Vantagens da transposição de cirurgias em regime de internamento para cirurgias em regime de ambulatório no sector privado

Este trabalho insere-se no âmbito de redução de custos no pagamento aos hospitais privados portugueses. Como gestores devemos promover a eficiência produtiva dos hospitais criando incentivos que levem os diferentes prestadores de cuidados de saúde destas instituições a minimizar os custos e a manter, simultaneamente um determinado padrão de qualidade. O enorme sucesso das cirurgias efectuadas em regime de ambulatório no sector público foi o impulsionador para a realização deste trabalho. O trabalho tem como objectivo verificar se a produção hospitalar nos hospitais privados em regime de ambulatório é mais benéfica ao nível dos custos e sem prejuízo da qualidade. São abordados os custos inerentes ao pagamento de cirurgias efectuadas em internamento que poderiam fazer parte de custos de cirurgias realizadas em ambulatório

Applications of Genome-Wide Screening and Systems Biology Approaches in Drug Repositioning

Simple Summary Drug repurposing is an accelerated route for drug development and a promising approach for finding medications for orphan and common diseases. Here, we compiled databases that comprise both computationally- or experimentally-derived data, and categorized them based on quiddity and origin of data, further focusing on those that present high throughput omic data or drug screens. These databases were then contextualized with genome-wide screening methods such as CRISPR/Cas9 and RNA interference, as well as state of art systems biology approaches that enable systematic characterizations of multi-omic data to find new indications for approved drugs or those that reached the latest phases of clinical trials. Modern drug discovery through de novo drug discovery entails high financial costs, low success rates, and lengthy trial periods. Drug repositioning presents a suitable approach for overcoming these issues by re-evaluating biological targets and modes of action of approved drugs. Coupling high-throughput technologies with genome-wide essentiality screens, network analysis, genome-scale metabolic modeling, and machine learning techniques enables the proposal of new drug-target signatures and uncovers unanticipated modes of action for available drugs. Here, we discuss the current issues associated with drug repositioning in light of curated high-throughput multi-omic databases, genome-wide screening technologies, and their application in systems biology/medicine approaches

Characterization of different fat depots in NAFLD using inflammation-associated proteome, lipidome and metabolome

Non-alcoholic fatty liver disease (NAFLD) is recognized as a liver manifestation of metabolic syndrome, accompanied with excessive fat accumulation in the liver and other vital organs. Ectopic fat accumulation was previously associated with negative effects at the systemic and local level in the human body. Thus, we aimed to identify and assess the predictive capability of novel potential metabolic biomarkers for ectopic fat depots in non-diabetic men with NAFLD, using the inflammation-associated proteome, lipidome and metabolome. Myocardial and hepatic triglycerides were measured with magnetic spectroscopy while function of left ventricle, pericardial and epicardial fat, subcutaneous and visceral adipose tissue were measured with magnetic resonance imaging. Measured ectopic fat depots were profiled and predicted using a Random Forest algorithm, and by estimating the Area Under the Receiver Operating Characteristic curves. We have identified distinct metabolic signatures of fat depots in the liver (TAG50:1, glutamate, diSM18:0 and CE20:3), pericardium (N-palmitoyl-sphinganine, HGF, diSM18:0, glutamate, and TNFSF14), epicardium (sphingomyelin, CE20:3, PC38:3 and TNFSF14), and myocardium (CE20:3, LAPTGF-beta 1, glutamate and glucose). Our analyses highlighted non-invasive biomarkers that accurately predict ectopic fat depots, and reflect their distinct metabolic signatures in subjects with NAFLD.Peer reviewe

Study of pathophysiology of Pompe disease and identification of novel therapeutic targets and biomarkers

Le basi molecolari e biochimiche della malattia di Pompe sono ormai ben note ma la fisiopatologia della malattia, ad oggi, resta ancora sconosciuta e la stessa terapia ha un’efficacia limitata nei pazienti. Il lavoro è focalizzato sullo studio delle interazioni proteiche dell’alfa glucosidasi acida (GAA), sulla presenza dello stress ossidativo nella malattia e nell’identificazione di nuovi biomarcatori per l’individuazione della diagnosi e per seguire la progressione della malattia. I risultati ottenuti offrono nuovi spunti per la comprensione dei meccanismi alla base della malattia e per l'individuazione di nuovi possibili target terapeutici

Multi-omics insights into host-viral response and pathogenesis in Crimean-Congo hemorrhagic fever viruses for novel therapeutic target.

