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Tese de doutoramento em Medicina, Buruli ulcer (BU) is the third most common mycobacteriosis affecting immunocompetent humans worldwide. It is caused by Mycobacterium ulcerans, which produces a dermonecrotic toxin, responsible for causing mutilating ulcerative skin lesions. However, not all people exposed to M. ulcerans develop BU and few among those that do, are actually able to recover from disease associated lesions, without the aid of any treatment. These and other findings suggest that there is a genetic component associated to the development of BU, which we sought to better understand in the present thesis. To such aim, we performed an expression profiling analysis of the Hartley Cavia porcellus and FVB/N Mus musculus animal models, known to mimic the aforementioned spontaneous healing phenotype exhibited by humans, and combined it with data from a recent Genome-Wide Association Study involving BU patients. We found that each species controls infection using previously underappreciated pathways, some of which are common to the three models. The obtained results fostered the pursuit for a clinical correlation over known targets of M. ulcerans infection. We thus expanded a national cohort of Beninese individuals from M. ulcerans-endemic areas to 309 BU cases and 309 age- and gender-matched controls, in order to carry out candidate gene association studies. We found a genetic variant in the intronic region of the BCL2L11 gene, rs13421194, to be associated with the odds of development of ulcerative forms of the disease, potentially due to differences in the transcription factors that are predicted to bind to the region. Finally, we present another genetic association between rs1800872 alleles in the IL10 promoter region and the odds of development of most severe BU presentations, WHO category III lesions. This association was accompanied by in vitro and in vivo functional studies using Il10 knockout and transgenic mice, demonstrating that high production of IL-10 is detrimental during the earlier stages of M. ulcerans infection. The present thesis therefore provides a valid framework to leverage future studies on the mechanisms of resistance to BU, opening novel and exciting horizons of research., A úlcera de Buruli (UB) é a terceira micobacteriose mais comum em humanos imunocompetentes em todo o mundo. É causada pela Mycobacterium ulcerans, produtora de uma toxina dermonecrótica, responsável por causar lesões cutâneas ulcerativas mutilantes. Contudo, nem todas as pessoas expostas à M. ulcerans desenvolvem UB e algumas das que a desenvolvem conseguem recuperar das suas lesões sem auxílio de qualquer tratamento. Estes e outros achados sugerem que existe uma componente genética associada ao desenvolvimento da UB, o qual nos propusemos a explorar na presente tese. Para tal, analisámos o perfil de expressão genética dos modelos animais Hartley de Cavia porcellus e FVB/N de Mus musculus, capazes de mimetizar o fenótipo de cura espontânea supramencionado exibido pelos humanos, e combinámo-los com os dados de um estudo recente de associação do genoma em doentes com UB. Descobrimos que cada espécie controla a infeção utilizando vias previamente subapreciadas, algumas das quais comuns aos três modelos. Os resultados obtidos fomentaram a busca por uma correlação clínica sobre alvos conhecidos da infeção por M. ulcerans. Expandimos assim uma coorte de indivíduos beninenses provenientes de áreas endémicas de M. ulcerans para 309 casos de UB e 309 controlos emparelhados por idade e sexo, de forma a realizarmos estudos de associação de genes candidatos. Encontrámos uma variante genética na região intrónica do gene BCL2L11, rs13421194, que se associa à probabilidade de desenvolvimento de formas ulcerativas da doença, provavelmente devido a diferenças nos fatores de transcrição preditos de se ligarem à região. Por fim, apresentamos outra associação genética entre os alelos do rs1800872 na região promotora da IL-10 e a probabilidade de desenvolvimento das apresentações mais graves de UB, as lesões da categoria III da OMS. Esta associação foi acompanhada por estudos funcionais in vitro e in vivo com murganhos transgénicos e knockout para Il10, demonstrando que a alta produção de IL-10 é prejudicial durante as fases iniciais da infeção por M. ulcerans. A presente tese oferece, portanto, uma estrutura válida para alavancar estudos futuros sobre os mecanismos de resistência à UB, abrindo novos e entusiasmantes horizontes de investigação., This work was developed in the Microbiology and Infection Research Domain of the Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, in collaboration with the Centre de Dépistage et de Traitement de l’Ulcère de Buruli (CDTUB) in Allada, Benin. Financial support derived from project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER); FEDER, through the Competitiveness Factors Operational Programme (COMPETE) and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145- FEDER-007038; Infect-ERA grant Infect-ERA/0002/2015. Personal sustainability was attentively conceded by the FCT through the PhD grant PD/BD/113699/2015.

