Evaluation of the potentials for optical coherence tomography (OCT) to detect early signs of retinal neurodegeneration

Among neuroretinal degenerations, glaucoma and age-related macular degeneration (AMD) have become the most frequent reasons for irreversible blindness globally. Among the causes of the elderly and senile dementia, Alzheimer’s disease (AD) has the leading position, the early ocular symptoms of which can potentially be a prognostic factor. The aim of this thesis was the early in vivo ligand-free detection of degenerative changes in the inner and outer retinal layers, which was possible using high-resolution optical coherence tomography (OCT) with the machine learning (ML) algorithms: support vector machine (SVM) and principal component analysis (PCA). Prior to the application of SVM and PCA for the classification of human OCT images, evaluation of the classifiers was performed in the classification of optical phantoms, the accuracy of which was in the range of 82-100%. This was the first attempt to measure the textural properties of various polystyrene and silica beads optical phantoms. To identify optical changes that characterise early apoptosis, OCT imaging of axotomised retinal ganglion cells (RGCs) in ex vivo retinal murine explants was performed. Substantial optical alterations in RGC dendrites in the early stages of apoptosis (up to 2 hours) were detected. ML algorithms correctly classified the retinal texture of the inner plexiform layer (IPL) of transgenic AD mice in all cases, indicating the potential for further investigation in in vivo animal and human studies. Not only the optical signature but also the transparency of the dissected murine retinal explants was investigated. Moreover, ML classification of 3xTg mice IPL layer was studied in terms of optical changes due to the RGD dendritic atrophy. ML classifiers’ accuracy in the detection of early and neovascular AMD was 93-100% for the texture of retinal pigment epithelium, 69-67% for the outer nuclear layer, 70% for the inner segment and 60-90% for the outer segment of photoreceptors. Classification of AMD stages and comparison with the age-matched healthy controls was carried out in the outer retina and RPE. Grey-level co-occurrence, run-length matrices, local binary patterns features were extracted from the IPL of the macula to classify glaucoma OCT images. The accuracy of linear and non-linear SVMs, linear and quadratic discriminant analyses, decision tree and logistic regression was between 55-70%. Based on the classifiers’ precision, recall and F1-score, Gaussian SVM outperformed other ML techniques. In this study, the observation of early glaucomatous subtle optical changes of human IPL was conducted. Also, the significance of various supervised ML algorithms was investigated. Understanding the optical signature of cumulative inherent speckle of OCT scans arising from apoptotic retinal ganglion cells and photoreceptors may provide vital information for the prevention of retinal neurodegeneration

Longitudinal normative OCT retinal thickness data for wild-type mice, and characterization of changes in the 3×Tg-AD mice model of Alzheimer's disease

Mice are widely used as models for many diseases, including eye and neurodegenerative diseases. However, there is a lack of normative data for retinal thickness over time, especially at young ages. In this work, we present a normative thickness database from one to four-months-old, for nine layers/layer-aggregates, including the total retinal thickness, obtained from the segmentation of spectral-domain optical coherence tomography (SD-OCT) data from the C57BL6/129S mouse strain. Based on fifty-seven mice, this normative database provides an opportunity to study the ageing of control mice and characterize disease models' ageing, such as the triple transgenic mouse model of Alzheimer's disease (3×Tg-AD) used in this work. We report thickness measurements, the differences in thickness per layer, demonstrate a nasal-temporal asymmetry, and the variation of thickness as a function to the distance to the optic disc center. Significant differences were found between the transgenic group's thickness and the normative database for the entire period covered in this study. Even though it is well accepted that retinal nerve fiber layer (RNFL) thinning is a hallmark of neurodegeneration, our results show a thicker RNFL-GCL (RNFL-Ganglion cell layer) aggregate for the 3×Tg-AD mice until four-months-old.This study was supported by The Portuguese Foundation for Science and Technology (FCT) through PTDC/ EMD-EMD/28039/2017, UIDB/04950/2020, Pest-UID/ NEU/04539/2019, and by FEDER-COMPETE through POCI-01-0145-FEDER-028039.info:eu-repo/semantics/publishedVersio

Investigation of the Retinal Biomarkers of Alzheimer’s Disease and Atherosclerosis Using Hyperspectral Images