The pathogenesis and host-viral interactions of the Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) are convoluted and not well evaluated. Application of the multi-omics system biology approaches, including biological network analysis in elucidating the complex host-viral response, interrogates the viral pathogenesis. The present study aimed to fingerprint the system-level alterations during acute CCHFV-infection and the cellular immune responses during productive CCHFV-replication in vitro. We used system-wide network-based system biology analysis of peripheral blood mononuclear cells (PBMCs) from a longitudinal cohort of CCHF patients during the acute phase of infection and after one year of recovery (convalescent phase) followed by untargeted quantitative proteomics analysis of the most permissive CCHFV-infected Huh7 and SW13 cells. In the RNAseq analysis of the PBMCs, comparing the acute and convalescent-phase, we observed system-level host's metabolic reprogramming towards central carbon and energy metabolism (CCEM) with distinct upregulation of oxidative phosphorylation (OXPHOS) during CCHFV-infection. Upon application of network-based system biology methods, negative coordination of the biological signaling systems like FOXO/Notch axis and Akt/mTOR/HIF-1 signaling with metabolic pathways during CCHFV-infection were observed. The temporal quantitative proteomics in Huh7 showed a dynamic change in the CCEM over time and concordant with the cross-sectional proteomics in SW13 cells. By blocking the two key CCEM pathways, glycolysis and glutaminolysis, viral replication was inhibited in vitro. Activation of key interferon stimulating genes during infection suggested the role of type I and II interferon-mediated antiviral mechanisms both at the system level and during progressive replication

Systems Biology in ELIXIR: modelling in the spotlight

In this white paper, we describe the founding of a new ELIXIR Community - the Systems Biology Community - and its proposed future contributions to both ELIXIR and the broader community of systems biologists in Europe and worldwide. The Community believes that the infrastructure aspects of systems biology - databases, (modelling) tools and standards development, as well as training and access to cloud infrastructure - are not only appropriate components of the ELIXIR infrastructure, but will prove key components of ELIXIR\u27s future support of advanced biological applications and personalised medicine. By way of a series of meetings, the Community identified seven key areas for its future activities, reflecting both future needs and previous and current activities within ELIXIR Platforms and Communities. These are: overcoming barriers to the wider uptake of systems biology; linking new and existing data to systems biology models; interoperability of systems biology resources; further development and embedding of systems medicine; provisioning of modelling as a service; building and coordinating capacity building and training resources; and supporting industrial embedding of systems biology. A set of objectives for the Community has been identified under four main headline areas: Standardisation and Interoperability, Technology, Capacity Building and Training, and Industrial Embedding. These are grouped into short-term (3-year), mid-term (6-year) and long-term (10-year) objectives