Tese de doutoramento em Medicina, O manuseio da via aérea é um componente central dos cuidados anestésicos. Todos os doentes submetidos a procedimentos anestésicos podem estar em risco de terem ventilação e oxigenação insuficientes, se não for conseguida uma via aérea segura. Existem indicadores clínicos que podem prever um manuseio difícil da via aérea, mas globalmente têm baixa sensibilidade e moderada especificidade, pelo que é relevante encontrar melhores preditores de forma a conseguir uma abordagem segura da via aérea a cada paciente. A recente divulgação da utilização da ultrassonografia (US) no cuidado do doente, inclui a sua aplicação à avaliação da via aérea. Numerosos parâmetros US têm sido estudados como preditores de via aérea difícil; no entanto, ainda não é claro quais os parâmetros que devem ser considerados para uso clínico. Na presente Tese, através dos resultados de uma revisão sistemática e meta-análise caracterizamos inicialmente o preditor US mais relevante na previsão de uma laringoscopia difícil. A distância hiomentoniana (DHM) em posição neutral revelou-se o preditor mais consistente para ser utilizado na clínica. Tratando-se de uma competência técnica, o uso da US é dependente do operador e requere treino e avaliação através de escalas objetivas e estruturadas. Assim, no segundo trabalho desta Tese criámos um programa de ensino e de treino da utilização da DHM por US. Simultaneamente, avaliamos a competência de participantes utilizando a escala Objective Structured Assessment Ultrasound Skills (OSAUS) e analisamos as suas propriedades psicométricas. Os resultados mostraram que a escala é válida e confiável na avaliação do desempenho técnico. Finalmente, testamos uma nova aplicação clínica para a US da via aérea na intubação/exclusão seletiva pulmonar, num modelo animal pediátrico. Este trabalho mostrou resultados promissores para a translação para a população pediátrica. Em conclusão, os trabalhos de investigação desta Tese permitiram identificar um preditor confiável de laringoscopia difícil, que pode ser avaliado clinicamente por US, para o qual é necessário o desenvolvimento e avaliação da competência. Simultaneamente, apresentamos a aplicação inovadora da US na intubação seletiva no modelo animal pediátrico que pode contribuir para uma mudança do paradigma da abordagem da via aérea nesta população., Airway management is a core component of anesthesia care. Every patient submitted to anesthesia procedures is at risk of insufficient ventilation and oxygenation if a secure airway could not be achieved. Many clinical indicators can predict difficult airway but globally with low sensibility and moderate specificity. Therefore, is relevant to find better predictors to achieve a safe airway approach. The recent widespread use of ultrasound in patient care includes airway assessment. Numerous ultrasound measurements have been studied as predictors of difficult airway; nevertheless, it is still unclear which parameters should be considered for routine clinical use. In the present Thesis, we have characterized the most relevant ultrasound predictors of difficult laryngoscopy and difficult intubation throughout a systematic review and meta-analysis. Hyomental distance (HMD) was the most consistent factor in the clinical setting. As a technical skill that is operator dependent, ultrasound requires training and assessment with objective and structured scales. So, the second work of our Thesis, we created a teaching and training program to use ultrasound HMD. Simultaneously, we assessed participants’ competence using the Objective Structured Assessment Ultrasound Skills (OSAUS) scale and analyzed the scale’s psychometric properties. Results showed that OSAUS scale is a valid and reliable tool that can be used to assess performance. Finally, we tested a new clinical application of ultrasound in selective lung intubation/exclusion in a pediatric animal model. This work showed promising results for translation to the pediatric population. In conclusion, the research work of this Thesis allowed the identification of a reliable ultrasound predictor of difficult laryngoscopy, that can be trained and assessed. Simultaneously, we presented an innovative application of ultrasound in selective intubation in pediatric animal model that can contribute to change the paradigm of airway management in this population., The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), University of Minho. Financial Support was provided by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020

Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder (ND). While most research in NDs has been following a neuron-centric point of view, microglia are now recognized as crucial in the brain. Previous work revealed alterations that point to an increased activation state of microglia in the brain of CMVMJD135 mice, a MJD mouse model that replicates the motor symptoms and neuropathology of the human condition. Here, we investigated the extent to which microglia are actively contributing to MJD pathogenesis and symptom progression. For this, we used PLX3397 to reduce the number of microglia in the brain of CMVMJD135 mice. In addition, a set of statistical and machine learning models were further implemented to analyze the impact of PLX3397 on the morphology of the surviving microglia. Then, a battery of behavioral tests was used to evaluate the impact of microglial depletion on the motor phenotype of CMVMJD135 mice. Although PLX3397 treatment substantially reduced microglia density in the affected brain regions, it did not affect the motor deficits seen in CMVMJD135 mice. In addition to reducing the number of microglia, the treatment with PLX3397 induced morphological changes suggestive of activation in the surviving microglia, the microglia of wild-type animals becoming similar to those of CMVMJD135 animals. These results suggest that microglial cells are not key contributors for MJD progression. Furthermore, the impact of PLX3397 on microglial activation should be taken into account in the interpretation of findings of ND modification seen upon treatment with this CSF1R inhibitor., This work was supported by Fundação para a Ciencia e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MEDOUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/127828/2016).B.C. was also supported by FCT (2020.03898.CEECIND). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT-project UIDB/50026/2020 and UIDP/50026/2020.

Circulating phagocytic cells often serve as cellular targets for a large number of pathogens such as Leishmania parasites. Studying primary human cells in an infectious context requires lengthy procedures for cell isolation that may affect the analysis performed. Using whole blood and a no-lyse and no-wash flow cytometric assay (NoNo assay), we monitored the Leishmania infantum infection of primary human cells. We demonstrated, using fluorescent parasites, that among monocyte cell populations, L. infantum preferentially infects classical (CD14+CD16−) and intermediate (CD14+CD16+) primary human monocytes in whole blood. Because classical monocytes are the preponderant population, they represent the larger L. infantum reservoir. Moreover, we also found that, concomitantly to monocyte infection, a subset of PMNs is infected early in whole blood. Of interest, in whole blood, PMNs are less infected compared to classical monocytes. Overall, by using this NoNo assay, we provided a novel avenue in our understanding of host–leishmania interactions., European Community’s Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED) and Infect-ERA ERA-NET (Project InLeish) to J.E. J.E. also thanks the Canada Research Chair program for financial assistance. S.A. is supported by a post-doctoral fellowship granted by Fédération pour la Recherche Médicale (FRM: SPF20160936115) and by Infect-Era (ANR-16-IFEC-0002). This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013) and the Fundação para a Ciência e Tecnologia (FCT) (contract CEECIND/00185/2020 to RS).

Colorectal cancer (CRC) is one of the most common malignancies and one of the leading causes of cancer-related death worldwide, urging the need for new and more efficient therapeutic approaches. Ruthenium complexes have emerged as attractive alternatives to traditional platinum-based compounds in the treatment of CRC. This work aims to evaluate anti-CRC properties, as well as to identify the mechanisms of action of ruthenium complexes with the general formula [Ru(η5-C5H4R)(PPh3)(4,4'-R'-2,2'-bipyridine)][CF3SO3], where R = CH3, CHO or CH2OH and R' = H, CH3, CH2OH, or dibiotin ester. The complexes (Ru 1-7) displayed high bioactivity, as shown by low IC50 concentrations against CRC cells, namely, RKO and SW480. Four of the most promising ruthenium complexes (Ru 2, 5-7) were phenotypically characterized and were shown to inhibit cell viability by decreasing cell proliferation, inducing cell cycle arrest, and increasing apoptosis. These findings were in accordance with the inhibition of MEK/ERK and PI3K/AKT signaling pathways. Ruthenium complexes also led to a decrease in cellular clonogenic ability and cell migration, which was associated with the disruption of F-actin cytoskeleton integrity. Here, we demonstrated that ruthenium complexes, especially Ru7, have a high anticancer effect against CRC cells and are promising drugs to be used as a new therapeutical strategy for CRC treatment., Fundação para a Ciência e Tecnologia (FCT), I.P./MCTES through national funds (PIDDAC)—UIDB/00100/2020, UIDB/04050/2020 and through PTDC/QUI-QIN/28662/2017. A.V. acknowledges the CEECIND 2017 Initiative (CEECCIND/01974/2017). A.R.B. and R.G.T. thank FCT for their Ph.D. Grants (SFRH/BD/139271/2018 and SFRH/BD/135830/2018, respectively)

