1,419 research outputs found
Ocular and systemic vascular function in human ageing
The importance of vascular risk factors in various age-related pathologies has been extensively researched. Nevertheless, the haemodynamic disturbance occurring in various ocular and systemic vascular beds to impact upon ocular function remained largely unknown. The purpose of the following studies was to explore the presence and impact of both ocular and systemic vascular dysregulation as well as biochemical vascular risk factors in healthy elderly individuals and patients with age-related macular degeneration. Furthermore, the possible role was played by circulatory oxidative stress and its relationship with endothelial dysfunction at both ocular and systemic levels has also been investigated. There were four principal sections to the present work: 1. To assess the relationship between ocular and systemic anti-oxidative defence in healthy individuals The principal findings of this work were: -It has been shown that MPOD significantly and positively related with circulatory GSH levels. 2. To investigate macro- and microcirculation and oxidative stress in early AMD patients without overt systemic disease The principal findings of this work were: -AMD patients exhibit abnormal macrocirculation compared to the controls. -Blood GSSG level was significantly higher in early AMD patients than controls. -AMD patients showed abnormal microcirculation at retinal level compared. -In early AMD patients, retinal venous RT positively correlated with blood GSSG levels. 3. To assess the relationship between ocular vascular function and circulatory markers of endothelial dysfunction and CVD risk The principal findings of this work were: -Age had a positive effect on ET-1, vWF and slope of retinal arterial constriction in otherwise healthy individuals. -Even in otherwise healthy individuals, retinal arterial vascular function showed a significant correlation with circulatory markers of endothelial dysfunction and CVD risk. 4. To assess age-related changes in ANS and vascular function, and their relationship to retinal vascular function parameters The principal findings of this work were: -Elderly individuals demonstrated abnormal circadian changes of PSNS activity compared to middle-aged group. -Elderly groups showed higher ET-1 and vWF level as well as C-IMT and AIx, and also impaired retinal vascular function compared to the middle-aged group. -In the elderly group, impaired retinal vascular function significantly correlated with the dysregulation of PSNS activity
Autosomal recessive retinitis pigmentosa, identification and partial characterisation of a novel gene implicated in RP25
The purpose of this project is to identify the causative gene for one type of autosomal
recessive retinitis pigmentosa, RP25. Through CGH (comparative genome
hybridisation) and mutation screening, independent mutations were identified in arRP
affected Spanish families mapping to RP25. These mutations were identified within a
cluster of uncharacterised gene transcripts all which have EGF-like repeat domains;
Q5T669, Q5T1H1, Q9H557_human, Q5TEL3_human, Q5TEL4_human,
Q5VVG4_human, and Q5T3C8. Through 5` and 3` RACE PCR analysis, the full
length gene was revealed to incorporate the EGFL11 gene. On assembling all
available data we noted that RP25 gene encompasses 30 exons belonging to nine
previously predicted genes and 13 newly identified exons, totaling 43 exons and
spanning the interval between 64,487,835 and 66,473,839 on chromosome 6q12.
The RP25 full length gene transcript is retinal specific. The genomic
length covers over 2.0 MB in size and is therefore the largest eye specific gene
identified to date. It is also the fifth largest gene in the human genome to date.