Active and passive defenses against oxidative stress: a computational study

Tese de doutoramento em Biologia Experimental e Biomedicina, na especialidade de Biologia Molecular, Celular e do Desenvolvimento, apresentada ao Instituto de Investigação Interdisciplinar da Universidade de CoimbraThis thesis addresses two complementary means of defense against oxidative stress — active and passive. First, we aimed to clarify the role of peroxiredoxin 2 (Prx2) – an active defense – in hydrogen peroxide (H2O2) metabolism. Peroxiredoxins play key roles in antioxidant protection and redox signaling in most cells, but their quantitative contribution to H2O2 clearance and signaling mechanisms are unclear. As a starting point to clarify these issues we thoroughly surveyed the available data about H2O2 metabolism in human erythrocytes, the cells for which this process has been most scrutinized. We then set up a mathematical model that accurately represents the present understanding of this process. Comparing model predictions to experimental observations of the behavior of intact erythrocytes we flagged a strong and hitherto unappreciated discrepancy. Namely, whereas the kinetic parameters determined for Prx2 purified from human erythrocytes imply that this protein should clear >99% of the H2O2, experiments show that it has a much lower contribution to H2O2 clearance. Nevertheless, Prx2 is quickly and quantitatively oxidized when erythrocytes are exposed to H2O2 boluses. A computational assessment of alternative explanations showed that models considering that the cellular concentration of Prx2 had been overestimated were inconsistent with the experimental observations. In contrast, quantitative agreement was achieved by considering an effective peroxidase activity of Prx2 ~1% of that determined for the purified protein, either due to a quickly reversible inhibition or to overestimation of Prx2’s intrinsic activity. The latter possibility is unlikely, because distinct methods yielded consistent determinations. Inhibition by covalent modification is excluded by energetic considerations and because purified Prx2 is not extensively modified. Further, the hypothetical inhibitor must virtually titrate Prx2, which is the third most abundant protein in human erythrocytes (~0.6 mM). This leaves few candidates: two proteins and a few metabolites, most of which also abundant in other cells. It is thus likely that Prx2’s peroxidase activity is also inhibited in other cells. The results above indicate that Prx2 is abundant in human erythrocytes for reasons other than minimizing H2O2 concentrations. And they raise the question of what advantages could a high Prx2 concentration combined with a strong quickly reversible inhibition of its peroxidase activity have. Seeking to clarify this question, we surveyed the available information about the physiology and dynamics of erythrocyte’s exposure to H2O2 in vivo. We then used the refined mathematical model to predict the responses of the Prx2 / thioredoxin 1 / thioredoxin reductase to physiologically plausible H2O2 stimuli. These analyses indicate that the low effective peroxidase activity of Prx2 in vivo endows this system with desirable redox signaling properties and spares NADPH. Finally, based on our results and in face of the interactions of Prx2 with many other proteins we proposed a conceptual model of Prx2 activation by regulated recruitment. The second part of the work focused on passive antioxidant defense. Namely, we analyzed if evolutionary adaptation of microorganisms to O2-rich environments modulated relative aminoacyl compositions (AAC) so as to decrease proteins’ reactivity with the hydroxyl radical (•OH). The work was based on comparisons of aerotolerant (AT; aerobic or facultative aerobic) to aerointolerant (AI; microaerophilic or anaerobic) organisms across 1099 genomes and proteomes of unicellulars from the three phylogenetic domains. In all phyla examined, the mean •OH-reactivity per aminoacyl residue (q) is lower for the proteins from AT organisms than for those from AI organisms. The lower q values in AT organisms are mainly due to a rarefaction of tyrosine, cysteine and methionine residues, which contribute majorly for proteins’ •OHreactivity. However, the very •OH-reactive tryptophan, histidine and arginine residues are enriched, and the relatively •OH-unreactive lysine residues are consistently and substantially rarefied in the proteins from AT organisms. This rarefaction may be due to lysine’s high reactivity with electrophilic autoxidation products. Interestingly, tryptophan, histidine and arginine are among the most selectively favored substituents for tyrosine and lysine residues, due to their similar physical-chemical properties. Statistical models accounting for the effects of aerotolerance, coding sequence G+C content, thermophily, protein size, and phylogeny highlight that aerotolerance is the main factor explaining variability in q values and cysteinyl frequency, and an important explanatory factor for the variability in most residues’ frequencies. Finally, we showed that the cost differentials in synthesizing the aminoacids in fermentative vs. respiratory conditions cannot explain the observed variation in AAC.Esta tese analisa dois modos complementares de defesa antioxidante – ativo e passivo. Primeiro, analisámos o papel da peroxiredoxina 2 (Prx2) – uma defesa ativa – no metabolismo de peróxido de hidrogénio (H2O2). As peroxiredoxinas são importantes na proteção antioxidante e sinalização redox em muitas células, mas a sua contribuição para o consumo de H2O2 e os seus mecanismos de sinalização não são claros. Como ponto de partida para clarificar estas questões revimos a informação disponível sobre o metabolismo de H2O2 em eritrócitos humanos, as células onde este processo foi mais estudado. Isto permitiu-nos contruir um modelo matemático que representa o conhecimento atual sobre este processo. A comparação das previsões deste modelo com observações experimentais em eritrócitos humanos permitiu identificar uma discrepância importante: os parâmetros cinéticos determinados para a Prx2 purificada de eritrócitos humanos implicam que esta proteína consumiria >99% do H2O2 nestas células, mas experiências com eritrócitos intactos indicam uma contribuição muito menor. No entanto, a Prx2 é rápida e substancialmente oxidada quando eritrócitos são expostos a pulsos de H2O2. Uma avaliação computacional de possíveis explicações desta discrepância demonstra que modelos onde se assume que a concentração de Prx2 foi sobrestimada são inconsistentes com as observações experimentais. Por outro lado, é conseguida uma concordância quantitativa com as observações quando se considera que a atividade efetiva de peroxidase da Prx2 é ≈1% da estimada para a proteína purificada, devido a uma inibição rapidamente reversível ou à sobrestimativa da reatividade da Prx2 purificada. A segunda destas possibilidades é improvável, pois metodologias distintas originaram resultados mutuamente consistentes. A inibição da Prx2 por modificação covalente é excluída por considerações energéticas e porque a Prx2 purificada não se encontra modificada. O hipotético inibidor terá pois de ser suficientemente abundante para titular a Prx2, que é a terceira proteína mais abundante em eritrócitos humanos (≈0.6 mM). Isto deixa como candidatos: apenas duas proteínas e poucos metabolitos, a maioria dos quais são abundantes também noutras células. Portanto, é provável que a atividade de peroxidase da Prx2 esteja também inibida noutras células. Os resultados acima indicam que minimizar a concentração de H2O2 não é a razão para a grande abundância da Prx2 em eritrócitos humanos. Que vantagens poderão conferir uma elevada concentração e inibição rapidamente reversível da atividade de peroxidase da Prx2? Para clarificar esta questão avaliámos a fisiologia e dinâmica de exposição de eritrócitos a H2O2 in vivo, e utilizámos um modelo matemático para prever as respostas do sistema Prx2 / tioredoxina 1 / tioredoxina reductase a estímulos fisiológicos de H2O2. Estas análises indicam que a baixa atividade de peroxidase da Prx2 in vivo melhora várias propriedades de sinalização do sistema e poupa NADPH. Finalmente, com base nos resultados computacionais e nas interações da Prx2 com outras proteínas propomos um modelo conceptual da ativação da Prx2 por recrutamento regulado. A segunda parte do trabalho foca-se em mecanismos passivos de defesa. Nomeadamente, analisámos se a adaptação evolutiva de microrganismos a ambientes ricos em O2 afeta a composição relativa de aminoácidos (AAC), diminuindo a reatividade de proteínas com o radical hidroxilo (•OH). Este trabalho foi baseado na comparação de genomas e proteomas de 1099 organismos aerotolerantes (AT; aeróbios e facultativos) e aerointolerantes (AI; microaerófilos e anaeróbios), dos três domínios filogenéticos. Em todos os filos examinados, a reatividade média com •OH por resíduo (q) é menor em proteínas de AT do que em AI. Esta diferença é fundamentalmente devida à menor utilização de tirosina, cisteína e metionina, os resíduos com maior contribuição para a reatividade de proteínas com •OH. No entanto, as proteínas de AT são enriquecidas em triptofano, histidina e arginina – resíduos muito reativos com •OH – e empobrecidas em lisina – resíduo pouco reativo com •OH. Este empobrecimento poderá ser devido à grande reatividade da lisina com produtos de autoxidação. Curiosamente, o triptofano, histidina e arginina são resíduos substituintes de tirosina e lisina, evolutivamente favorecidos devido às propriedades físico-químicas semelhantes com estes últimos. Modelos estatísticos que consideram os efeitos da aerotolerância, conteúdo G+C da sequência codificante, termofilia, tamanho de proteína, e filogenia indicam que a aerotolerância é o principal fator explicativo da variabilidade nos valores de q e de frequência de cisteína. A aerotolerância é também um fator importante para explicar a variabilidade das frequências da maioria dos aminoácidos. Finalmente, demonstramos que custos diferenciais de biossíntese de aminoácidos em condições de fermentação vs respiração não explicam os padrões de AAC observados