Melanoma is the most aggressive and life-threatening skin cancer type. The melanoma genome is the most frequently mutated, with the BRAF mutation present in 40–60% of melanoma cases. BRAF-mutated melanomas are characterized by a higher aggressiveness and progression. Adjuvant targeted treatments, such as BRAF and MEK inhibitors, are added to surgical excision in BRAF-mutated metastatic melanomas to maximize treatment effectiveness. However, resistance remains the major therapeutic problem. Interest in natural products, like propolis, for therapeutic applications, has increased in the last years. Propolis healing proprieties offer great potential for the development of novel cancer drugs. As the activity of Portuguese propolis has never been studied in melanoma, we evaluated the antitumoral activity of propolis from Gerês (G18.EE) and its fractions (n-hexane, ethyl acetate (EtOAc), and n-butanol) in A375 and WM9 melanoma cell lines. Results from DPPH•/ABTS• radical scavenging assays indicated that the samples had relevant antioxidant activity, however, this was not confirmed in the cell models. G18.EE and its fractions decreased cell viability (SRB assay) and promoted ROS production (DHE/Mitotracker probes by flow cytometry), leading to activation of apoptotic signaling (expression of apoptosis markers). Our results suggest that the n-BuOH fraction has the potential to be explored in the pharmacological therapy of melanoma., Foundation for Science and Technology (FCT)—projects UIDB/04050/2020, UDBI/04033/2020, UIDB/50026/2020, UIDP/50026/2020, UIDB/50006/2020, UIDP/50006/2020, and by the project NORTE-01-0145-FEDER-000055, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). S.P.C., C.B.M. and A.S.F. are recipients of FCT grants (2020.05779.BD, SFRH/BD/145955/2019, and PD/BD/128276/2017, respectively)

Pancreatic cancer evades most of the current therapies and there is an urgent need for new treatments that could efficiently eliminate this aggressive tumor, such as the blocking of routes driving cell proliferation. In this work, we propose the use of small interfering RNA (siRNA) to inhibit the combined expression of FOSL-1 and YAP, two signaling proteins related with tumor cell proliferation and survival. To improve the efficacy of cell transfection, DODAB:MO (1:2) liposomes were used as siRNA nanocarriers, forming a complex denominated siRNA-lipoplexes. Liposomes and lipoplexes (carrying two siRNA for each targeted protein, or the combination of four siRNAs) were physico-chemically and biologically characterized. They showed very good biocompatibility and stability. The efficient targeting of FOSL-1 and YAP expression at both mRNA and protein levels was first proved in vitro using mouse pancreatic tumoral cell lines (KRASG12V and p53 knockout), followed by in vivo studies using subcutaneous allografts on mice. The peri-tumoral injection of lipoplexes lead to a significant decrease in the tumor growth in both Athymic Nude-Foxn1nu and C57BL/6 mice, mainly in those receiving the combination of four siRNAs, targeting both YAP and FOSL-1. These results open a new perspective to overcome the fast tumor progression in pancreatic cancer., government of Spain (ref. BIO2017-84974-R) and the Xunta de Galicia (Grupo de Referencia Competitiva [ED431C 2020/02]) and Centro singular de investigacion de Galicia and the European Regional Development Fund (ERDF)-[ED431G2019/06]. This work was also supported by grants from the CRIS Cancer Foundation and the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00) to M.B. M.B. is a recipient of an Endowed Chair from the AXA Research Fund.

Tese de doutoramento em Medicina, A reconstrução capsular superior artroscópica (RCSA) é uma opção de tratamento cirúrgico recente para roturas irreparáveis da coifa dos rotadores (RICR). O primeiro estudo clínico sobre RCSA reportou melhorias significativas dos resultados clínicos e imagiológicos com autoenxerto de fáscia lata proximal (AFLP) colhido por via aberta. Preocupações com a morbilidade da zona dadora levaram a modificações da RCSA e ao uso do aloenxerto dérmico humano (ADH), mas foram reportados resultados clínicos inferiores e uma menor taxa de sobrevivência do ADH. Modificámos a RCSA e utilizámos autoenxerto de fáscia lata do terço médio da coxa (AFLM) colhido por via minimamente invasiva (MI). A importância da escolha do tipo de enxerto, visando maximizar os resultados clínicos, permaneceu por esclarecer. Esta tese teve como objetivo determinar os resultados da RCSA com autoenxerto de fáscia lata (AFL) ou ADH, comparar as propriedades biomecânicas do AFLP e AFLM, avaliar os resultados clínicos e de ressonância magnética aos 3 anos da RCSA com AFLM, e determinar a importância clínica da integridade do enxerto. As hipóteses eram que a RCSA com AFL teria resultados superiores à RCSA com ADH; as propriedades biomecânicas do AFLP e AFLM não seriam significativamente diferentes; os resultados clínicos melhorariam significativamente desde o pré-operatório até aos 3 anos e seriam significativamente piores em pacientes que tivessem uma rotura completa do enxerto aos 3 anos. Verificámos que a RCSA com AFL ou ADH resultou em melhorias clínicas significativas aos 12 meses; os valores médios da rigidez e do módulo de Young não diferiram significativamente entre o AFLP e o AFLM; o impacto da morbilidade da zona dadora da colheita MI do AFLM foi baixo, os resultados clínicos da RCSA usando o AFLM foram bons, e as roturas completas do enxerto correlacionaram-se com diminuição da força de rotação externa e maior grau de degenerescência gorda dos músculos infraespinhoso e pequeno redondo aos 3 anos. O presente trabalho mostra que a integridade do enxerto é importante para a manutenção de melhores resultados clínicos a médio prazo, e que o AFLM pode ser uma boa alternativa ao AFLP. As nossas observações biomecânicas podem contribuir para futuros trabalhos para determinar o papel do AFL, abrindo caminho para novas estratégias para melhorar a técnica, a cicatrização do enxerto e os resultados clínicos. Este trabalho contribuiu para aumentar o conhecimento sobre a RCSA e demonstrou ter um papel relevante na melhoria da qualidade de vida dos pacientes submetidos a RCSA por RICR., Arthroscopic superior capsular reconstruction (ASCR) is a recent surgical treatment option for irreparable rotator cuff tears (IRCTs). The first clinical study on ASCR reported significant improvements in clinical and imaging results using an open harvested proximal fascia lata autograft (FLA). Concerns about donor site morbidity led to modifications of ASCR and to the use of the human dermal allograft (HDA). However, inferior clinical outcomes and decreased allograft survival rates were reported. The importance of the choice of the type of graft for each patient, aiming to maximize clinical outcomes in ASCR, remained unclear. We modified ASCR and used a minimally invasively (MI) harvested mid-thigh FLA (MFLA) aiming to reproduce the good clinical results of ASCR using the open harvested proximal FLA (PFLA). This thesis aimed to determine the clinical outcomes of ASCR using either the FLA or HDA, compare the biomechanical properties of the PFLA and MFLA, assess the 3-year clinical and MRI outcomes of ASCR using the MFLA, and determine the clinical importance of graft integrity. The hypotheses were that the ASCR using FLA would produce superior clinical outcomes and a higher graft survival rate than ASCR using the HDA; the biomechanical properties of the PFLA and MFLA would not significantly differ; the clinical outcomes would improve significantly from preoperatively to 3 years and would be significantly worse in patients who had a complete graft tear 3 years after ASCR. We found that ASCR using either FLA or HDA resulted in significant clinical improvements at a minimum follow-up of 12 months; the average values of the stiffness and Young’s modulus did not significantly differ between the PFLA and MFLA; the impact of the donor site morbidity of the MI harvesting of the MFLA was low and the clinical outcomes of ASCR using the MFLA were good at 3 years; and complete graft tears were correlated with significantly decreased external rotation strength and increased grades of infraspinatus and teres minor fatty degeneration. The present work shows that graft integrity is important for maintaining improved clinical shoulder outcomes at medium-term and that the MFLA may be a good alternative to the PFLA. Our biomechanical observations may assist in future research designed to determine the role of the FLA, paving the way to finding new strategies to improve the technique, graft healing, and clinical outcomes of ASCR. Finally, this work contributed to increase the knowledge about ASCR and proved to have a relevant role in contributing to the improvement of the quality of life of the patients who undergo ASCR for IRCTs., Financial support was provided by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020

