The role of LRRK2 in Parkinson’s disease : from function to dysfunction

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2015Parkinson’s disease (PD) belongs to the group of neurodegenerative disorders and it is currently considered the most common progressive movement disorder. Neurodegenerative disorders, such as Alzheimer’s, Huntington’s, frontotemporal dementia and amyotrophic lateral sclerosis, share several dysfunctional molecular pathways and impairments in basic cell mechanisms. Despite intense efforts to understand to decipherthe triggers underlying these disorders, to date, there is no effective cure. This results in a growing number of cases and, consequently, in a complex social and economic problem. Therefore, it is of extreme importance to understand the common biological mechanisms involved in the pathogenesis of this devastating group of diseases, in order to develop effective therapies. The majority of the PD cases are sporadic, however, in the last decades, it has been recognized that rare genetic mutations are patholgical for PD in a number of inherited cases. Futhermore, these mutations can be as well a risk factor for sporadic PD, supporting the idea that familial and sporadic PD can share common pathlogical mechanisms. This study focused on a key player protein in PD, Leucine-rich repeat kinase 2 (LRRK2). Mutations in LRRK2 gene are the most frequent cause of autosomal dominant forms of PD and they are also consider a risck factor for sporadic cases. A central catalytic GTPase and kinase core, flanked by protein interaction domains, composes this large and complex multi-domain protein. The most frequent LRRK2 PD-related mutation occurs at the animoacid 2019, a glycine subtitution for a serine (G2019S), precisely on the kinase domain of the protein resulting in its toxic gain of function. LRRK2 is known to play a role in distinct cellular mechanisms such as vesicular trafficking, microtubule network regulation and mitochondrial morphology. However, the function of LRRK2 in these important mechanisms and their related pathways is not fully understood, which is crucial for developing new therapeutic targets. Here, we investigated LRRK2 function by characterizing/identifying its protein interactors and, in particular, by exploring its relationship with two central proteins in neurodegenerative disorders, α-synuclein and Tau. In PD brain samples, we show that levels of LRRK2 are positively correlated to an increase in α-synuclein phosphorylation and aggregation in affected brain regions, where both proteins co-localize in neurons and Lewy body inclusions. In a cell line model, this co-localization also occurs in α-synuclein inclusions and knocking down LRRK2 promotes formation of smaller inclusions. Moreover, we show an interaction between α-synuclein and LRRK2 under endogenous and over-expression conditions. These results shed light on the complex interaction of these two central PD proteins and, in particular, on underlying molecular mechanisms involved in a disease scenario. Furthermore, we demonstrate that LRRK2 also interacts with Tau protein in a cell line model, in which co-expression of both proteins promotes accumulation of Tau protein. This accumulation occurs independently of LRRK2 kinase activity and it gives rise to formation of high molecular weight Tau species and increased levels of Tau secretion. Moreover, we suggest that these effects are a consequence of an impairment of proteasomal Tau degradation and that this impairment is promoted by LRRK2. Consistently, a LRRK2-knockout mouse displayed lower levels of Tau in the brain, when compared with transgenic animals expressing human wild-type LRRK2. Our results highlight the compromised status of cellular and molecular neurodegenerative mechanisms. The identification of LRRK2 interactors is crucial to placing the protein in known biochemical pathways. To that end, we performed a screen to identify LRRK2-interacting proteins. The results obtained confirmed that this is a multifaceted protein, involved in a variety of molecular functions and biochemical pathways. α-synuclein and Tau are two proteins present in the list of interactors, which validates previously reported results. The role of LRRK2 on the cytoskeleton is also highlighted by the presence of several protein interactors linked to microtubule dynamics, which lead us to explore the effect of LRRK2 on mechanical properties of the cell. Applying a combined microscopy tecniques in cell indentation experiments, we confirmed that different distribution patterns of LRRK2 result in differential states of cell stiffness. We found that the stiffest cells exhibit a diffuse pattern of LRRK2 distribution, such that LRRK2 is dispersed throughout the entire cell, interacting with microtubule-related proteins and compromising cytoskeletal dynamics. The identification of novel interactos resulted in a better understanding of LRRK2 patho-physiological role. Taken together, our results presented in this thesis provide novel insight into the function of LRRK2 and its particular role in neurodegenerative diseases. Ultimately, this knowledge is essential for the understanding of the molecular underpinnings of PD and for the development of novel therapeutics.A doença de Parkinson (DP) pertence ao grupo das doenças neurodegenerativas, sendo atualmente considerada a doença neurodegenerativas motora progressiva mais comum. As doenças neurodegenerativas, como a doença de