New Challenges to Study Heterogeneity in Cancer Redox Metabolism

Reactive oxygen species (ROS) are important pathophysiological molecules involved in vital cellular processes. They are extremely harmful at high concentrations because they promote the generation of radicals and the oxidation of lipids, proteins, and nucleic acids, which can result in apoptosis. An imbalance of ROS and a disturbance of redox homeostasis are now recognized as a hallmark of complex diseases. Considering that ROS levels are significantly increased in cancer cells due to mitochondrial dysfunction, ROS metabolism has been targeted for the development of efficient treatment strategies, and antioxidants are used as potential chemotherapeutic drugs. However, initial ROS-focused clinical trials in which antioxidants were supplemented to patients provided inconsistent results, i.e., improved treatment or increased malignancy. These different outcomes may result from the highly heterogeneous redox responses of tumors in different patients. Hence, population-based treatment strategies are unsuitable and patient-tailored therapeutic approaches are required for the effective treatment of patients. Moreover, due to the crosstalk between ROS, reducing equivalents [e.g., NAD(P)H] and central metabolism, which is heterogeneous in cancer, finding the best therapeutic target requires the consideration of system-wide approaches that are capable of capturing the complex alterations observed in all of the associated pathways. Systems biology and engineering approaches may be employed to overcome these challenges, together with tools developed in personalized medicine. However, ROS- and redox-based therapies have yet to be addressed by these methodologies in the context of disease treatment. Here, we review the role of ROS and their coupled redox partners in tumorigenesis. Specifically, we highlight some of the challenges in understanding the role of hydrogen peroxide (H2O2), one of the most important ROS in pathophysiology in the progression of cancer. We also discuss its interplay with antioxidant defenses, such as the coupled peroxiredoxin/thioredoxin and glutathione/glutathione peroxidase systems, and its reducing equivalent metabolism. Finally, we highlight the need for system-level and patient-tailored approaches to clarify the roles of these systems and identify therapeutic targets through the use of the tools developed in personalized medicine