Advanced strategies to bioengineer a fibrocartilaginous tissue to restore the function of the meniscus are necessary. Currently, 3D bioprinting technologies have been employed to fabricate clinically relevant patient-specific complex constructs to address unmet clinical needs. In this study, a highly elastic hybrid construct for fibrocartilaginous regeneration is produced by coprinting a cell-laden gellan gum/fibrinogen (GG/FB) composite bioink together with a silk fibroin methacrylate (Sil-MA) bioink in an interleaved crosshatch pattern. We characterize each bioink formulation by measuring the rheological properties, swelling ratio, and compressive mechanical behavior. For in vitro biological evaluations, porcine primary meniscus cells (pMCs) are isolated and suspended in the GG/FB bioink for the printing process. The results show that the GG/FB bioink provides a proper cellular microenvironment for maintaining the cell viability and proliferation capacity, as well as the maturation of the pMCs in the bioprinted constructs, while the Sil-MA bioink offers excellent biomechanical behavior and structural integrity. More importantly, this bioprinted hybrid system shows the fibrocartilaginous tissue formation without a dimensional change in a mouse subcutaneous implantation model during the 10-week postimplantation. Especially, the alignment of collagen fibers is achieved in the bioprinted hybrid constructs. The results demonstrate this bioprinted mechanically reinforced hybrid construct offers a versatile and promising alternative for the production of advanced fibrocartilaginous tissue., United States National Institutes of Health (1P41EB023833-346 01) and the Portuguese Foundation for Science and Technology (PTDC/BBB-ECT/2690/2014 and PTDC/EMD-EMD/ 31367/2017). FCT/MCTES is acknowledged for the Ph.D. scholarship attributed to J.B.C. (PD/BD/113803/2015) and the financial support provided to J.S.-C. (IF/00115/2015) and J.M.O. (IF/01285/2015) under the program “Investigador FCT”

Background: Patients with obsessive–compulsive disorder (OCD) employ ritualistic behaviours to reduce or even neutralize the anxiety provoked by their obsessions. The presence of excessive rumination and indecision has motivated the view of OCD as a disorder of decision-making. Most studies have focused on the “cold,” cognitive aspects of decision-making. This study expands current understanding of OCD by characterizing the abnormalities associated with affective, or “hot” decision-making. Methods: We performed a functional MRI study in a sample of 34 patients with OCD and 33 sex-and age-matched healthy controls, during which participants made 2-choice gambles taking varying levels of risk. Results: During risky decisions, patients showed significantly reduced task-related activation in the posterior cingulum, lingual gyrus and anterior cingulate cortex. We identified significant group × risk interactions in the calcarine cortex, precuneus, amygdala and anterior cingulate cortex. During the outcome phase, patients with OCD showed stronger activation of the orbitofrontal cortex, anterior cingulate cortex and putamen in response to unexpected losses. Limitations: The group of patients not receiving medication was very small (n = 5), which precluded us from assessing the effect of medication on risk-taking behaviour in these patients. Conclusion: Obsessive–compulsive disorder is associated with abnormal brain activity patterns during risky decision-making in a set of brain regions that have been consistently implicated in the processing of reward prediction errors. Alterations in affective “hot” processes implicated in decision-making may contribute to increased indecisiveness and intolerance to uncertainty in patients with OCD., FCT fellow-ship grants (PhD-iHES program) with the references PDE/BDE/113601/2015 and PDE/BDE/113604/2015, respectively. P. Marques was funded by the Fundação Calouste Gulbenkian (Con-tract grant number P-139977, project “Better mental health during aging based on temporal prediction of individual brain aging trajec-tories [TEMPO]”). A. Coelho is supported by a scholarship from the project NORTE-08-5639-FSE-000041 (Norte Portugal Regional Opera-tional Programme, NORTE 2020; UMINHO/BD/51/2017). The pres-ent work was supported by SwitchBox-FP7-HEALTH-2010 grant 259772-2 and cofinanced by the Portuguese North Regional Opera-tional Program (ON.2 – O Novo Norte) under the National Strategic Reference Framework (QREN), through the European Regional De-velopment Fund (FEDER). H. Siebner holds a 5-year professorship in precision medicine at the Faculty of Health Sciences and Medicine, University of Copenhagen, which is sponsored by the Lundbeck Foundation (grant no. R186-2015-2138).