Homologs of the RP25 gene to Drosophila eys/eys-shut (Spacemaker) were
identified, leading to the annotation of the name EYS (SPAM). An apparently intact
eys gene is found across the mammalian clade, including monotremes (platypus) and
marsupials (opossum). However, despite the mutations and the presumed loss of
function associated with human disease, this gene has been dispensed with on at least
four separate occasions in the last 100 million years of mammalian evolution
including in the armadillo (Dasypus novemcinctus), little brown bat (Myotis
lucifugus) and ruminant (cattle and sheep) lineages. EYS has acquired several (<3)
reading-frame disruptions in three rodents (mouse, rat and guinea pig) representing two of the three major rodent clades. Through immunohistochemical and electron
microscopy analysis, a signal for SPAM was identified in the outer segments of
photoreceptor cells
Functional and molecular characterisation of a unique double homozygous variant causing ABCA4-related retinopathy
Masteroppgave i molekylærbiologiMOL399MAMN-MO
Clinical and Molecular-Genetic Insights into the Role of Oxidative Stress in Diabetic Retinopathy: Antioxidant Strategies and Future Avenues
Reactive oxygen species (ROS) overproduction and ROS-signaling pathways activation attack the eyes. We evaluated the oxidative stress (OS) and the effects of a daily, core nutritional supplement regimen containing antioxidants and omega 3 fatty acids (A/ω3) in type 2 diabetics (T2DM). A case-control study was carried out in 480 participants [287 T2DM patients with (+)/without (−) diabetic retinopathy (DR) and 193 healthy controls (CG)], randomly assigned to a daily pill of A/ω3. Periodic evaluation through 38 months allowed to outline patient characteristics, DR features, and classic/OS blood parameters. Statistics were performed by the SPSS 24.0 program. Diabetics displayed significantly higher circulating pro-oxidants (p = 0.001) and lower antioxidants (p = 0.0001) than the controls. Significantly higher plasma malondialdehyde/thiobarbituric acid reactive substances (MDA/TBARS; p = 0.006) and lower plasma total antioxidant capacity (TAC; p = 0.042) and vitamin C (0.020) was found in T2DM + DR versus T2DM-DR. The differential expression profile of solute carrier family 23 member 2 (SLC23A2) gene was seen in diabetics versus the CG (p = 0.001), and in T2DM + DR versus T2DM − DR (p < 0.05). The A/ω3 regime significantly reduced the pro-oxidants (p < 0.05) and augmented the antioxidants (p < 0.05). This follow-up study supports that a regular A/ω3 supplementation reduces the oxidative load and may serve as a dietary prophylaxis/adjunctive intervention for patients at risk of diabetic blindness
Melanin-binding colorants: updating molecular modeling, staining and labeling mechanisms, and biomedical perspectives
Melanin and melanoma tumors are two fields of increasing interest in biomedical research.
Melanins are ubiquitous biopigments with adaptive value and multiple functions, and occur in the
malignant melanoma. Although several chemical structures have been proposed for eumelanin,
molecular modeling and orbitals indicate that a planar or spiral benzoquinone-porphycene polymer
would be the model that better explains the broad-band light and ultrasound absorption, electric
conductivity, and graphite-like organization shown by X-ray crystallography and electron microscopy.
Lysosomes and melanosomes are selectively labeled by vital probes, and melanin also binds to
metal cations, colorants, and drugs, with important consequences in pharmacology, pathology,
and melanoma therapy. In addition to traditional and recent oncologic treatments, photodynamic,
photothermal, and ultrasound protocols represent novel modalities for melanoma therapy. Since
eumelanin is practically the ideal photothermal and ultrasound sensitizer, the vibrational decay
from photo-excited electrons after NIR irradiation, or the electrochemical production of ROS and
radicals after ultrasound absorption, induce an efficient heating or oxidative response, resulting in
the damage and death of tumor cells. This allows repetitive treatments due to the remaining melanin
contained in tumoral melanophages. Given that evolution and prognosis of the advanced melanoma
is still a concern, new biophysical procedures based on melanin properties can now be developed
and applie
Deep learning for diabetic retinopathy analysis : a review, research challenges, and future directions
Deep learning (DL) enables the creation of computational models comprising multiple processing layers that learn data representations at multiple levels of abstraction. In the recent past, the use of deep learning has been proliferating, yielding promising results in applications across a growing number of fields, most notably in image processing, medical image analysis, data analysis, and bioinformatics. DL algorithms have also had a significant positive impact through yielding improvements in screening, recognition, segmentation, prediction, and classification applications across different domains of healthcare, such as those concerning the abdomen, cardiac, pathology, and retina. Given the extensive body of recent scientific contributions in this discipline, a comprehensive review of deep learning developments in the domain of diabetic retinopathy (DR) analysis, viz., screening, segmentation, prediction, classification, and validation, is presented here. A critical analysis of the relevant reported techniques is carried out, and the associated advantages and limitations highlighted, culminating in the identification of research gaps and future challenges that help to inform the research community to develop more efficient, robust, and accurate DL models for the various challenges in the monitoring and diagnosis of DR
Desenvolvimento de testes genéticos por PCR em tempo-real para diagnóstico rápido de LHON e surdez
Mitochondrial cytopathies are a set of diseases caused by a disturbance in the cell energy production. Mitochondrial dysfunction impairs efficiency of the mitochondrial respiratory chain (MRC) and ATP production, affecting the organism’s energetic equilibrium. Pathogenic sequence variants in mitochondrial DNA (mtDNA) that lead to these pathologies are more frequent in tissues that need higher energy levels to function.