Le fait que l'oeil puisse être visualisé de manière non invasive ouvre des possibilités de mesure de biomarqueurs pour le diagnostic de conditions à long terme. Selon de nombreuses études, plusieurs maladies cardiovasculaires et neurodégénératives telles que la maladie d’Alzheimer (AD) et l’athérosclérose (ATH) se manifestent dans la rétine sous forme de modifications morphologiques pathologiques et / ou vasculaires. Des méthodes d'imagerie oculaire en deux dimensions et des techniques de tomographie par cohérence optique (OCT) en trois dimensions ont été développées pour fournir des descriptions des structures rétiniennes. Cependant, les images acquises par ces techniques permettent principalement de mesurer les caractéristiques spatiales et pas la variance relative de l’intensité des pixels sur différentes longueurs d’onde, de sorte que d’importantes caractéristiques liées aux tissus peuvent encore rester à découvrir. Dans cette étude, une caméra rétinienne métabolique hyperspectrale (MHRC) a été utilisée pour permettre l'acquisition d'une série d'images rétiniennes obtenues à des longueurs d'onde spécifiques couvrant le spectre du visible au proche infrarouge (NIR). Dans cette technique, le facteur de transmission, l'absorption et la diffusion de la lumière sont reflétés dans le spectre de la lumière émise par le tissu. Par conséquent, non seulement les caractéristiques spatiales communes mais également les « signatures spectrales » de biomolécules pourraient être révélées. Cela aide à trouver une plus grande variété de caractéristiques spatiales / spectrales pour une investigation plus précise des biomarqueurs rétiniens des maladies. En ce qui concerne les coûts et les limites associés aux diagnostics actuels de l’AD et de l’ATH, le but de cette thèse était d’analyser le contenu en informations d’images rétiniennes hyperspectrales riches en données dans le but de caractériser des informations discriminantes cachées liées aux tissus afin d’identifier des biomarqueurs possibles de ces deux maladies. À cette fin, une combinaison de caractéristiques vasculaires et de mesures de textures spatiales-spectrales ont été extraites de différentes régions anatomiques de la rétine. Dans le contexte de la maladie d'Alzheimer, des images rétiniennes de 20 cas présentant une altération cognitive et de 26 cas normaux cognitivement ont été acquises à l'aide de la caméra MHRC. Le statut amyloïde cérébral a été déterminé à partir de lectures binaires effectuées par un panel de 3 experts noteurs ayant participé à des études de TEP au 18F-Florbetaben. Des caractéristiques de l’image rétinienne ont été calculées, notamment la tortuosité et le diamètre des vaisseaux, ainsi que les mesures de textures spatiales-spectrales sur les artérioles, les veinules et le tissu environnant. Les veinules rétiniennes des sujets amyloïdes positifs (Aβ +) ont présenté une tortuosité moyenne plus élevée par rapport aux sujets amyloïdes négatifs (Aβ-). Le diamètre artériolaire des sujets Aβ + s'est avéré supérieur à celui des sujets Aβ- dans une zone adjacente à la tête du nerf optique. De plus, une différence significative entre les mesures de texture construites sur les artérioles rétiniennes et leurs régions adjacentes a été observée chez les sujets Aβ + par rapport aux Aβ-. Dans le contexte de l'ATH, 60 images rétiniennes de 30 ATH probables sur le plan clinique et 30 cas de contrôle ont été acquises. Les critères d'inclusion pour les sujets souffrant d'ATH comprenaient: l'infarctus du myocarde; angiographie coronaire montrant au moins une sténose coronaire (plus de 50%); et / ou une angioplastie coronaire; et /ou pontage coronaire. Les artérioles rétiniennes des sujets ATH ont montré un rétrécissement significatif par rapport aux sujets témoins. En outre, une différence significative entre les mesures de textures d'images prises sur les artérioles et les veinules rétiniennes et leurs régions adjacentes a été trouvée entre les sujets ATH et les sujets témoins. Nos études transversales ont montré que l’analyse hyperspectrale des images rétiniennes pouvait discerner avec une précision acceptable l’AD et l’ATH des sujets témoins correspondants.----------ABSTRACT The fact that eye can be visualized non-invasively, opens up possibilities to measure biomarkers for diagnosis of long-term conditions. A significant body of literature has demonstrated that many of the neurodegenerative and cardiovascular diseases such as Alzheimer’s disease (AD) and atherosclerosis (ATH) manifest themselves in retina as pathological and/or vasculature morphological changes. Methods for two-dimensional fundus imaging and techniques for three-dimensional optical coherence tomography (OCT) have been developed to provide descriptions of retinal structures. However, images acquired by these techniques mostly allow for measuring the spatial characteristics of the tissue and lack of the relative variances across differing wavelengths, thus important spectral features may remain uncovered. In this study, a Metabolic Hyperspectral Retinal Camera (MHRC) was used that permits the acquisition of a series of retinal images obtained at specific wavelengths covering the visible and near infrared (NIR) spectrum. In this technique, light transmittance, absorption, and scatter are reflected in the spectrum of light emitted from the tissue. Use of MHRC in this study was aimed to extract not only the common spatial features but also “spectral signatures” of biomolecules in retinal tissue. Regarding the costs and limitations of the current diagnostic methods for AD and ATH, the purpose of this thesis was to analyze the information content of data-rich hyperspectral retinal images to characterize tissue-related discriminatory information to identify possible biomarkers of Alzheimer’s disease and atherosclerosis. To this end, a combination of vascular features and spatial/spectral texture measures were extracted from different anatomical regions of the retina. In the context of AD, retinal images from 20 cognitively impaired and 26 cognitively unimpaired cases were acquired using MHRC. The cerebral amyloid status was determined from binary reads by a panel of three expert raters on 18F-Florbetaben PET studies. Our approach did not aim to visualize directly Aβ deposits in the retina but rather to determine a likely amyloid status based on sets of retinal image features highly correlated with the cerebral amyloid status. Retinal image features were calculated including vessels’ tortuosity and diameter. Spatial/spectral texture measures over arterioles, venules, and tissue around were also extracted. Retinal venules of amyloid positive subjects (Aβ+) showed a higher mean tortuosity compared to the amyloid negative (Aβ-) subjects. Arteriolar diameter of Aβ+ subjects was found to be higher than the Aβ- subjects in a zone adjacent to the optical nerve head. Furthermore, a significant difference between spatial/spectral texture measures built over retinal arterioles and surrounding tissues were observed in Aβ+ subjects when compared to the Aβ-. In the context of ATH, 60 retinal images from 30 clinically probable ATH and 30 control cases were acquired. Inclusion criteria for subjects suffering from ATH included: myocardial infarction; coronary angiography showing at least one coronary stenosis (more than 50%); and/or coronary angioplasty; and/or coronary bypass. Retinal arterioles of ATH subjects showed a significant narrowing when compared to control subjects. Moreover, a significant difference between image texture measures taken over retinal arterioles and retinal venules and their adjacent regions was observed between ATH subjects and control subjects. Our cross-sectional studies have shown that hyperspectral retinal image analysis could be used to discriminate AD and ATH from corresponding control subjects based on a non-invasive eye scan