It is generally accepted that metabolism is able to shape the immune response. Only recently we are gaining awareness that the metabolic crosstalk between different tumor compartments strongly contributes to the harsh tumor microenvironment (TME) and ultimately impairs immune cell fitness and effector functions. The major aims of this review are to provide an overview on the immune system in cancer; to position oxygen shortage and metabolic competition as the ground of a restrictive TME and as important players in the anti-tumor immune response; to define how immunotherapies affect hypoxia/oxygen delivery and the metabolic landscape of the tumor; and vice versa, how oxygen and metabolites within the TME impinge on the success of immunotherapies. By analyzing preclinical and clinical endeavors, we will discuss how a metabolic characterization of the TME can identify novel targets and signatures that could be exploited in combination with standard immunotherapies and can help to predict the benefit of new and traditional immunotherapeutic drugs., M. Mazzone was supported by European Research Council (ERC) Consolidator Grant 773208, Fonds Wetenschappelijk Onderzoek (Flemish Research Foundation) Grants G0D1717N and G066515N, and Stichting Tegen Kanker (Fondation Contre le Cancer) Grant 2014197. C. Riera-Domingo was supported by Fonds Wetenschappelijk Onderzoek (Flemish Research Foundation) Grant 1108919N. S. Granja received a fellowship from the Foundation for Science and Technology (FCT) Ref. No. SFRH/BPD/117858/2016. F. Baltazar worked under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020) under the Portugal Partnership Agreement, through the European Regional Development Fund (FEDER), and through the Competitiveness Factors Operational Programme (COMPETE) and by National funds, through the FCT, under the scope of the project POCI-01-0145-FEDER-007038. F. Baltazar also acknowledges financial support from the projects Ref. No. NORTE-01-0145-FEDER-031142 and UTAP-EXPL/NTec/0038/2017. C. Stockmann was supported by Swiss National Fund (179235), the Swiss Cancer Research (KFS-4397-02-2018), and the National Center of Compentence in Research (NCCR): Kidney Control of Homeostasis (183774). P.-C. Ho was supported in part by the SNSF Project Grant 31003A_182470 and ERC Staring Grant 802773-Mito-Guide.

Small bowel capsule endoscopy (SBCE) is the reference standard tool for diagnosing small bowel bleeding (SBB). The "TOP 100", which performs an automatic selection of the 100 images that mostly likely contain abnormalities, emerged as a new functionality of the RAPID Reader® software in 2017. Background Small bowel capsule endoscopy (SBCE) is the reference standard tool for diagnosing small bowel bleeding (SBB). The “TOP 100”, which performs an automatic selection of the 100 images that mostly likely contain abnormalities, emerged as a new functionality of the RAPID Reader® software in 2017. Aim To compare the concordance of findings between the standard reading (SR) and the use of TOP 100 in suspected SBB. Methods Retrospective study, including consecutive patients submitted to SBCE for suspected SBB. Two experienced readers performed SR and reported the most important findings. Another experienced reader, who was blinded to the SR results, reviewed all the SBCE videos using TOP 100 and reported the most important findings. The relevant findings were defined as the presence of high bleeding potential lesions (P2). Results 97 patients were included. The TOP 100 detected 81/97(83.5%) of the P2 lesions, in particular 64/67(95.5%) of the angioectasias and 17/30(56.7%) of the ulcers. The TOP 100 identified all sites of active bleeding (n = 9). Conclusion The TOP 100 identified all sites of active bleeding, as well as the vast majority of significant lesions (83.5%); in particular, it detected over 95% of the angioectasias. Although SR remains the reference standard in the SBCE review, these findings demonstrate that TOP 100 allows for a quick preview reading constituting an important asset in the identification of lesions that may require priority full review and intervention planning

Mutation testing of the key genes involved in melanoma oncogenesis is now mandatory for the application of targeted therapeutics. However, knowledge of the mutational profile of melanoma remains largely unknown in Brazil. PURPOSE Mutation testing of the key genes involved in melanoma oncogenesis is now mandatory for the application of targeted therapeutics. However, knowledge of the mutational profile of melanoma remains largely unknown in Brazil. PATIENTS AND METHODS In this study, we assessed the mutation status of melanoma driver genes BRAF, NRAS, TERT, KIT, and PDGFRA in a cohort of 459 patients attended at Barretos Cancer Hospital between 2001 and 2012. We used polymerase chain reaction followed by Sanger sequencing to analyze the hot spot mutations of BRAF exon 15 (V600E), NRAS (codons 12/13 and 61), TERT (promoter region), KIT (exons 9, 11, 13, and 17), and PDGFRA (exons 12, 14, and 18) in tumors. The mutational profile was investigated for associations with demographic, histopathologic, and clinical features of the disease. RESULTS The nodular subtype was most frequent (38.9%) followed by the superficial spreading subtype (34.4%). The most frequent tumor location was in the limbs (50.0%). The mutation rates were 34.3% for TERT and 34.1% for BRAF followed by NRAS (7.9%), KIT (6.2%), and PDGFRA (2.9%). The BRAF (P = .014) and TERT (P = .006) mutations were associated with younger patients and with different anatomic locations, particularly in the trunk, for the superficial spreading and nodular subtypes, respectively (P = .0001 for both). PDGFRA mutations were associated with black skin color (P = .023) and TERT promoter mutations with an absence of ulceration (P = .037) and lower levels of lactate dehydrogenase. There was no association between patient survival rates and mutational status. CONCLUSION The similar mutational profile we observe in melanomas in Brazil compared with other populations will help to guide precision medicine in this country., CAPES -Coordenação de Aperfeiçoamento de Pessoal de Nível Superior(2012/04194-1)