Alzheimer, a demência frontotemporal ou a esclerose lateral amiotrófica, partilham várias disfuncionalidades em importantes vias de sinalização molecular e mecanismos celulares. Apesar dos esforços desenvolvidos para compreender os factores que estão na origem e na progressão destas doenças, presentemente ainda não foi encontrada uma cura eficaz. O resultante crescente número de casos destas doenças, consequentemente contribui para um complexo problema socioeconómico. É assim de extrema importância identificar os mecanismos biológicos envolvidos na patogénese deste devastador grupo de doenças, a fim de desenvolver terapias eficazes para o combate das mesmas. A maioria dos casos de DP são esporádicos, no entanto nas últimas décadas têm sido identificadas várias mutações genéticas ligadas a casos hereditários. Estas mutações podem ainda ser consideradas um factor de risco para o desenvolvimento de casos esporádicos da DP, o que suporta a ideia que os casos hereditários e esporádicos partilham os mesmos mecanismos patológicos. Este estudo foca-se numa proteína chave na DP, Leucine-rich repeat kinase 2 (LRRK2). Mutações na proteína LRRK2 são consideradas a causa mais frequente em casos autossómicos dominantes da doença, ocorrendo também em casos esporádicos. Esta grande e complexa proteína com múltiplos domínios, é composta por um núcleo catalítico central de GTPase e quinase, flanqueado por vários domínios de interação proteica. A mutação mais frequente em LRRK2 é a substituição de uma glicina por uma serina, que ocorre no aminoácido 2019 (G2019S). Esta mutação localiza-se precisamente no domínio da quinase da proteína, promovendo um tóxico ganho de função da mesma. É conhecido o envolvimento de LRRK2 em distintos mecanismos celulares como o tráfego vesicular, regulação da rede de microtúbulos e morfologia mitocondrial. No entanto, não é completamente conhecido o papel de LRRK2 nestes importantes mecanismos e suas vias de sinalização, o que é crucial para o desenvolvimento de novos alvos terapêuticos. Neste trabalho investigamos a função de LRRK2 através da caracterização/identificação de proteínas interatuantes, em particularexplorando a sua relação com duas proteínas centrais em doenças neurodegenerativas, alpha-sinucleína (α-sinucleína) e Tau. Em amostras de cérebro de pacientes com DP, mostramos que os níveis de LRRK2 são positivamente corelacionados com um aumento de fosforilaçao e agregação de α- sinucleína fosforilada e agregada, nas regiões do cérebro mais afectadas. Também nas regiões de cérebro mais afectadas, se verifica uma co-localização destas duas proteínas em neurónios e em inclusões de corpos de Lewy. Num modelo de linha celular, esta co-localização também ocorre em inclusões de α- sinucleína, onde o knockdown de LRRK2 promove a formação de inclusões mais pequenas. A interação entre α-sinucleína e LRRK2 é ainda confirmada em condições endógenas e de sobre-expressão. Estes resultados contribuem para uma melhor compreensão sobre a complexa interação destas duas proteínas centrais na DP, em particular sobre os mecanismos moleculares subjacentes, envolvidos num cenário de doença. Em seguida e usando um modelo celular, demostramos que a LRRK2 interatua com a Tau, sendo que a co-expressão destas proteínas promove uma acumulação de Tau. Esta acumulação ocorre independentemente da atividade de quinase da LRRK2, e promove a formação de espécies de Tau com elevado peso molecular, bem como um aumento de secreção de Tau. Estes efeitos serão a consequência de uma falha ao nível da degradação de Tau pelo proteassoma, que por sua vez será promovida pela LRRK2. Em cérebros de ratinhos knockout para LRRK2, verifica-se um decréscimo dos níveis de Tau, quando comparado com animais transgénicos para LRRK2 humana. Estes resultados realçam a disfunção de mecanismos celulares e moleculares, envolvidos nas doenças neurodegenerativas. A identificação de proteínas interatuantes com LRRK2 é crucial para posicionar esta proteína nas conhecidas vias de sinalização bioquímica. Com este objectivo, desenvolvemos um screen para identificar novas proteínas interatuantes com LRRK2. Os resultados obtidos confirmam que esta é uma proteína multifacetada, envolvida em várias funções moleculares e vias de sinalização bioquímicas. A presença de α-sinucleína e Tau nesta lista de proteínas interatuantes, vem validar os resultados acima descritos. Também a presença de várias proteínas relacionadas com a dinâmica de microtúbulos, vem realçar o papel de LRRK2 ao nível do citoesqueleto celular, o que nos levou a explorar o efeito de LRRK2 nas propriedades mecânicas das células. Aplicando uma técnica combinada em microscopia celular, confirmámos que diferentes padrões de distribuição de LRRK2, resultam em diferentes estados de rigidez celular. Descobrimos que as células com maior rigidez são as que exibem um padrão difuso de distribuição de LRRK2, onde a proteína está dispersa por toda a célula, interagindo com proteínas relacionadas com os microtúbulos, comprometendo assim a dinâmica do citoesqueleto. A identificação de novas proteínas interatuantes resulta num melhor conhecimento da função pato-fisiológica de LRRK2. Em resumo, os resultados apresentados nesta tese, fornecem novos conhecimentos sobre as funções da LRRK2 e o seu particular papel nas doenças neurodegenerativas. Por fim, estes conhecimentos são essenciais para a compreensão das bases moleculares da DP e consequentemente para o desenvolvimento de novas terapêuticas