Midget retinal ganglion cell dendritic and mitochondrial degeneration is an early feature of human glaucoma

Glaucoma is characterized by the progressive dysfunction and loss of retinal ganglion cells. However, the earliest degenerative events that occur in human glaucoma are relatively unknown. Work in animal models has demonstrated that retinal ganglion cell dendrites remodel and atrophy prior to the loss of the cell soma. Whether this occurs in human glaucoma has yet to be elucidated. Serial block face scanning electron microscopy is well established as a method to determine neuronal connectivity at high resolution but so far has only been performed in normal retina from model animals. To assess the structure-function relationship of early human glaucomatous neurodegeneration, regions of inner retina assessed to have none-to-moderate loss of retinal ganglion cell number were processed using serial block face scanning electron microscopy (n = 4 normal retinas, n = 4 glaucoma retinas). This allowed detailed 3D reconstruction of retinal ganglion cells and their intracellular components at a nanometer scale. In our datasets retinal ganglion cell dendrites degenerate early in human glaucoma, with remodeling and redistribution of the mitochondria. We assessed the relationship between visual sensitivity and retinal ganglion cell density and discovered that this only partially conformed to predicted models of structure-function relationships, which may be affected by these early neurodegenerative changes. In this study, human glaucomatous retinal ganglion cells demonstrate compartmentalized degenerative changes as observed in animal models. Importantly, in these models, many of these changes have been demonstrated to be reversible, increasing the likelihood of translation to viable therapies for human glaucoma

