However, as glial cells are generally born distant from the place where they settle, molecular cues are important to direct their migration. Glial cell migration is important in both normal development and disease, thus current research in the laboratory has been focused on dissecting regulatory events underlying that crucial process. With this purpose, the Drosophila eye imaginal disc has been used as a model. In response to neuronal photoreceptor differentiation, glial cells migrate from the CNS into the eye disc where they act to correctly wrap axons. To ensure proper development, attractive and repulsive signals must coordinate glial cell migration. Importantly, one of these signals is Bnl, a Fibroblast Growth Factor (FGF) ligand expressed by retinal progenitor cells that was suggested to act as a non-autonomous negative regulator of excessive glial cell migration (overmigration) by binding and activating the Btl receptor expressed by glial cells. Through the experimental results described in chapter 3 we gained a detailed insight into the function of bnl in eye disc growth, photoreceptor development, and glia migration. Interestingly, we did not find a direct correlation between the defects on the ongoing photoreceptors and the glia overmigration phenotype; however, bnl knockdown caused apoptosis of eye progenitor cells what was strongly correlated with glia migration defects. Glia overmigration due to Bnl down-regulation in eye progenitor cells was rescued by inhibiting the pro-apoptotic genes or caspases activity, as well as, by depleting JNK or Dp53 function in retinal progenitor cells. Thus, we suggest a cross-talk between those developmental signals in the control of glia migration at a distance. Importantly, these results suggest that Bnl does not control glial migration in the eye disc exclusively through its ability to bind and activate its receptor Btl in glial cells. We also discuss possible biological roles for the glia overmigration in the bnl knockdown background. Previous results in the lab showed an interaction between dMyc, a master regulator of tissue growth, and Dpp, a Transforming Growth Factor-β important for retinal patterning and for accurate glia migration into the eye disc. Thus, we became interested in understanding putative relationships between Bnl and dMyc. In chapter 4, we show that they positively cooperate in order to ensure proper development of the eye disc. This work highlights the importance of the FGF signaling in eye disc development and reveals a signaling network where a range of extra- and intra-cellular signals cooperate to non-autonomously control glial cell migration. Therefore, such inter-relations could be important in other Drosophila cellular contexts, as well as in vertebrate tissue development.

Recombinant human endostatin reduces hypertrophic scar formation in rabbit ear model through down- regulation of VEGF and TIMP-1.

Background: Recombinant human endostatin (Endostar) has been widely used to suppress angiogenesis in carcinoma patients. Hypertrophic scar (HS) tissue, much like a carcinoma, is often associated with angiogenesis. However, there have been few studies conducted on the effects of Endostar on HS or its mechanism. Objective: This paper investigated the effects Endostar on the HS of rabbit ears and studied the effects of Endostar on VEGF and TIMP-1 expression. Methods: Sixteen New Zealand white rabbits were used to establish HS models. Then, rabbit ears containing HS were randomly assigned to either the Endostar group or the control group. The changes of appearance and histology were evaluated using the naked eye, hematoxylin eosin staining, and a scar elevation index. The VEGF and TIMP-1 expressions were detected by immunohistochemical staining, RT-PCR, and western blot. Results: The thickness of the connective tissue in the Endostar group were thinner, the numbers of micro vessels and fibroblasts were fewer, and the collagen fibers were smoother. Moreover, the mRNA and protein expressions of VEGF and TIMP-1 in the Endostar group were significantly lower than those in the control group. Conclusion: The results suggested that Endostar reduced the formation of HS by down-regulation of VEGF and TIMP-1 expressions.

The Use of Mucoadhesive Polymers to Enhance the Hypotensive Effect of a Melatonin Analogue, 5-MCA-NAT, in Rabbit Eyes

Purpose.: 5-Methoxy-carbonylamino-N-acetyltryptamine (5-MCA-NAT, a melatonin receptor agonist) produces a clear intraocular pressure (IOP) reduction in New Zealand White rabbits and glaucomatous monkeys. The goal of this study was to evaluate whether the hypotensive effect of 5-MCA-NAT was enhanced by the presence of cellulose derivatives, some of them with bioadhesive properties, as well as to determine whether these formulations were well tolerated by the ocular surface. Methods.: Formulations were prepared with propylene glycol (0.275%), carboxymethyl cellulose (CMC, 0.5% and 1.0%) of low and medium viscosity and hydroxypropylmethyl cellulose (0.3%). Quantification of 5-MCA-NAT (100 μM) was assessed by HPLC. In vitro tolerance was evaluated by the MTT method in human corneal-limbal epithelial cells and normal human conjunctival cells. In vivo tolerance was analyzed by biomicroscopy and specular microscopy in rabbit eyes. The ocular hypotensive effect was evaluated measuring IOP for 8 hours in rabbit eyes. Results.: All the formulations demonstrated good in vitro and in vivo tolerance. 5-MCA-NAT in CMC medium viscosity 0.5% was the most effective at reducing IOP (maximum IOP reduction, 30.27%), and its effect lasted approximately 7 hours. Conclusions.: The hypotensive effect of 5-MCA-NAT was increased by using bioadhesive polymers in formulations that are suitable for the ocular surface and also protective of the eye in long-term therapies. The use of 5-MCA-NAT combined with bioadhesive polymers is a good strategy in the treatment of ocular hypertension and glaucoma.