Airway and Respiratory Devices in the Prevention of Ventilator-Associated Pneumonia

Ventilator-associated pneumonia (VAP) is the most common ICU-acquired infection among patients under mechanical ventilation (MV). It may occur in up to 50% of mechanically ventilated patients and is associated with an increased duration of MV, antibiotic consumption, increased morbidity, and mortality. VAP prevention is a multifaceted priority of the intensive care team. The use of specialized artificial airways and other devices can have an impact on the prevention of VAP. However, these devices can also have adverse effects, and aspects of their efficacy in the prevention of VAP are still a matter of debate. This article provides a narrative review of how different airway and respiratory devices may help to reduce the incidence of VAP.publishersversionpublishe

Understanding pseudo-albinism in sole (Solea senegalensis): a transcriptomics and metagenomics approach

Pseudo-albinism is a pigmentation disorder observed in flatfish aquaculture with a complex, multi-factor aetiology. We tested the hypothesis that pigmentation abnormalities are an overt signal of more generalised modifications in tissue structure and function, using as a model the Senegalese sole and two important innate immune barriers, the skin and intestine, and their microbiomes. Stereological analyses in pseudo-albino sole revealed a significantly increased mucous cell number in skin (P < 0.001) and a significantly thicker muscle layer and lamina propria in gut (P < 0.001). RNA-seq transcriptome analysis of the skin and gut identified 573 differentially expressed transcripts (DETs, FDR < 0.05) between pseudo-albino and pigmented soles (one pool/tissue from 4 individuals/phenotype). DETs were mainly linked to pigment production, skin structure and regeneration and smooth muscle contraction. The microbiome (16 S rRNA analysis) was highly diverse in pigmented and pseudo-albino skin but in gut had low complexity and diverged between the two pigmentation phenotypes. Quantitative PCR revealed significantly lower loads of Mycoplasma (P < 0.05) and Vibrio bacteria (P < 0.01) in pseudo-albino compared to the control. The study revealed that pseudo-albinism in addition to pigmentation changes was associated with generalised changes in the skin and gut structure and a modification in the gut microbiome.Agência financiadora H2020 European Funds MSCA-RISE project 691102 Portuguese national funds from FCT - Foundation for Science and Technology UID/Multi/04326/2019 Portuguese national funds from the operational programme CRESC Algarve 2020 EMBRC. PT ALG-01-0145-FEDER-022121 Portuguese national funds from the operational programme COMPETE 2020 EMBRC. PT ALG-01-0145-FEDER-022121 European Union (EU) 654008 Fundacao para a Ciencia e a Tecnologia (FCT) SFRH/BPD/84033/2012 Portuguese Institute for Employment and Vocational Training 0068/ET/18info:eu-repo/semantics/publishedVersio

Impact of dementia on informal care : a systematic review of family caregivers' perceptions

