Understanding of retinal degeneration through the lens of high throughput gene expression

The retina is comprised of an intricate network of neurons, including the light-sensing photoreceptors, and supporting glia. Retinal glia maintain tissue homeostasis, while the underlying retinal-pigmented epithelium (RPE) and vascular choroid synergise to ensures a constant nutrient supply and waste removal from the highly metabolically active photoreceptors. Photoreceptor degeneration is central to almost all retinal degenerative diseases, including the increasingly prevalent and currently untreatable Age-Related Macular Degeneration (AMD). Oxidative stress and retinal inflammation are established as drivers of degeneration and are instigated by retinal glia and immune cells from circulation and choroid. Although reductionist approaches and histological observations have unravelled some of the molecular players driving photoreceptor degeneration, the application of high-throughput methods is required to penetrate the bewildering molecular complexity of retinal degeneration. To this end, the work presented in this thesis leverages powerful RNA profiling technologies to uncover novel gene expression patterns in mRNA and regulatory microRNA (miRNA) underpinning retinal degeneration. This thesis integrates data from 7 bulk miRNA/mRNA datasets and 3 single-cell RNA sequencing datasets (scRNAseq) to probe the transcriptomes of (1) the whole degenerating retina, as well as (2) effector cells driving degeneration including Muller glia and choroidal melanocytes and (3) extracellular vesicles (EV) as follow: Published results [1] presented in Chapter 3 describe changes in the mRNA and miRNA in the mouse retina following retinal degeneration induced by photo-oxidative damage. miRNA are short, non-coding RNAs working within a protein complex (RNA-induced silencing complex - RISC) to repress the translation of their mRNA targets. This chapter demonstrates that retinal degeneration is underpinned by a shift in miRNA and mRNA expression towards a proinflammatory state. Simultaneously, retinal degeneration alters miRNA binding sites within pro-inflammatory glial mRNAs allowing targeting by RISC-bound, neuronal miRNA. Published results [2] in Chapter 4 explore the role of EVs in retinal degeneration. EVs are membrane-bound vesicles secreted by nearly all cells as a form of intercellular communication between anatomically separated cells. This chapter developed the first published method for EV isolation from mouse retinas. Using this method, retinal EVs were found to be enriched with neuronal miRNA, and retinal degeneration results in a depletion of EV bioavailability. Lastly, this chapter demonstrates that inhibition of EV biogenesis accelerates retinal degeneration and results in impaired miRNA trafficking. Chapter 5 the explores miRNA-mRNA interactions in Muller glia using data integration from miRNA expression and scRNAseq. These results demonstrate that Muller glia activate a transient gene expression programme driving proliferation and pluripotency early in degeneration, then a shift towards a sustained pro-inflammatory programme follows. Published work [3] in Chapter 6 focuses on the response of choroidal melanocytes to inflammation. The choroid is a reservoir of and entry portal for immune cells in retinal degeneration, yet the relevance of the melanocytes (non-immune, highly abundant choroidal cells) in retinal degeneration is unknown. This chapter demonstrates that choroidal melanocytes have extensive immunomodulatory properties that are activated by both inflammatory challenge and retinal degeneration. In summary, this thesis uncovers novel tissue-level and cell-level gene expression patterns and regulatory networks altered in retinal degeneration, potentially providing the impetus for future therapeutics tacking complex retinal degenerations such as AMD

Instalación Eléctrica para Albergue Aislado

Dimensionado de una instalación Fotovoltaica para un albergue Aislado, que pueda satisfacer la demanda energética en las condiciones mas desfavorables. Incluyendo el estudio energético del emplazamiento basado en el recurso solar disponible y las posibles perdidas del generador. Asi como todos los planos necesarios tanto de la instalación eléctrica en baja tension del Albergue como la localización y conexión de los distintos equipos necesarios para la puesta en marcha

Bent-Core Liquid Crystals: Structures and Mesomorphic Properties

Bent-core (BC) molecules became an attractive liquid crystal class due to their potential use in smart displays and photonic devices. In contrast to calamitic mesogens, bent-shaped mesogens are self-organized superstructures with remarkable properties, given the presence of polar order in mesophases, although the molecules themselves are not chiral. A particular interest represents the biaxial nematic liquid crystal materials that are used in display technology and allow a faster switching response, compared to calamitic liquid crystals, with considerably reduced costs. This chapter briefly reviews the bent-core liquid crystals with three different core units in the structure: (1) 2,5-disubstituted oxadiazole, (2) 1,3-disubstituted benzene, and (3) 2,7-disubstituted naphthalene. To the central bent units (BUs) containing reactive functional groups of phenolic or aminic type, various mesogenic groups are symmetrically or asymmetrically connected, via esterification or condensation reactions. The obtained compounds showed biaxial nematic and/or smectic mesophases with high transition temperatures in the case of oxadiazole derivatives or cholesteric and banana-type mesophases with lower transition temperatures in the case of benzene and naphthalene derivatives