The presented work looks into two such diseases: Leber’s Hereditary Optic Neuropathy and mitochondrial non-syndromic Hearing Loss (MNSHL).
LHON is characterized by presence of genetic alterations in mtDNA, with three main primary pathogenic sequence variants existing, which represent 90-95% of LHON cases with an identified genetic cause: m.3460G>A, in ND1 subunit gene; m.11778G>A, in ND4 subunit gene; and m.14484T>C, in ND6 subunit gene. All of these are subunits of the MRC’s complex I. These mtDNA variations lead to mitochondrial dysfunction in complex I, creating ATP depletion, reactive oxygen species (ROS) increase and oxidative stress.
LHON is commonly characterized by a sequential vision loss and, within 1 year of symptoms starting, 97% of patients with vision loss in one eye develop loss in the second. Therapy administration yields good outcomes, if done in a short-time span after first vision loss. It is essential to quickly and reliably scan for pathogenic sequence variants, in order to act timely and rescue function.
Mitochondrial non-syndromic hearing loss and deafness (MNSHL) is characterized by sensorineural hearing loss (SNHL). This type of hearing loss, particularly when induced by aminoglycosides, has also three primary pathogenic sequence variants associated with ototoxicity: m.1494C>T and m.1555A>G, both in the MTRNR1 gene, and m.7445A>G, in the MTCO1 and MTTS1 genes. These are responsible for ATP depletion, an increase of ROS and oxidative stress, due to alterations in the mitochondrial ribosome or tRNA.
In MNSHL, the cochlea is the affected tissue. With this disorder the principal modifier factor is the administration of aminoglycosides, a type of antibiotics, which trigger a cascade, that leads the individual permanently deaf. The best course of action is prevention, and to ensure clinical action is not dramatically slowed down, results that show whether administration is safe or not need to be quick.
The aim of this work, for both diseases, is the development of a screening method characterized by fast and reliable approach for genetic assessment, to be used for clinical guidance, particularly in therapeutics.
For LHON, the screening method is based on real-time PCR with High-Resolution Melting (HRM) analysis, for detection of the TOP-3 pathogenic sequence variants, by assessing the amplicon’s Tm. In this case, 94 samples were analyzed, including LHON suspected patients, relatives, other mitochondrial disease patients and healthy controls. All samples were previously
classified by another method, having then been blinded before the performance of this work.
For analysis, Real-Time PCR was run in triplicates, to allow for a more robust HRM analysis. The software had the ability to classify samples as different variants, wild-type or mutant; information which was then crossed with the previous classification of the sample to assess the success of the software classification. Samples were correctly assigned.
This approach provides results in a quick fashion that guides clinical action in a timely fashion. The presence of other polymorphisms in the amplicons might be a hindrance to the robustness of the results provided by this technique and their effect on variant classification needs to be considered. For this, a predictive in-silico analysis was performed, regarding all described variants’ presence in the sequences in analysis. Accordingly, an additional complementary method may be necessary for assurance of result’s specificity.