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.Introduction: Caregivers play a major role in providing all the support and care in daily activities for their relatives with dementia. To fully describe the influence of dementia caregiving on family caregivers’ life, we conducted a systematic review including caregivers’ perceptions about the positive and negative aspects of caring and the expressed factors. Materials and methods: We conducted a systematic review including articles from January 1998 to July 2020. Qualitative studies reporting family caregivers’ perceptions about their experiences and the effects/impact of dementia caregiving were eligible. Two authors extracted the data independently, and the analysis focused on the positive and negative aspects of dementia caregiving in caregivers’ life. Results: Eighty-one studies with 3347 participants were included in this review. The positive aspects of caregiving in caregivers’ life encompass personal accomplishment and strengthening relationships, which were enhanced by good medical counselling/formal care support and family/friends support. The negative aspects included emotional and social aspects experienced by caregivers. Other factors such as inappropriate medical/formal care support, illness progression and the costs of dementia contributed to negative appraisal. Discussion and implications: The findings provide insights into the holistic experience of caring for a person with dementia revelling the major positive and negative aspects underlying the caregiver role. The evidence emphasises the need ‘to focus on positive aspects’ and targeted interventions aimed at reducing the negative impact of caregiving, which has serious consequences on caregivers’ quality of life. A multicomplex intervention for dementia informal caregiving should be developed, committing the society to promote mental health, address these community needs and improve the quality of life of the person with dementia and their family caregivers.This review was funded by the 'Fundação para a Ciência e Tecnologia (FCT)' Portugal and Merck Sharp & Dohme Portugal (grant number PDE/BDE/120498/2016).info:eu-repo/semantics/publishedVersio

Let’s review the gut microbiota in systemic lupus erythematosus

© The Author(s) 2022. This is an Open Access article licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, andSystemic lupus erythematosus (SLE) is a chronic, immune-mediated disease associated with significant morbidity and mortality. New evidence suggests that diet, gut microbiota, intestinal permeability, and endotoxemia may modulate chronic inflammation and disease activity in SLE. This review focus on what is known about the gut microbiota in lupus mouse models and SLE patients and the possible mechanisms that connect the gut microbiota with SLE. It included 29 studies (12 animal studies, 15 human studies, and 2 included data on both), with variable results regarding alpha and beta-diversity and gut microbiota composition between lupus-mouse models and SLE patients. Ruminococcus ( R.) gnavus was significantly increased in lupus nephritis (LN) in one study, but this was not corroborated by others. Despite the different results, mechanistic lupus mouse model studies have shown that gut microbiota can modulate disease activity. Interestingly, pathobiont translocation in monocolonized and autoimmune-prone mice induced autoantibodies and caused mortality, which could be prevented by a vaccine targeting the pathobiont. Moreover, studies on fecal transplants and diet on different lupus mouse models showed an effect on disease activity. In SLE patients, a higher adherence to the Mediterranean diet was associated with lower disease activity, which may be explained by the connection between diet and gut microbiota. Although gut dysbiosis has been observed in SLE patients and lupus mouse models, it remains to clarify if it is a cause or a consequence of the disease or its treatments. Further studies with larger and well-characterized populations will undoubtedly contribute to deciphering the role of gut microbiota in SLE development, progression, and outcome.info:eu-repo/semantics/publishedVersio

Atualizações das guidelines da American Heart Association (AHA) em reanimação pediátrica

Abordam-se as principais mudanças nas diretrizes de 2020 da AHA para a ressuscitação cardiopulmonar (RCP), relativas à atuação em lactentes e crianças.info:eu-repo/semantics/publishedVersio

Conhecimentos e práticas na gestão de resíduos hospitalares em postos de colheitas de material biológico, enquanto fatores promotores da otimização dos processos e procedimentos de gestão

Mestrado em Gestão e Avaliação de Tecnologias em SaúdeOs Resíduos Hospitalares são um dos resultados imediatos e inevitáveis da prestação de cuidados de saúde, mas diferem na sua diversidade, perigosidade e grau de risco, sendo necessárias diferentes formas de tratamento e eliminação. Num sistema de gestão de qualidade em saúde e na procura da melhoria contínua, faz sentido identificar os fatores que condicionam o processo em estudo e quais as ações corretivas que permitem evitar o desperdício e as más práticas, onde o conhecimento e as práticas dos profissionais são relevantes. A metodologia incluiu o levantamento bibliográfico sobre o tema e a formulação da pergunta de investigação: Os conhecimentos e práticas dos profissionais de saúde têm influência na gestão de resíduos hospitalares? Nesta sequência, foram definidos objetivos e foram utilizados dois instrumentos de recolha de dados, consistindo na aplicação de uma grelha de auditoria de gestão de Resíduos Hospitalares a um posto de colheitas de material biológico e no envio de um questionário online a 119 profissionais de saúde, da rede continental de postos de colheitas, entre janeiro e fevereiro de 2022. Após o tratamento dos dados conseguiu-se confirmar que a ausência ou errados conhecimentos dos profissionais de saúde levam a erros nas práticas influenciando diretamente a gestão de resíduos hospitalares.ABSTRACT - Hospital Waste is one of the immediate and inevitable results of the provision of health care, but it differs in its diversity, danger, and degree of risk, requiring different forms of treatment and disposal. In a health quality management system and in the search for continuous improvement, it makes sense to identify the factors that condition the process under study and which corrective actions allow to avoid waste and bad practices, where the knowledge and practices of professionals are relevant. The methodology included a bibliographic survey on the topic and the formulation of the research question: Do the knowledge and practices of health professionals influence the management of hospital waste? In this sequence, objectives were defined and two data collection instruments were used, consisting of the application of a Hospital Waste Management audit grid to a biological material collection point and the sending of an online survey to 119 health professionals, from the continental network of the collection post, in between January and February 2022. After processing the data, it was possible to confirm that the absence or wrong knowledge of health professionals leads to errors in practices, directly influencing the management of hospital waste.N/