Discovery of new biomarkers of mild cognitive impairment and Alzheimer\u27s disease risk in buccal cells using laser scanning cytometry

Previous studies have shown that mild cognitive impairment (MCI) may reflect the early stages of more pronounced neurodegenerative disorders such as Alzheimer’s disease (AD). In clinical practice, patients with AD are not usually identified until the disease has progressed to a stage when primary prevention is no longer possible. Therefore there is a need for a minimally invasive and inexpensive diagnostic to identify those who exhibit cellular pathology indicative of MCI and AD risk so that they can be prioritised for primary prevention. Human buccal cells are accessible in a minimally invasive manner, and exhibit cytological and nuclear morphologies that may be indicative of accelerated ageing or neurodegenerative disorders such as AD. The hypothesis that a minimally invasive approach using isolated buccal mucosa cells can be used to identify individuals diagnosed with MCI or AD was therefore tested using laser scanning cytometry (LSC). LSC combines the principles of flow cytometry, quantitative imaging and immunohistochemistry with high-content, multi-color fluorescence analysis, and can be used to identify specific cells in a heterogeneous population as well as scoring unique molecular events within them. This study aimed at investigating buccal cell types (buccal cell cytome) by the use of high-content LSC analysis and to detect potential biomarkers of MCI and AD risk i.e. buccal cell types, nuclear DNA content, intracellular neutral lipids, Tau protein and amyloid-β (Aβ) protein. Buccal cells were sampled from the South Australian Alzheimer’s Nutrition & DNA Damage study (SAND) or the The Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL), fixed and stained with labelled fluorescent antibodies (for detection of Aβ and Tau) and/or DAPI, Fast Green and Oil Red O dyes. In an initial study an LSC protocol was developed to identify and measure differences in buccal cell types and nuclear DNA content as well as a significant increase in micronuclei measured in AD (n=10) and Down’s syndrome (n=10) compared to their respective controls (n=20). Another LSC protocol measured a significant increase in DNA content and hyperdiploidy (as measured by DAPI fluorescence) as well as a significant decrease in neutral lipid content (measured by Oil Red O staining) in buccal cells of MCI (n=22) and AD (n=15) compared to controls (n=37) from the SAND study. Using another novel LSC protocol a significant increase in Aβ was measured in buccal cells from AD (n=20) compared to controls (n=20) from the AIBL study. Immunocytochemistry and ELISA experiments showed no significant differences in putative buccal cell Tau protein. The diagnostic value of parameters examined in these studies, individually or in combination was assessed and reported as specificity and sensitivity scores. In these studies, LSC has proven to be an efficient and useful technology for high-content analysis of buccal cells. Moreover, the changes in the buccal cell cytome observed using LSC may reflect alterations in the metabolism, cellular kinetics, gene expression, genome stability or structural profile of the buccal mucosa, and may prove useful as potential biomarkers in identifying individuals with a high risk of developing MCI and eventually AD