Alantolactona în tulpina, frunzele şi inflorescenţele de Tagetes Erecta

The genus Tagetes (Asteraceae) comprises species with a wide arrays of uses. Previous studies have been focused on the distribution of flavonoids in the genus but little has been done on the identification of the sesquiterpenlactones in the medicinal, cosmetic and aromatic species. The significance of the distribution and accumulation of this compound thorough the plant is not yet clear. In the current study, the alantolactone content of aerial parts of Tagetes erecta was investigated, during budding and full flowering stages. The TLC and HPLC methods confirmed the presence of alantolactones, greater in budding stage sampes than in full flowering ones (0.2309 μg % in buds, 0.5097 μg % in leaves). The fertilized plant inflorescences contain greater amounts of studied metabolites than unfertilized plants. The leaves content is not influenced by fertilization. The presence of this metabolite alerts to the allergenic potential of plant, especially in budding stag

Memory-enhancing activities of the aqueous extract of Albizia adianthifolia leaves in the 6-hydroxydopamine-lesion rodent model of Parkinson’s disease

BACKGROUND: Albizia adianthifolia (Schumach.) W. Wright (Fabaceae) is a traditional herb largely used in the African traditional medicine as analgesic, purgative, anti-inflammatory, antioxidant, antimicrobial and memory-enhancer drug. This study was undertaken in order to evaluate the possible cognitive-enhancing and antioxidative effects of the aqueous extract of A. adianthifolia leaves in the 6-hydroxydopamine-lesion rodent model of Parkinson’s disease. METHODS: The effect of the aqueous extract of A. adianthifolia leaves (150 and 300 mg/kg, orally, daily, for 21 days) on spatial memory performance was assessed using Y-maze and radial arm-maze tasks, as animal models of spatial memory. Pergolide - induced rotational behavior test was employed to validate unilateral damage to dopamine nigrostriatal neurons. Also, in vitro antioxidant activity was assessed through the estimation of total flavonoid and total phenolic contents along with determination of free radical scavenging activity. Statistical analyses were performed using two-way analysis of variance (ANOVA). Significant differences were determined by Tukey’s post hoc test. F values for which p < 0.05 were regarded as statistically significant. Pearson’s correlation coefficient and regression analysis were used in order to evaluate the association between behavioral parameters and net rotations in rotational behavior test. RESULTS: The 6-OHDA-treated rats exhibited the following: decrease of spontaneous alternations percentage within Y-maze task and increase of working memory errors and reference memory errors within radial arm maze task. Administration of the aqueous extract of A. adianthifolia leaves significantly improved these parameters, suggesting positive effects on spatial memory formation. Also, the aqueous extract of A. adianthifolia leaves showed potent in vitro antioxidant activity. Furthermore, in vivo evaluation, the aqueous extract of A. adianthifolia leaves attenuated the contralateral rotational asymmetry observed by pergolide challenge in 6-OHDA-treated rats. CONCLUSIONS: Taken together, our results suggest that the aqueous extract of A. adianthifolia leaves possesses antioxidant potential and might provide an opportunity for management neurological abnormalities in Parkinson’s disease conditions

Short exposure to photo-oxidative damage triggers molecular signals indicative of early retinal degeneration

IntroductionAge-related macular degeneration (AMD) is the leading cause of blindness in the developed world, currently affecting over 350 billion people globally. For the most prevalent late-stage form of this disease, atrophic AMD, there are no available prevention strategies or treatments, in part due to inherent difficulties in early-stage diagnosis. Photo-oxidative damage is a well-established model for studying inflammatory and cell death features that occur in late-stage atrophic AMD, however to date has not been investigated as a potential model for studying early features of disease onset. Therefore, in this study we aimed to determine if short exposure to photo-oxidative damage could be used to induce early retinal molecular changes and advance this as a potential model for studying early-stage AMD.MethodsC57BL/6J mice were exposed to 1, 3, 6, 12, or 24h photo-oxidative damage (PD) using 100k lux bright white light. Mice were compared to dim-reared (DR) healthy controls as well as mice which had undergone long periods of photo-oxidative damage (3d and 5d-PD) as known timepoints for inducing late-stage retinal degeneration pathologies. Cell death and retinal inflammation were measured using immunohistochemistry and qRT-PCR. To identify retinal molecular changes, retinal lysates were sent for RNA sequencing, following which bioinformatics analyses including differential expression and pathway analyses were performed. Finally, to investigate modulations in gene regulation as a consequence of degeneration, microRNA (miRNA) expression patterns were quantified using qRT-PCR and visualized using in situ hybridization.ResultsShort exposure to photo-oxidative damage (1-24h-PD) induced early molecular changes in the retina, with progressive downregulation of homeostatic pathways including metabolism, transport and phototransduction observed across this time-course. Inflammatory pathway upregulation was observed from 3h-PD, preceding observable levels of microglia/macrophage activation which was noted from 6h-PD, as well as significant photoreceptor row loss from 24h-PD. Further rapid and dynamic movement of inflammatory regulator miRNA, miR-124-3p and miR-155-5p, was visualized in the retina in response to degeneration.ConclusionThese results support the use of short exposure to photo-oxidative damage as a model of early AMD and suggest that early inflammatory changes in the retina may contribute to pathological features of AMD progression including immune cell activation and photoreceptor cell death. We suggest that early intervention of these inflammatory pathways by targeting miRNA such as miR-124-3p and miR-155-5p or their target genes may prevent progression into late-stage pathology

