Protein conformational plasticity and aggregation : transthyretin and δ-toxin as two case studies

Tese de doutoramento em Química (Química Biológica), apresentada à Faculdade de Ciências e Tecnologia da Universidade de CoimbraOs organismos vivos possuem atributos comuns que os distinguem do mundo inorgânico. Uma rede complexa de interacções entre moléculas orgânicas propicia a existência e a perpetuação da vida. As proteínas são uma das mais extraordinárias classes de biomoléculas, participando activamente em todos os processos biológicos. Esta ubiquidade das proteínas deve-se à sua relativa plasticidade conformacional, um requisito importante nas reacções bioquímicas, processos dinâmicos por definição. A maioria das proteínas tem uma estrutura tridimensional bem definida, essencial para a sua função biológica. Muitas das doenças hoje conhecidas têm a sua origem num incorrecto funcionamento das proteínas. O estudo da relação existente entre a estrutura de uma dada biomolécula e a sua dinâmica, é um dos tópicos mais importantes na compreensão dos mecanismos moleculares de múltiplas patologias. Relativamente a esta temática, duas proteínas foram o sujeito de estudo na elaboração desta tese. Foram utilizadas técnicas experimentais bem como protocolos computacionais, onde diferentes aspectos biofísicos foram abordados. A bactéria Staphylococcus aureus é responsável por inúmeros casos de septicémia. Umas das toxinas excretadas pela bactéria para o plasma, a d-toxina, é responsável pela desintegração membranar em organismos eucarióticos. Durante o mecanismo infeccioso, o peptídeo experiencia a influência de diferentes ambientes químicos, e por conseguinte com impacto na sua conformação tridimensional. A estrutura anfipática helical do peptídeo foi estudada por ressonância magnética nuclear (NMR). Simulações moleculares foram também produzidas no estudo da plasticidade conformacional e propensão à agregação do peptídeo em solução. A interacção do peptídeo com membranas foi também estudado por dinâmica molecular. Os resultados evidenciam que o estado de agregação do peptídeo pode ser relevante para o processo lítico. A transtirretina (TTR) humana é uma das muitas proteínas envolvidas em doenças amilóides. Dependendo da amiloidogeneceidade da variante proteica, a estabilidade do complexo tetramérico nativo é alterado, ocorrendo dissociação e formação de espécies monoméricas com tendência para agregar. Os agregados solúveis e as fibras amilóides insolúveis são responsáveis pelas alterações morfológicas e morte celular observadas nestas patologias. Um método computacional baseado em acoplagem molecular (“docking”) utilizando dados experimentais foi desenvolvido nesta tese. O objectivo do protocolo centrou-se na modelação molecular de um modelo protofilamentar, a unidade elementar das fibras amilóides. Os modelos polimórficos obtidos foram agrupados de acordo com a sua periodicidade helical, para posterior análise das interfaces proteicas e qualidade estereoquímica das estruturas. Os modelos produzidos poderão ter um impacto significativo em estudos terapêuticos com o objectivo de produzir fármacos que inibam a formação das fibras amilóides.All living organisms possess common attributes that distinguishes them from the surrounding inorganic matter. A complex network of interacting organic molecules are responsible for the existence and perpetual of life. Among the most extraordinary biomolecules are proteins. Proteins are the workhorses of life, they actively participate in all biological processes. This is accomplished through an intrinsic conformational plasticity, a requisite in all biochemical reactions inherently dynamic processes. For most proteins a correct three dimensional structure is essential for its biological function. It is a fact that many diseases can be traced back to malfunction of proteins. Understanding the underlying connection between the biomolecule structure and its dynamics, is thus essential for current day medicine. Throughout this thesis, two proteins were the subject of study using both experimental and computational techniques. Different biophysical aspects were focused stressing the importance of the usage of different approaches to get complementary views. d-lysin is a small peptide excreted by the bacteria Staphylococcus aureus, responsible for the disruption of eukaryotic membranes in infection conditions. On its way to the membrane, the peptide experience subtle chemical environment changes with consequences on peptide conformation. The amphyphatic helical structure of this peptide was studied using nuclear magnetic resonance (NMR), and its conformational plasticity and propensity for aggregation in solution was assessed through molecular dynamics (MD) simulations and NMR. The first stages of the lytic process were also modeled by analyzing MD trajectories describing the interaction of the peptide with a zwiterionic membrane. The results show that peptide aggregation may play a key role in the process. Transthyretin (TTR) is one of the many proteins known to be involved in human amyloid diseases. Depending on the protein variant amyloidogenicity, the native tetrameric complex dissociates into monomeric species with high tendency for aggregation. Soluble aggregates and insoluble amyloid fibrils are responsible for the morphological alterations and cell observed in these pathologies. We developed a computational method using protein docking driven by experimental data, to build a high resolution molecular model of the elementary units that constitute the fibrils. We obtained a polymorphic set of protofilament models, further characterized in terms of helical periodicities and interface match. The overall stereochemical quality of the structures was assessed. This models may be a valuable instrument in the rational design of compounds with therapeutic potential to inhibit amyloid fibril formation.Financial support for the execution and publication of this thesis was provided by Fundação para a Ciência e Tecnologia through grant SFRH/BD/1354/200

Morphological and Postural changes in the foot during pregnancy and puerperium : a longitudinal study

The aim of this study is to observe the morphological and postural changes to the foot that take place during pregnancy and the puerperium. Method: In this descriptive, observational, longitudinal study, we analysed 23 pregnant women, with particular attention to morphological and postural aspects of the foot, at three time points during and after pregnancy: in weeks 9-13 of gestation, weeks 32-35 of gestation and weeks 4-6 after delivery. The parameters considered were changes in foot length, the Foot Posture Index (FPI) and the Hernández Corvo Index, which were analysed using a pedigraph and taking into account the Body Mass Index (BMI). The same procedure was conducted in each review. Results: The statistical analyses obtained for each foot did not differ significantly between the three measurement times. A pronator-type footprint was most frequently observed during the third trimester of pregnancy; it was predominantly neutral during the postpartum period. Statistically significant differences between the measurement times were obtained in the right foot for cavus vs. neutral foot type (between the first and third trimesters and also between the first trimester and the puerperium) (in both cases, p < 0.0001). Conclusions: Foot length increases in the third trimester and returns to normal in the puerperium. According to FPI findings, the third trimester of pregnancy is characterised by pronation, while the posture returns to neutrality during the postpartum period. During pregnancy, the plantar arch flattens, and this persists during the puerperium. The incidence of cavus foot increases significantly in the third trimester and in the puerperium

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND: Disorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021. METHODS: We estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined. FINDINGS: Globally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer. INTERPRETATION: As the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BackgroundDisorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021.MethodsWe estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined.FindingsGlobally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer.InterpretationAs the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42·4% vs 44·2%; absolute difference -1·69 [-9·58 to 6·11] p=0·67; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5-8] vs 6 [5-8] cm H2O; p=0·0011). ICU mortality was higher in MICs than in HICs (30·5% vs 19·9%; p=0·0004; adjusted effect 16·41% [95% CI 9·52-23·52]; p&lt;0·0001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0·80 [95% CI 0·75-0·86]; p&lt;0·0001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

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