NASA Space Radiation Program Element: Research Overview

No abstract availabl

Development of nanocarriers for Alzheimer’s disease treatment

Dissertação de mestrado em Biofísica e BionanossistemasPlaying a central role in all the body’s functions, the brain is perhaps the best protected organ in the organism. Although this is a huge advantage before all potential pathogens and harmful molecules that could put its integrity at risk, it is also a very difficult organ to target therapeutically. Being highly selective to all molecules, the blood brain barrier is the primary and most difficult barrier to overcome when one aims to deliver a specific molecule to the brain. While it is well protected, the brain is not immune to disease, and some of the molecules involved in the most threatening disorders, like amyloid-β and TAU in Alzheimer’s disease, and alpha synuclein in Parkinson’s disease, are thought to be produced in the brain itself. Moreover, these diseases have another hallmark in common, which is the accumulation of reactive oxygen species in the brain. This happens because, being a highly energy demanding organ, the brain is the most metabolically active organ in the human body. In order to overcome these two major debilitating hallmarks, a dual therapy is being developed by our group. By encapsulating siRNA against TAU protein into exosome-like liposomes we aim to reduce its expression, diminishing its harmful effects due to excessive accumulation in neurons, in Alzheimer’s disease. Furthermore, the encapsulation of a molecule with antioxidant properties, such as curcumin, that reduces the accumulation of amyloid-β, another important hallmark in this disease, would most likely have considerably interesting results in the attempt to slow disease progression. For the encapsulation of these molecules, a liposome carrier, with similar constitution and properties of the exosomes, was chosen. Along with its low cytotoxicity verified in two cell lines, mouse L929 fibroblasts and human neuroblastoma SH-SY5Y cells, this carrier has features compatible with the uptake across the blood brain barrier and therefore could directly influence disease progression. It was also found that curcumin-loaded liposomes had greater neuroprotective effects than free curcumin after induction of oxidative stress.Desempenhando um papel central em todas as funções do corpo, o cérebro é talvez o órgão mais bem protegido em todo o corpo. Embora isso seja uma enorme vantagem, devido a todos os patógenos perigosos e moléculas prejudiciais que poderiam colocar a sua integridade em risco, este torna-se assim também num órgão muito difícil de intervencionar em termos clínicos. Terapeuticamente, o primeiro passo limitante para entregar uma substância ativa no cérebro é a passagem dessa molécula através da barreira hematoencefálica. Embora esteja bem protegido, o cérebro não é imune a todos os distúrbios, uma vez que algumas das moléculas envolvidas nas patologias mais graves, tais como as proteínas Aβ-amiloide e TAU na doença de Alzheimer, e alfa-sinucleína na doença de Parkinson, parecem ser produzidas no próprio cérebro. Além disso, essas doenças têm outra característica em comum: a acumulação de espécies reativas de oxigénio no cérebro. Com o encapsulamento de siRNA contra a proteína TAU em lipossomas do tipo exossomal, pretendemos reduzir a expressão dessa proteína, cuja acumulação em células nervosas é uma característica da doença de Alzheimer. Além disso, o encapsulamento de uma molécula com propriedades antioxidantes, como a curcumina, poderá contribuir para a diminuição da acumulação de-β-amiloide no cérebro, contribuindo para uma progressão mais lenta desta doença. Foi conseguido, com sucesso, o encapsulamento de ambas as moléculas nos lipossomas miméticos de exossomas, com eficiências de encapsulamento elevadas. Conseguida a otimização da formulação para garantir que os lipossomas produzidos têm o tamanho necessário para conseguir ultrapassar a barreira hematoencefálica, também se verificou que os lipossomas vazios ou carregados com curcumina não apresentavam toxicidade significativa em duas linhas celulares: fibroblastos de ratinho L929 e linha celular de neuroblastoma humano SH-SY5Y. Adicionalmente demonstramos que curcumina veiculada em lipossomas tinha efeito neuroprotetor maior do que a curcumina livre, após a indução de stress oxidativo

