Diabetes Impairs the Aldehyde Detoxifying Capacity of the Retina

Purpose: We studied whether the accumulation of advanced lipoxidation end-products (ALEs) in the diabetic retina is linked to the impairment of lipid aldehyde detoxification mechanisms.

Methods: Retinas were collected from nondiabetic and diabetic rats and processed for conventional and quantitative RT-PCR (qRT-PCR), Western blotting, immunohistochemistry, and aldehyde dehydrogenase (ALDH) activity assays. The effect of the ALDH1a1 inhibitor, NCT-501, on ALE accumulation and cell viability in cultured Müller glia also was investigated.

Results: The rat retina expressed a range of lipid aldehyde detoxifying ALDH and aldo-keto reductase (AKR) genes. In diabetes, mRNA levels were reduced for 5 of 9 transcripts tested. These findings contrasted with those in the lens and cornea where many of these enzymes were upregulated. We have reported previously accumulation of the acrolein (ACR)-derived ALE, FDP-lysine, in retinal Müller glia during diabetes. In the present study, we show that the main ACR-detoxifying ALDH and AKR genes expressed in the retina, namely, ALDH1a1, ALDH2, and AKR1b1, are principally localized to Müller glia. Diabetes-induced FDP-lysine accumulation in Müller glia was associated with a reduction in ALDH1a1 mRNA and protein expression in whole retina and a decrease in ALDH1a1-immunoreactivity specifically within these cells. No such changes were detected for ALDH2 or AKR1b1. Activity of ALDH was suppressed in the diabetic retina and blockade of ALDH1a1 in cultured Müller glia triggered FDP-lysine accumulation and reduced cell viability.

Conclusions: These findings suggest that downregulation of ALDH and AKR enzymes, particularly ALDH1a1, may contribute ALE accumulation in the diabetic retina.

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.

Regulation of Glucose uptake in mammalian cells by phosphorylation networks

Objectives: Characterize the role of protein kinase WNK1 in the phosphorylation network regulating cellular glucose uptake

Fish nodavirus lytic cycle and semipermissive expression in mammalian and fish cell cultures

In this study, Dicentrarchus labrax encephalitis virus (DIEV), which causes sea bass encephalitis, was propagated in cell culture, thus allowing study of its lytic cycle, DIEV infection of mammalian and fish cells induced different patterns of expression of capsid proteins, which were assembled as virus-like particles, accumulating in the cytoplasm either as diffuse masses or in vesicles, as shown by electron microscopy, These particles correspond to virions, as shown by their ability to induce Secondary infection, Fish cells proved to be more permissive for DIEV than mammalian cells, although virus yield remained low, RNA analysis of infected sea bass cells revealed DIEV RNA3, in addition to genomic RNA1 and RNA2, and the presence of the RNA;! minus strand, thus demonstrating the replication of the DIEV genome, In addition, DIEV RNA-dependent RNA polymerase was associated with mature virions even after purification by a CsCl gradient, but it was dissociated when capsids were destabilized, In addition to providing more information about the relatedness of DIEV to the members of the family Nodaviridae, this study shows that fish nodaviruses may not be able to infect as wide a variety of cells as insect nodaviruses can.

Investigations of the Toxic Effect of Silver Nanoparticles on Mammalian Cell Lines

Silver nanoparticles are widely used for many applications. In this study silver nanoparticles have been tested for their toxic effect on fibroblasts (NIH-3T3), on a human lung adenocarcinoma epithelial cell line (A-549), on PC-12-cells, a rat adrenal pheochromocytoma cell line, and on HEP-G2-cells, a human hepatocellular carcinoma cell line. The viability of the cells cultivated with different concentrations of silver was determined by the MTT assay, a photometric method to determine cell metabolism. Dose-response curves were extrapolated and IC50, total lethal concentration (TLC), and no observable adverse effect concentration (NOAEC) values were calculated for each cell line. As another approach, ECIS (electric-cell-substrate-impedance-sensing) an automated method to monitor cellular behavior in real-time was applied to observe cells cultivated with silver nanoparticles. To identify the type of cell death the membrane integrity was analyzed by measurements of the lactate dehydrogenase releases and by determination of the caspase 3/7 activity. To ensure that the cytotoxic effect of silver nanoparticles is not traced back to the presence of Ag+ ions in the suspension, an Ag+ salt (AgNO3) has been examined at the same concentration of Ag+ present in the silver nanoparticle suspension that is assuming that the Ag particles are completely available as Ag+ ions.