Although guidelines exist for advanced and variant bladder cancer management, evidence is limited/conflicting in some areas and the optimal approach remains controversial. Background: Although guidelines exist for advanced and variant bladder cancer management, evidence is limited/conflicting in some areas and the optimal approach remains controversial. Objective: To bring together a large multidisciplinary group of experts to develop consensus statements on controversial topics in bladder cancer management. Design: A steering committee compiled proposed statements regarding advanced and variant bladder cancer management which were assessed by 113 experts in a Delphi survey. Statements not reaching consensus were reviewed; those prioritised were revised by a panel of 45 experts before voting during a consensus conference. Setting: Online Delphi survey and consensus conference. Participants: The European Association of Urology (EAU), the European Society for Medical Oncology (ESMO), experts in bladder cancer management. Outcome measurements and statistical analysis: Statements were ranked by experts according to their level of agreement: 1–3 (disagree), 4–6 (equivocal), 7–9 (agree). A priori (level 1) consensus was defined as 70% agreement and 15% disagreement, or vice versa. In the Delphi survey, a second analysis was restricted to stakeholder group(s) considered to have adequate expertise relating to each statement (to achieve level 2 consensus). Results and limitations: Overall, 116 statements were included in the Delphi survey. Of these, 33 (28%) statements achieved level 1 consensus and 49 (42%) statements achieved level 1 or 2 consensus. At the consensus conference, 22 of 27 (81%) statements achieved consensus. These consensus statements provide further guidance across a broad range of topics, including the management of variant histologies, the role/limitations of prognostic biomarkers in clinical decision making, bladder preservation strategies, modern radiotherapy techniques, the management of oligometastatic disease and the evolving role of checkpoint inhibitor therapy in metastatic disease. Conclusions: These consensus statements provide further guidance on controversial topics in advanced and variant bladder cancer management until a time where further evidence is available to guide our approach, The authors would like to thank Peter E. Clark from Atrium Health, Levine Cancer Institute, Charlotte, NC, USA, for his contribution to the Delphi survey. Angela Corstorphine of Kstorfin Medical Communications Ltd provided medical writing support with the preparation of this manuscript; this support was funded jointly by EAU and ESMO.

Epstein-Barr virus (EBV) has been associated with about 9% of all gastric carcinomas, but its role in gastric carcinogenesis remains unclear since there is lack of evidence of EBV presence in pre-neoplastic lesions of gastric mucosa. This study intends to determine the prevalence of EBV in gastric dysplasia and superficial neoplasia to clarify whether EBV infection is an early or late event in gastric cancer development. This retrospective study included a total of 242 gastric lesions from 199 consecutive patients who were referred for endoscopic resection. The histological classification of lesions includes 137 low- and high-grade dysplasia and 105 superficial carcinomas. EBV infection was investigated by EBER-ISH. Results showed that EBV was not detected in any epithelial cells of any case with dysplasia or superficial carcinomas, although we observed the presence of a small number of EBV-infected lymphocytes in 2.1% of all lesions. These results showed that EBV is not present in gastric dysplasia neither in superficial carcinomas suggesting that EBV carcinogenesis is a late event in well/moderately differentiated gastric carcinogenesis., Joana Ribeiro was granted with a PhD Scholarship (SFRH/BD/107740/2015) from FCT-Fundação para Ciência e Tecnologia.

Tese de Doutoramento em Ciências da Saúde, O disco intervertebral é a unidade responsável por absorver o stress mecânico e proporcionar flexibilidade à coluna. Para isso, a composição e organização da matriz extracelular torna.se essencial para preservar o equilíbrio biomecânico deste. Contudo, esta patologia não é devidamente compreendida dada a sua complexidade e falta de modelos experimentais. A degeneração do disco compreende uma vasta apresentação clínica durante o envelhecimento tais como: fibrose, calcificação, hérnia ou apenas degeneração a nível molecular. Para além disto, a relação entre o fundo genético, o envelhecimento e o fenótipo patológico apresentado ainda não foram estabelecidos. Assim, caracterizamos o fenótipo do disco intervertebral de 3 raças de ratinhos com diferentes capacidades regenerativas na cartilagem aos 6 e 23 meses de idade: C57BL/6 (BL6), LG/J e SM/J. Cada espécie de ratinho apresentou uma progressão única de degeneração do disco a nível morfológico e do transcriptoma. Os LG/J mostraram uma grande prevalência de calcificação no disco intervertebral. Por outro lado, os SM/J apresentaram uma degeneração acelerada do compartimento, com desorganização da matriz, morte celular e aumento da deposição de tecido fibrótico em comparação com os BL6. Estes resultados, mostram que a composição genética é um fator essencial no desenvolvimento degeneração do disco e do seu fenótipo durante o envelhecimento. Estudos recentes mostraram também uma associação importante senescência, degeneração do disco e catabolismo de matiz celular, quer em modelos animais quer em humanos. De facto, a eliminação de células positivas para o marcador de senescência, p16Ink4a, diminuiu a patologia associada a diferentes doenças do envelhecimento, incluindo a degeneração do disco intervertebral. Assim, caracterizamos pela primeira vez a evolução da senescência nas células do disco intervertebral de ratinhos jovens e envelhecidos e avaliamos o fenótipo de ratinhos envelhecidos p16Ink4a “condition knockout” (cKO). Adicionalmente, exploramos o potencial terapêutico da combinação de Dasatinib (D) e Quercetina (Q), na diminuição de células senescentes no disco e na prevenção da degeneração do mesmo durante o envelhecimento. Estes resultados sugerem que a regulação da senescência é importante na promoção e manutenção da homeostasia das células do disco, e que pode ser usado com alvo terapêutico para a prevenção e tratamento da degeneração do disco durante o envelhecimento., The intervertebral disc is a critical structure of the spinal column, responsible for mechanical loads and flexibility. Importantly, disc extracellular matrix (ECM) is complex and highly contributes to the biomechanical properties of the disc. However, it is still not well understood due to its complexity and lack of experimental models. Disc degeneration presents a broad spectrum of clinically degenerative phenotypes during aging, such as fibrosis, calcification, herniation, and mild degeneration. Moreover, the interaction between genetic background and aging, degenerative outcomes has not been established. Hence, we characterized the spinalphenotype of 3 mouse strains with varying cartilage-regenerative potential at 6- and 23-months: C57BL/6 (BL6), LG/J, and SM/J. Interestingly, each strain presented an unique aging phenotype and transcriptome modulation during intervertebral disc aging. LG/J mice showed a high prevalence of dystrophic disc calcification in caudal discs, while SM/J evidenced accelerated degeneration compared to BL6, with cellular disorganization, cell loss together, and fibrosis of the NP. These studies suggested that disc aging and degeneration depends on the genetic background and involves changes in various molecular pathways, which might help to explain the diverse phenotypes seen during disc disease. Additionally, recent studies have shown that senescence may play a role in age-related disc degeneration and matrix catabolism in humans and mouse models. Clearance of p16Ink4a, one of the main markers of cell senescence, positive cells reduce the degenerative phenotype in age-associated diseases, including disc degeneration. Therefore, we first characterized the senescence status of discs in young and old mice and accessed the degeneration status of aged p16Ink4a conditional knockout mice. Additionally, we explored the potential of Dasatinib and Quercetin combination in targeting senescence in disc compartment and preventing progression of the disease with age. These results suggested that senescence plays an essential role in governing disc homeostasis during aging and can be used as a therapeutic target to treat disc disease., Financial support for this PhD work was provided by Fundação para a Ciência e a Tecnologia (FCT) fellowship (PD/BD/128077/2016) from MD/PhD Program of the University of Minho. Additionally, this study was supported by the grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01AR055655, R01AR064733 and R01AR074813 to Makarand V. Risbud.