For MNSHL, the screening method was also real-time PCR based, but this one was performed with Amplification-Refractory Mutation System (ARMS) primers, designed for the pathogenic sequence variants previously associated in literature for the MNSHL. Discrimination of results was done based on amplification in positive cases and lack of it in negative cases. This approach analyzed 32 samples, including MNSHL suspected patients, their relatives, other mitochondrial disease patients and healthy controls, but only results concerning the m.1555A>G were obtained timely. All samples were previously classified by another method, having then been blinded before performance of this work.
For optimization, Real-Time PCR was run in duplicates, to increase robustness of analysis. The Real-Time software showed if samples amplified as wild-type or mutant, with classification following. This data was crossed with previous known classification of the samples to assess the success of the approach. All analyzed samples were correctly identified with this approach. However, two of the three pathogenic sequence variants did not achieve implementation within the timeframe necessary for their inclusion, namely m.1494C>T and m.7445A>G. The optimization of their screening was not possible and further work is necessary to optimize and implement the approach concerning the analysis for these variants.
In conclusion, it was possible to implement an analysis method for LHON’s TOP-3 pathogenic sequence variants within 24h, which represents a big step in precision medicine for diagnosis of this disease.
On the other hand, although the implementation was not concluded, a similar approach was started for MNSHL – that, when concluded, will have an enormous impact in preventing aminoglycoside induced HL.
This work represents a high impact scientific contribution in reverse translational research.As citopatias mitocondriais são um conjunto de doenças causadas por um distúrbio na produção de energia celular. A disfunção mitocondrial prejudica a eficiência da cadeia respiratória mitocondrial (CRM) e a produção de ATP, afetando o equilíbrio energético do organismo. As variações de sequência patogénicas no DNA mitocondrial (mtDNA) que levam a estas patologias são mais frequentes em tecidos que necessitam de maiores níveis de energia para funcionar.
O presente trabalho explora duas dessas doenças: Neuropatia ótica hereditária de Leber (LHON) e Surdez mitocondrial induzida por aminoglicosídeos.
A LHON é caracterizada pela presença de alterações genéticas do mtDNA, existindo três variações de sequência patogénicas primárias principais, que representam 90-95% de casos de LHON com identificação da causa genética: m.3460G>A, no gene que codifica a subunidade ND1; m.11778G>A, no gene que codifica a subunidade ND4; e m.14484T>C, no gene que codifica a subunidade ND6. Todas estas subunidades pertencem ao complexo I da CRM. Estas alterações no mtDNA levam a disfunção mitocondrial no complexo I, criando depleção de ATP, aumento de espécies reativas de oxigénio (ROS) e stresse oxidativo.
A LHON é comummente caracterizada pela perda sequencial de visão e, 1 ano após o início dos sintomas, 97% dos casos com perda de visão num olho desenvolvem perda de visão no segundo. A administração de terapia produz bons resultados, quando realizada num curto período de tempo após a primeira perda de visão. Assim, é essencial pesquisar variações de sequência patogénicas genéticas de forma rápida e fiável, para atuar rapidamente e recuperar a função visual.
A Surdez mitocondrial não-sindrómica (MNSHL), em particular a induzida por aminoglicosídeos, tem também três mutações principais associadas à perda de audição: m.1494C>T e m.1555A>G, ambas no gene MTRNR1, e m.7445A>G, nos genes MTCO1 e MTTS1. Estas são responsáveis pela depleção de ATP, aumento de ROS e stresse oxidativo, devido a alterações no ribossoma ou no tRNA mitocondrial.
Aqui, o tecido afetado é a cóclea. Nesta doença, o fator modificador em destaque é a administração de antibióticos de tipo aminoglicosídeos, que despoletam uma cascata de acontecimentos, levando à surdez permanente. A melhor estratégia passa pela prevenção, enquanto ao mesmo tempo se garante que a ação clínica não sofre atrasos. Desta forma, são necessários resultados rápidos, que demonstrem se a administração será segura ou não.