Multifactorial approach to non-viral gene therapy: development of an efficient system for the retina

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk. Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen because of its similarity to rhPA in terms of structure. The PR-mAb was prepared by hybridoma technology and screened by ELISA-elution assay. Screening antibody was performed using rhPA milk in an ELISA-elution assay. The antibody clone C4-PR-mAb was selected for immunoaffinity chromatography. The rhPA was effectively bound to immobilized C4-PR-mAb on the column and was eluted with Tris buffer comprising 0.75 mol/L ammonium sulfate and 40n% propanediol (pH7.9). The rhPA was further purified by passing through Chromdex75 gel filtration column. Results: There were 12 hybridoma strains selected into the polyol-responsive mAbs screen step and three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). The rhPA can be purified from transgenic rabbit milk and maintained a higher thrombolytic activity in vitro by FAPA. Conclusion: The results demonstrate the suitability of the alternative approach used in this study. Using immunoaffinity chromatography and gel filtration column is feasible and convenient for extracting rhPA from milk, and should be useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

Características del desprendimiento de retina regmatógeno en pacientes atendidos en la Clínica Oftalmológica OFTALMOLASER, Cuenca- Ecuador en el período 2013-2015

El presente estudio tuvo como objetivo determinar las características epidemiológicas del desprendimiento regmatógeno de retina en la población atendida en la Clínica Oftalmológica Oftalmolaser en el periodo 2013 - 2015. Se realizó un estudio cuantitativo descriptivo basado en la revisión y análisis del registro de historias clínicas, obteniendo los siguientes resultados: la frecuencia del desprendimiento regmatógeno de retina fue mayor en el sexo masculino con 54%; el mayor numero de casos se presento en la sexta década de la vida con 30%, la edad promedio de la población en estudio fue 55 años, los antecedentes patológicos mas frecuentes fueron miopía y pseudofaquia con un 27,4% para cada uno, el 51,08% de la población no presentó ningún antecedente quirúrgico oftalmológico, el 36,02% refirió haber tenido cirugía de catarata, el 63,40% de pacientes presentaron alteración de la agudeza visual como manifestación sintomática mientras que defectos del campo visual se presentaron en un 20,85%, el ojo derecho fue el mas afectado con 56%, el 56% de los pacientes acudieron luego de cuatro semanas del inicio de la sintomatología, el 82,78% presentaron agudeza visual de 20/400 al momento del examen físico y el 56,11% presentaron PIO normal mientras que el 40% presento hipotonía ocular. La localización más frecuente fue la temporal superior con un 30,56% y el 71% presentaron afección de la mácula evidenciada en la retinoscopía

Distribution of Light in the Human Retina under Natural Viewing Conditions

Age-related macular degeneration (AMD) is the leading cause of blindness inAmerica. The fact that AMD wreaks most of the damage in the center of the retina raises the question of whether light, integrated over long periods, is more concentrated in the macula. A method, based on eye-tracking, was developed to measure the distribution of light in the retina under natural viewing conditions. The hypothesis was that integrated over time, retinal illumination peaked in the macula. Additionally a possible relationship between age and retinal illumination was investigated. The eye tracker superimposed the subject’s gaze position on a video recorded by a scene camera. Five informed subjects were employed in feasibility tests, and 58 naïve subjects participated in 5 phases. In phase 1 the subjects viewed a gray-scale image. In phase 2, they observed a sequence of photographic images. In phase 3 they viewed a video. In phase 4, they worked on a computer; in phase 5, the subjects walked around freely. The informed subjects were instructed to gaze at bright objects in the field of view and then at dark objects. Naïve subjects were allowed to gaze freely for all phases. Using the subject’s gaze coordinates, and the video provided by the scene camera, the cumulative light distribution on the retina was calculated for ~15° around the fovea. As expected for control subjects, cumulative retinal light distributions peaked and dipped in the fovea when they gazed at bright or dark objects respectively. The light distribution maps obtained from the naïve subjects presented a tendency to peak in the macula for phases 1, 2, and 3, a consistent tendency in phase 4 and a variable tendency in phase 5. The feasibility of using an eye-tracker system to measure the distribution of light in the retina was demonstrated, thus helping to understand the role played by light exposure in the etiology of AMD. Results showed that a tendency for light to peak in the macula is a characteristic of some individuals and of certain tasks. In these situations, risk of AMD could be increased. No significant difference was observed based on age.