Intervertebral disc degeneration is an important contributor to chronic low back and neck pain. Although many environmental and genetic factors are known to contribute to disc degeneration, age is still the most significant risk factor. Recent studies have shown that senescence may play a role in age-related disc degeneration and matrix catabolism in humans and mouse models. Clearance of p16Ink4a-positive senescent cells reduces the degenerative phenotype in many age-associated diseases. Whether p16Ink4a plays a functional role in intervertebral disc degeneration and senescence is unknown. We first characterized the senescence status of discs in young and old mice. Quantitative histology, gene expression and a novel p16tdTom reporter mice showed an increase in p16Ink4a, p21 and IL-6, with a decrease in Ki67 with aging. Accordingly, we studied the spinal-phenotype of 18-month-old mice with conditional deletion of p16Ink4a in the disc driven by Acan-CreERT2 (cKO). The analyses of discs of cKO and age-matched control mice showed little change in cell morphology and tissue architecture. The cKO mice exhibited changes in functional attributes of aggrecan as well as in collagen composition of the intervertebral disc. While cKO discs exhibited a small decrease in TUNEL positive cells, lineage tracing experiments using ZsGreen reporter indicated that the overall changes in cell fate or numbers were minimal. The cKO mice maintained expression of NP-cell phenotypic markers CA3, Krt19 and GLUT-1. Moreover, in cKO discs, levels of p19Arf and RB were higher without alterations in Ki67, γH2AX, CDK4 and Lipofuscin deposition. Interestingly, the cKO discs showed lower levels of SASP markers, IL-1β, IL-6, MCP1 and TGF-β1. These results show that while, p16Ink4a is dispensable for induction and maintenance of senescence, conditional loss of p16Ink4a reduces apoptosis, limits the SASP phenotype and alters matrix homeostasis of disc cells., National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) AR055655 and AR064733 to MVR and the National Institute on Aging F32 AG050399 to BOD (BOD). Part of this research was conducted while Brian Diekman was an Arthritis and Aging Research Grant recipient from the Arthritis National Research Foundation and the American Federation for Aging Research. EJN receives a PhD fellowship (PD/BD/128077/2016) from MD/PhD Program of the University of Minho funded by the Fundação para a Ciência e a Tecnologia (FCT)

BTB/POZ domain-containing 3 (BTBD3) was identified as a potential risk gene in the first genome-wide association study of obsessive-compulsive disorder (OCD). BTBD3 is a putative transcription factor implicated in dendritic pruning in developing primary sensory cortices. We assessed whether BTBD3 also regulates neural circuit formation within limbic cortico-striato-thalamo-cortical circuits and behaviors related to OCD in mice. Behavioral phenotypes associated with OCD that are measurable in animals include compulsive-like behaviors and reduced exploration. We tested Btbd3 wild-type, heterozygous, and knockout mice for compulsive-like behaviors including cage-mate barbering, excessive wheel-running, repetitive locomotor patterns, and reduced goal-directed behavior in the probabilistic learning task (PLT), and for exploratory behavior in the open field, digging, and marble-burying tests. Btbd3 heterozygous and knockout mice showed excessive barbering, wheel-running, impaired goal-directed behavior in the PLT, and reduced exploration. Further, chronic treatment with fluoxetine, but not desipramine, reduced barbering in Btbd3 wild-type and heterozygous, but not knockout mice. In contrast, Btbd3 expression did not alter anxiety-like, depression-like, or sensorimotor behaviors. We also quantified dendritic morphology within anterior cingulate cortex, mediodorsal thalamus, and hippocampus, regions of high Btbd3 expression. Surprisingly, Btbd3 knockout mice only showed modest increases in spine density in the anterior cingulate, while dendritic morphology was unaltered elsewhere. Finally, we virally knocked down Btbd3 expression in whole, or just dorsal, hippocampus during neonatal development and assessed behavior during adulthood. Whole, but not dorsal, hippocampal Btbd3 knockdown recapitulated Btbd3 knockout phenotypes. Our findings reveal that hippocampal Btbd3 expression selectively modulates compulsive-like and exploratory behavior., Brain Research Foundation seed grant (S.C.D.), a NARSAD Independent Investigator award (S.C.D.), R21-MH115395-01 (S.C.D.), Della Martin Foundation (J.A.K.), and training grants: T32 GM07839 (S.L.T.), and T32 DA07255 (S.L.T.).

Deficits in decoding rewarding (and aversive) signals are present in several neuropsychiatric conditions such as depression and addiction, emphasising the importance of studying the underlying neural circuits in detail. One of the key regions of the reward circuit is the nucleus accumbens (NAc). The classical view on the field postulates that NAc dopamine receptor D1-expressing medium spiny neurons (D1-MSNs) convey reward signals, while dopamine receptor D2-expressing MSNs (D2-MSNs) encode aversion. Here, we show that both MSN subpopulations can drive reward and aversion, depending on their neuronal stimulation pattern. Brief D1- or D2-MSN optogenetic stimulation elicited positive reinforcement and enhanced cocaine conditioning. Conversely, prolonged activation induced aversion, and in the case of D2-MSNs, decreased cocaine conditioning. Brief stimulation was associated with increased ventral tegmenta area (VTA) dopaminergic tone either directly (for D1-MSNs) or indirectly via ventral pallidum (VP) (for D1- and D2-MSNs). Importantly, prolonged stimulation of either MSN subpopulation induced remarkably distinct electrophysiological effects in these target regions. We further show that blocking κ-opioid receptors in the VTA (but not in VP) abolishes the behavioral effects induced by D1-MSN prolonged stimulation. In turn, blocking δ-opioid receptors in the VP (but not in VTA) blocks the behavioral effects elicited by D2-MSN prolonged stimulation. Our findings demonstrate that D1- and D2-MSNs can bidirectionally control reward and aversion, explaining the existence of controversial studies in the field, and highlights that the proposed striatal functional opposition needs to be reconsidered., FCT -Fundação para a Ciência e a Tecnologia(30/2016)