O objetivo deste trabalho, para ambas as doenças, é o desenvolvimento de um método de screening, caracterizado por uma abordagem rápida e fiável, usado para guiar a decisão clínica, particularmente na terapêutica.
Para a LHON, o método de screening é baseado em PCR em tempo-real com análise de High-Resolution Melting (HRM), para deteção das variantes
patogénicas TOP-3, avaliando as Tm dos amplicons. Neste caso, foram analisadas 94 amostras, incluindo doentes com suspeita de LHON, familiares, outros doentes com suspeita de outra doença mitocondrial e controlos saudáveis. Todas as amostras foram previamente classificadas por outro método, tendo sido sujeitas a anonimização antes da realização do trabalho.
Para a análise, a PCR em tempo-real foi realizada em triplicados, para permitir uma análise de HRM mais robusta. O software teve a capacidade de classificar amostras como diferentes variantes, ou seja, normal ou mutante. Esta informação foi cruzada com as classificações previamente existentes para avaliar o sucesso da classificação pelo software. As amostras foram corretamente classificadas.
Esta abordagem fornece resultados de forma rápida, podendo guiar a ação clínica em tempo útil. A presença de outros polimorfismos nos amplicons poderão obstruir a robustez dos resultados fornecidos por esta técnica e o seu efeito na classificação de variantes precisa de ser considerado. Por esta razão, foi realizada uma análise de previsão in-silico, considerando a presença de todas as variantes descritas. Nesse sentido, pode ser necessário um método complementar de análise para assegurar a especificidade dos resultados.
Para a Surdez mitocondrial não-sindrómica, o método de screening baseou-se também na PCR em tempo-real, mas foi realizada com primers de Amplification-Refractory mutation system (ARMS), desenhados para as variantes de sequência patogénicas associadas à MNSHL induzida por aminoglicosídeos, previamente descritas na literatura para esta doença. A discriminação de resultados foi feita com base na presença/ausência de amplificação para cada variante. Foram analisadas 32 amostras com esta abordagem, incluindo doentes com suspeita de MNSHL, seus familiares, doentes com suspeita de outra doença mitocondrial e controlos saudáveis, mas apenas foram obtidos resultados em tempo útil para a m.1555A>G. Todas as amostras tinham sido previamente classificadas por outro método, tendo sido anonimizadas antes da realização do trabalho.
Para a otimização, a PCR em tempo-real foi realizada em duplicados, aumentando a robustez da análise. O software de tempo-real mostrou quais as amostras que amplificaram como normais ou mutantes, permitindo a classificação das mesmas. Os dados foram comparados com as classificações previamente conhecidas, para avaliar o sucesso da abordagem em estudo. Todas as amostras em análise foram corretamente identificadas. No entanto, duas das três variantes patogénicas não foram implementadas em tempo útil para inclusão neste trabalho. Para a m.1494C>T e a m.7445A>G, a otimização não foi possível, e será necessário trabalho adicional no futuro, para a implementação da análise destas variantes.
Em conclusão, foi possível implementar um método da análise das variantes genéticas TOP-3 da LHON em 24h, o que representa um grande passo na medicina de precisão para diagnóstico desta doença.
Por outro lado, apesar de não ter sido concluída a implementação, iniciou-se uma abordagem semelhante para a MNSHL – que, quando for concluída, terá um enorme impacto para evitar a perda auditiva por exposição a aminoglicosídeos.
Este trabalho representa uma contribuição científica de alto impacto na investigação translacional reversa.O Laboratório de Biomedicina Mitocondrial e Teranóstica recebeu apoio financeiro da Santhera Pharmaceuticals que permitiu implementação do projeto nacional “Investigação Translacional Epidemiológica, Bigenómica e Funcional nas Atrofias Ópticas” (IP Professora Doutora Manuela Grazina).
Apoio financeiro do CNC.IBILI no âmbito do Plano Estratégico UID/NEU/04539/2019.Mestrado em Biologia Aplicad
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