Saliva as a non-invasive diagnostic tool: COVID-19 and T2DM as case-study

Saliva has nowadays a vast research background of how it can mirror the body's health status. Specific salivary biomarkers have been already suggested for multiple diseases and is particularly useful for detecting infectious diseases. We focused on the importance of having a non-invasive, painless and self-collected fluid to study different aspects of two known pandemic diseases: COVID-19 and Type 2 Diabetes Mellitus. COVID-19 is the most impacting global pandemic of all time requiring frequent testing of populations. The necessity to identify cost-effective strategies for the detection of SARS-CoV-2 outbreak became a priority. Nasopharyngeal samples were considered the sampling golden standard but require a healthcare professional to collect the sample causing discomfort and pain to the patient. As saliva has proved successful in SARS-CoV-2 detection, a pooling strategy could be a good approach to decrease the number of individual tests and hazardous material waste which is also beneficial for the environment. We have tested this strategy on two hundred and seventy-nine saliva samples with pools of 10 and 20 randomized samples through RT-PCR. Cycle Threshold of the genes detected was 29.7. Consecutive reactions analysis of positive samples showed an equivalent cycle threshold average (p 0.05). We concluded that saliva-pool samples allowed effective SARS-CoV-2 screening on 10-sample and 20-sample pools. Our strategy was successfully applied in population-wide testing of more than 2000 individuals, showing that it is possible to use pooled saliva as diagnostic fluid for SARS-CoV-2 infection. The SARS-CoV-2 detection is well established with reliable methodologies including saliva as a detection fluid. In the opposite direction are the molecular alterations induced by this infectious virus which remain elusive. We developed a hybrid proteomics and in silico interactomics strategy to establish a COVID-19 salivary protein profile. The most distinctive proteins between healthy and COVID-19 samples were defined with the Partial Least-Squares Discriminant Analysis and the enrichment analysis was performed with FunRich software. In parallel, Protein-Protein virus-host interactome was identified with OralInt algorithm. Five dysregulated biological processes were identified in the COVID-19 proteome profile: Apoptosis, Energy Pathways, Immune Response, Protein Metabolism, and Transport. We identified 10 proteins (KLK 11, IMPA2, ANXA7, PLP2, IGLV2-11, IGHV3-43D, IGKV2-24, TMEM165, VSIG10, and PHB2) that had never been associated with SARS-CoV-2 infection, representing new evidence for the molecular profile behind COVID-19. Interactomics analysis showed viral influence on the host immune response, mainly through interaction with the degranulation of neutrophils. From our results, we can conclude that the virus also alters the host’s energy metabolism and interferes with apoptosis mechanisms. Type 2 Diabetes Mellitus is a chronic metabolic disease and is a major health risk due to its characteristic long-term complications. It is estimated that about 537 million people live with diabetes worldwide and will continue to increase. Diagnosis and glucose monitoring in diabetes are well established. However, monitoring the many of diabetes complications remains a challenge, compromising patients' prognosis and quality of life. We established a hybrid strategy that identified salivary markers of T2DM and its complications. From the functional analysis we highlight metabolic processes, response to stimulus, immune system processes and signalling as disrupted biological processes by the known relation with T2DM. The enrichment analysis identified 11 deregulated biological processes emphasizing 20 proteins directly related to complications in diabetes. Diabetic retinopathy, metabolic syndrome, insulin resistance, molecular impact of glucose and insulin homeostasis dysregulation, atherosclerosis, diabetic foot ulcer, protein catabolism and salivary gland function are diabetic complication capable of being monitored using saliva. We conclude that saliva has the potential to identify several molecules altered in diabetic patients compared to non-diabetic patients and that may be biomarkers not only of diabetes but also of the different complications of this disease. In conclusion, with this research we have confirmed the potential of saliva as a fluid of interest in both diagnosing and discovering new insights into diseases. Saliva was crucial in demonstrating the flexibility and capacity of its use demonstrated by the development of a reliable detection of SARS-CoV-2, the identification and discussion of molecular aspects of viral infection in and with the host, and the discovery of new markers in the diagnosis and monitoring of Type 2 Diabetes Mellitus.A Saliva tem atualmente um vasto background de investigação sobre como pode espelhar o estado de saúde do corpo. Já foram identificados biomarcadores salivares específicos para múltiplas doenças e são particularmente úteis para a monitorização de medicamentos. A