The tourist experience: Exploring the relationship between tourist satisfaction and destination loyalty

Previous studies show that the quality of the tourist experience is affected by a large set of factors, many of which not directly related to the acquisition of specific products or services. Research also shows that tourist satisfaction is a determinant of destination loyalty. This study explores this topic by empirically analysing how the relationship between the satisfaction with the tourist experience and destination loyalty is affected by personal factors, such as socio-demographic characteristics and travelling motivations. This study is based on a small region representing an important destination in Algarve, Portugal, and uses data from a survey conducted on tourists of different nationalities during peak season. We identify the reasons leading to dissatisfaction with the tourism experience and, based on structural equation modelling, ascertain the relationship between satisfaction levels with the tourism experience and tourist destination loyalty. Then, a multiple group analysis provides a detailed overview of this relationship by establishing in which groups of tourists the relatedness between satisfaction and destination loyalty is stronger or weaker. In this sense, this study provides an important backdrop for destination managers looking to offer more effective and targeted marketing strategies

Aderência ao programa de integridade da lei anticorrupção brasileira e implantação de controles internos

The aim of this study is to investigate the adherence of Brazilian publicly listed companies to the parameters of the anticorruption law compliance program as well as its association with internal controls implementation. In light of the accounting scandals and corporate frauds reported in several countries, including Brazil, Law n. 12,846, known as the anti-corruption law, was approved on August 1st, 2013, and Decree n. 8,420, approved on March 18, 2013, regulated this law and defined criteria for the functioning of compliance programs. Considering that compliance programs can affect internal controls, it is important to understand the adherence of companies to their parameters as well as identifying the internal controls derived from them. The findings show a substantial degree of adherence among listed companies to the parameters of the compliance program. Additionally, the results revealed the existence of new internal controls, such as the emergence of the “compliance officer” function. A survey in the form of a questionnaire was adopted. The questionnaire was sent to companies associated with ABRASCA (Brazilian Association of Publicly Listed Companies). The final sample included 32 publicly listed companies. Paraconsistent logic was applied to analyze the data. This paper contributes by empirically showing that, in Brazil, publicly listed companies are adherent to the anti-corruption law. Furthermore, this paper reveals that higher levels of adherence to compliance programs are associated with greater implementation of internal controls.Este estudo investiga o grau de aderência das empresas brasileiras de capital aberto aos critérios do programa de integridade estabelecido na lei anticorrupção e, ademais, avalia a relação entre esse programa e a implantação de controles internos. Diante dos escândalos contábeis e fraudes corporativas reportados em diversos países, incluindo o Brasil, aprovou-se a Lei n. 12.846, de 1 de agosto de 2013, conhecida como lei anticorrupção, e o Decreto n. 8.420, de 18 de março de 2015, que regulamentou essa lei e definiu critérios para o funcionamento de programas de integridade. Considerando que os programas de integridade podem afetar os controles internos, é relevante compreender a aderência das empresas aos parâmetros neles estabelecidos, bem como identificar controles internos deles decorrentes. Os resultados do estudo apontam que as empresas de capital aberto têm substancial grau de aderência aos parâmetros do programa de integridade. Ademais, constataram-se novos controles internos como, por exemplo, o surgimento da função compliance officer. Um questionário foi enviado para empresas de capital aberto associadas à Associação Brasileira de Companhias Abertas (ABRASCA). A amostra final foi composta por 32 empresas. A lógica paraconsistente foi aplicada para análise dos dados. Evidências obtidas na pesquisa indicam que, no Brasil, as pessoas jurídicas, especificamente as empresas de capital aberto, mostram-se aderentes à lei anticorrupção e que maiores níveis na adesão aos programas de integridade relacionam-se a maiores níveis de implantação de controles internos