Small-Medium Extracellular Vesicles and Their miRNA Cargo in Retinal Health and Degeneration: Mediators of Homeostasis, and Vehicles for Targeted Gene Therapy

Photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (AMD). However, the molecular mechanisms underlying these biological processes are largely unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. EVs, including exosomes, encapsulate and transfer microRNA (miRNA) to recipient cells and in this way can modulate the environment of recipient cells. Dysregulation of EVs however is correlated to a loss of cellular homeostasis and increased inflammation. In this work we investigated the role of isolated retinal small-medium sized EV (s-mEV) which includes exosomes in both the healthy and degenerating retina. Isolated s-mEV from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and in situ hybridization of s-mEV-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of GW4869-induced exosome depletion. Results demonstrated an inverse correlation between s-mEV concentration and photoreceptor survivability, with a decrease in s-mEV numbers following degeneration. Small RNAseq revealed that s-mEVs contained uniquely enriched miRNAs in comparison to in whole retinal tissue, however, there was no differential change in the s-mEV miRNAnome following photo-oxidative damage. Exosome inhibition via the use of GW4869 was also found to exacerbate retinal degeneration, with reduced retinal function and increased levels of inflammation and cell death demonstrated following photo-oxidative damage in exosome-inhibited mice. Further, GW4869-treated mice displayed impaired translocation of photoreceptor-derived miR-124-3p to the inner retina during damage. Taken together, we propose that retinal s-mEV and their miRNA cargo play an essential role in maintaining retinal homeostasis through immune-modulation, and have the potential to be used in targeted gene therapy for retinal degenerative diseases.This work would not have been possible without the support of the National Health and Medical Research Council of Australia (NHMRC: 1127705), Retina Australia, The Gordon and Gretel Bootes Foundation, and The ANU Translational Fellowshi

Voluntary exercise modulates pathways associated with amelioration of retinal degenerative diseases

Background: Exercise has been shown to promote a healthier and longer life and linked to a reduced risk of developing neurodegenerative diseases including retinal degenerations. However, the molecular pathways underpinning exercise-induced cellular protection are not well understood. In this work we aim to profile the molecular changes underlying exercise-induced retinal protection and investigate how exercise-induced inflammatory pathway modulation may slow the progression of retinal degenerations. Methods: Female C57Bl/6J mice at 6 weeks old were given free access to open voluntary running wheels for a period of 28 days and then subjected to 5 days of photo-oxidative damage (PD)-induced retinal degeneration. Following, retinal function (electroretinography; ERG), morphology (optical coherence tomography; OCT) and measures of cell death (TUNEL) and inflammation (IBA1) were analysed and compared to sedentary controls. To decipher global gene expression changes as a result of voluntary exercise, RNA sequencing and pathway and modular gene co-expression analyses were performed on retinal lysates of exercised and sedentary mice that were subjected to PD, as well as healthy dim-reared controls. Results: Following 5 days of PD, exercised mice had significantly preserved retinal function, integrity and reduced levels of retinal cell death and inflammation, compared to sedentary controls. In response to voluntary exercise, inflammatory and extracellular matrix integrity pathways were significantly modulated, with the gene expression profile of exercised mice more closely trending towards that of a healthy dim-reared retina. Conclusion: We suggest that voluntary exercise may mediate retinal protection by influencing key pathways involved in regulating retinal health and shifting the transcriptomic profile to a healthy phenotype

STUDIES REGARDING THE INFLUENCE OF IRRIGATION ON FRUIT YIELD AT APPLE TREES IN IARA - TURDA, TRANSYLVANIA CONDITIONS

The paper presents the results of the experiments that were carried out during 2012-2014 period, in the Iara-Turda depressionary area conditions. The research`s general objective is the study of agroproductivity at Jonagold, Florina, Generos, Idared, Jonathan, Granny Smith and Golden Delicious apple varieties, all grafted on to a semi-vigourous M106 rootstock. The influence of the irrigation and fertilization regime was studied in an polifactorial experiment that aimed the study of the interactions of three factors and the specific and cumulative effects on growth and fructification parameters of the apple tree varieties in the pedoclimatic context of the studied area. The present paper refers only to the interaction and effect of the irrigation regime in relation to the studied factors