Improving protein yield from mammalian cells by manipulation of stress response pathways

Monoclonal antibodies are a class of therapeutic that is an expanding area of the lucrative biopharmaceutical industry. These complex proteins are predominantly produced from large cultures of mammalian cells; the industry standard cell line being Chinese Hamster Ovary (CHO) cells. A number of optimisation strategies have led to antibody titres from CHO cells increasing by a hundred-fold, and it has been proposed that a further bottleneck in biosynthesis is in protein folding and assembly within the secretory pathway. To alleviate this bottleneck, a CHO-derived host cell line was generated by researchers at the pharmaceutical company UCB that stably overexpressed two critical genes: XBP1, a transcription factor capable of expanding the endoplasmic reticulum and upregulating protein chaperones; and Ero1α, an oxidase that replenishes the machinery of disulphide bond formation. This host cell line, named CHO-S XE, was confirmed to have a high yield of secreted antibody. The work presented in this thesis further characterises CHO-S XE, with the aim of using the information gained to lead the generation of novel host cell lines with more optimal characteristics than CHO-S XE. In addition to antibodies, it was found that CHO-S XE had improved production of two other secreted proteins: one with a simple tertiary structure and one complex multi-domain protein; and higher levels of a number of endogenous protein chaperones. As a more controlled system of gene expression to unravel the specific roles of XBP1 and Ero1α in the secretory properties of CHO-S XE, CHO cells with inducible overexpression of XBP1, Ero1α, or a third gene involved in the Unfolded Protein Response, GADD34, were generated. From these cell lines, it was shown that more antibody was secreted by cells with induced overexpression of XBP1; however, Ero1α and GADD34 overexpression did not improve antibody yield. Further investigation revealed that endogenous XBP1 splicing was downregulated in the presence of an abundance of the active form of XBP1. This result indicated a novel aspect of the regulation of the activity of IRE1, the stress-induced endoribonuclease responsible for XBP1 splicing. Overall, the work described in this thesis confirms that the overexpression of XBP1 has an enhancing effect on the secretory properties of CHO cells; information which could contribute to the development of host cells with a greater capacity for antibody production.

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

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.

Dopamine and visually regulated eye growth in chick

Retinal image properties such as contrast and spatial frequency play important roles in the development of normal vision. For example, visual environments comprised solely of low contrast and/or low spatial frequencies induce myopia. The visual image is processed by the retina and it then locally controls eye growth. In terms of the retinal neurotransmitters that link visual stimuli to eye growth, there is strong evidence to suggest involvement of the retinal dopamine (DA) system. For example, effectively increasing retinal DA levels by using DA agonists can suppress the development of form-deprivation myopia (FDM). However, whether visual feedback controls eye growth by modulating retinal DA release, and/or some other factors, is still being elucidated. This thesis is chiefly concerned with the relationship between the dopaminergic system and retinal image properties in eye growth control. More specifically, whether the amount of retinal DA release reduces as the complexity of the image degrades was determined. For example, we investigated whether the level of retinal DA release decreased as image contrast decreased. In addition, the effects of spatial frequency, spatial energy distribution slope, and spatial phase on retinal DA release and eye growth were examined. When chicks were 8-days-old, a cone-lens imaging system was applied monocularly (+30 D, 3.3 cm cone). A short-term treatment period (6 hr) and a longer-term treatment period (4.5 days) were used. The short-term treatment tests for the acute reduction in DA release by the visual stimulus, as is seen with diffusers and lenses, whereas the 4.5 day point tests for reduction in DA release after more prolonged exposure to the visual stimulus. In the contrast study, 1.35 cyc/deg square wave grating targets of 95%, 67%, 45%, 12% or 4.2% contrast were used. Blank (0% contrast) targets were included for comparison. In the spatial frequency study, both sine and square wave grating targets with either 0.017 cyc/deg and 0.13 cyc/deg fundamental spatial frequencies and 95% contrast were used. In the spectral slope study, 30% root-mean-squared (RMS) contrast fractal noise targets with spectral fall-off of 1/f0.5, 1/f and 1/f2 were used. In the spatial alignment study, a structured Maltese cross (MX) target, a structured circular patterned (C) target and the scrambled versions of these two targets (SMX and SC) were used. Each treatment group comprised 6 chicks for ocular biometry (refraction and ocular dimension measurement) and 4 for analysis of retinal DA release. Vitreal dihydroxyphenylacetic acid (DOPAC) was analysed through ion-paired reversed phase high performance liquid chromatography with electrochemical detection (HPLC-ED), as a measure of retinal DA release. For the comparison between retinal DA release and eye growth, large reductions in retinal DA release possibly due to the decreased light level inside the cone-lens imaging system were observed across all treated eyes while only those exposed to low contrast, low spatial frequency sine wave grating, 1/f2, C and SC targets had myopic shifts in refraction. Amongst these treatment groups, no acute effect was observed and longer-term effects were only found in the low contrast and 1/f2 groups. These findings suggest that retinal DA release does not causally link visual stimuli properties to eye growth, and these target induced changes in refractive development are not dependent on the level of retinal DA release. Retinal dopaminergic cells might be affected indirectly via other retinal cells that immediately respond to changes in the image contrast of the retinal image.