saliva é um tipo de amostra particularmente favorável para a deteção de doenças infeciosas. Trata-se de um fluído não-invasivo, indolor e passível de auto recolha, ideal para estudar diferentes aspetos de duas doenças pandémicas conhecidas: COVID-19 e Diabetes Mellitus Tipo 2. A COVID-19 é uma das pandemias mais impactante de todos os tempos, exigindo testes frequentes às populações. A necessidade de identificar estratégias rentáveis para a deteção do surto de SARS-CoV-2 tornou-se uma prioridade global. As amostras nasofaríngeas foram consideradas como o padrão de amostragem, apesar de requerem um profissional de saúde qualificado para efetuar a colheita além de causar desconforto e dor ao doente. Uma vez que a saliva demonstrou ser bem-sucedida na deteção da SARS-CoV-2, o próximo passo seria criar uma estratégia de pools de amostras com vista a diminuir o número de testes individuais e de resíduos de materiais perigosos, o que também seria benéfico para o ambiente. Esta estratégia foi testada em duzentas e setenta e nove amostras de saliva com pools de 10 e 20 amostras aleatórias. O Cycle-Threshold médio dos genes detetados foi de 29,7. Reações de RT-PCT consecutivas em amostras positivas mostraram reprodutibilidade. A análise de comparação de amostras positivas individualizadas mostrou uma carga viral mediana mais elevada em amostras de saliva comparativamente a amostras nasofaríngeas. Concluímos que as amostras de saliva-pool permitiram um rastreio eficaz da SARS-CoV-2 em pools de 10 e de 20 amostras. Esta estratégia foi aplicada com sucesso em testes populacionais de mais de 2000 indivíduos, mostrando que é possível utilizar saliva em pool como líquido de diagnóstico para a infeção pelo SARS-CoV-2. Está demonstrado que a deteção de SARS-CoV-2 está bem estabelecida e com metodologias fiáveis, incluindo a saliva como fluido de deteção. No entanto as alterações moleculares induzidas por este vírus continuam por desvendar. Desenvolvemos uma estratégia proteómica híbrida para estabelecer um perfil proteico salivar COVID-19. As proteínas que mostram uma maior distinção entre amostras saudáveis e COVID-19 foram definidas através da análise PLS-DA e de enriquecimento funcional. Paralelamente, foram previstas as interações proteína-proteína entre o vírus e o hospedeiro. Foram identificados cinco processos biológicos desregulados no perfil do proteoma COVID-19: Apoptose, Vias de Energia, Resposta Imune, Metabolismo de Proteínas, e Transporte. Foram identificadas 10 proteínas (KLK 11, IMPA2, ANXA7, PLP2, IGLV2-11, IGHV3-43D, IGKV2-24, TMEM165, VSIG10 e PHB2) que nunca tinham sido associadas à infeção por SARS-CoV-2, representando novas provas do perfil molecular por detrás da COVID-19. A análise de interactómica mostrou influência do vírus na resposta imunitária do hospedeiro, principalmente através da interação com a desgranulação dos neutrófilos. A partir destes resultados, é possível concluir que o vírus altera não só a resposta imune, mas também o metabolismo energético do hospedeiro e interfere com os mecanismos de apoptose. A Diabetes Mellitus Tipo 2 é uma doença metabólica crónica e constitui um risco de saúde importante devido às suas complicações características a longo prazo. Estima-se que cerca de 537 milhões de pessoas vivem com diabetes em todo o mundo e continuarão a aumentar. O diagnóstico e a monitorização da glicose na diabetes estão atualmente bem estabelecidos. Contudo, a monitorização de muitas das complicações da diabetes continua a ser um desafio, comprometendo o prognóstico e a qualidade de vida dos pacientes. Tivemos como objetivo estabelecer uma estratégia híbrida que identificou os marcadores salivares do T2DM e as suas complicações. Da análise funcional destacamos processos metabólicos, resposta a estímulos, processos do sistema imunitário e sinalização como processos biológicos perturbados pela relação conhecida com o T2DM. A análise de enriquecimento identificou 11 processos biológicos desregulamentados em que demos destaque a 20 proteínas diretamente relacionadas com complicações na diabetes. A retinopatia diabética, síndrome metabólica, resistência à insulina, impacto molecular da glicose e desregulação da homeostase da insulina, aterosclerose, úlcera do pé diabético, catabolismo proteico e função da glândula salivar são complicações diabéticas capazes de serem monitorizadas usando saliva. Concluímos que a saliva tem o potencial de identificar proteínas alteradas em doentes diabéticos em comparação com não diabéticos e que podem ser biomarcadores não só da diabetes, mas também das diferentes complicações desta doença. Em conclusão, com esta investigação confirmamos o potencial da saliva como fluido de interesse tanto no diagnóstico como na descoberta de novos insights sobre doenças. A saliva foi crucial para demonstrar a flexibilidade e capacidade de seu uso demonstrada pelo desenvolvimento de uma deteção confiável de SARS-CoV-2, a identificação e discussão de aspetos moleculares da infeção viral no hospedeiro e com o hospedeiro, bem como a descoberta de novos marcadores no diagnóstico e monitorização da Diabetes Mellitus tipo 2