In Mexico, HPV vaccines available immunize against genotypes 16/18 and 16/18/6/11; however, there is limited surveillance about carcinogenic subtypes in different states of the country that allow evaluating the effectiveness of vaccination and cervical cancer screening programs. Here, we report the regional and age-specific prevalence of 14 hr-HPV genotypes as well as their prevalence in abnormal cytology (from ASCUS to cervical cancer) among Mexican women which were undergoing from cervical cancer screening in the Salud Digna clinics in 20 states of the country. This study includes women with social security from the majority of public health institutions (IMSS, ISSSTE, SEMAR, and PEMEX), and women without social security. For cervical cancer screening, we used the SurePath liquid-based cytology and the BD Onclarity HPV Assay. From December 1, 2016, to August 2, 2018, the hr-HPV prevalence among 60,135 women was 24.78%, the most prevalent types were HPV 16 (4.13%), HPV 31 (4.12%) and HPV 51 (3.39%), while HPV 18 (1.70%) was less prevalent among infected women. Interestingly, the genotypes not covered by current vaccines in Mexico were commonly found in precancerous lesions, evidencing their carcinogenic potential, so it is necessary to increase their surveillance and inclusion in cervical cancer screening triage., We gratefully acknowledge to Iromy Meza, Jessica Avitia, and Oswaldo Carrillo for their technical support in obtaining databases during this project. Also, we want to thanks the staff of the Salud Digna clinics and the National Reference Center of Salud Digna for their support during this work. This work was funding by Salud Digna.

Increasing multidrug-resistant tuberculosis (MDR-TB) incidence is a major threat against TB eradication worldwide. We aim to conduct a detailed MDR-TB study in Portugal, an European country with endemic TB, combining genetic analysis and epidemiological data, in order to assess the efficiency of public health containment of MRD-TB in the country., This work was developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and project PTDC/SAU-PUB/29521/2017. OO is supported by the project NORTE-08-5369-FSE-000041, financed by the Operational Program NORTE 2020 and co-financed by the European Social Fund through a doctoral grant (UMINHO/BD/47/2016). TR is supported by the Portuguese Foundation for Science and Technology (FCT) through a post-doctoral grant (SFRH/BPD/108126/2015). RG was partially supported by CMUP (UID/MAT/00144/2013), which is funded by FCT with national (MEC) and European structural funds (FEDER), under the partnership agreement PT2020.

Accepted Manuscript, The literature on gait analysis in Vascular Parkinsonism (VaP), addressing issues such as variability, foot clearance patterns, and the effect of levodopa, is scarce. This study investigates whether spatiotemporal, foot clearance and stride-to-stride variability analysis can discriminate VaP, and responsiveness to levodopa. Fifteen healthy subjects, 15 Idiopathic Parkinson's Disease (IPD) patients and 15 VaP patients, were assessed in two phases: before (Off-state), and one hour after (On-state) the acute administration of a suprathreshold (1.5 times the usual) levodopa dose. Participants were asked to walk a 30-meter continuous course at a self-selected walking speed while wearing foot-worn inertial sensors. For each gait variable, mean, coefficient of variation (CV), and standard deviations SD1 and SD2 obtained by Poincaré analysis were calculated. General linear models (GLMs) were used to identify group differences. Patients were subject to neuropsychological evaluation (MoCA test) and Brain MRI. VaP patients presented lower mean stride velocity, stride length, lift-off and strike angle, and height of maximum toe (later swing) (p, projects NORTE-01-0145-FEDER-000016 (NanoSTIMA) and NORTE-01-0145-FEDER-000026 (DeM), financed by the Regional Operational Program of the North (NORTE2020), under PORTUGAL2020 and FEDER.

Delayed parasite clearance and, consequently, reduced efficacy of artemisinin-based combination therapies have been linked with Plasmodium falciparum K13 gene SNPs in Southeast Asia. In Africa, significantly prolonged clearance has not yet been observed and the presently restricted variation in parasite clearance cannot be explained by K13 polymorphisms., Swedish Research Council (VR-2014-3134); the Swedish International Development Cooperation Agency(SWE-200-165); the Northern Portugal Regional Operational Programme(NORTE 2020), under the Portugal 2020 Partnership Agreement, throughthe European Regional Development Fund (NORTE-01-0145-FEDER-000013); and the Fundac ̧~ao para a Cieˆ ncia e Tecnologia (FCT) (SFRH/BD/129769/2017 to M. S., IF/00143/2015 to P. E. F., PD/BD/127826/2016 toC. C. and SFRH/BPD/76614/2011 to M. I. V.

Neurobiological models to explain vulnerability of major depressive disorder (MDD) are scarce and previous functional magnetic resonance imaging studies mostly examined "static" functional connectivity (FC). Knowing that FC constantly evolves over time, it becomes important to assess how FC dynamically differs in remitted-MDD patients vulnerable for new depressive episodes. Using a recently developed method to examine dynamic FC, we characterized re-emerging FC states during rest in 51 antidepressant-free MDD patients at high risk of recurrence (≥2 previous episodes), and 35 healthy controls. We examined differences in occurrence, duration, and switching profiles of FC states after neutral and sad mood induction. Remitted MDD patients showed a decreased probability of an FC state (p, NORTE‐01‐0145‐FEDER‐000023, by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Dr. G.D. is supported by the Spanish Research Project PSI2016‐75688‐P (AEI/FEDER) and by the European Union's Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement No. 785907 (Human Brain Project SGA2). Dr. M.L.K. is supported by the ERC Consolidator Grant: CAREGIVING (no. 615539) and Center for Music in the Brain, funded by the Danish National Research Foundation (DNRF117). Dr. H.G.R. is supported by a NWO/ZonMW VENI‐Grant #016.126.059. Dr. PE acknowledges support from the EPSRC award EP/N014529/1 funding the EPSRC Centre for Mathematics of Precision Healthcare at Imperial

Two key moments shaped the extant South Asian gene pool within the last 10 thousand years (ka): the Neolithic period, with the advent of agriculture and the rise of the Harappan/Indus Valley Civilisation; and Late Bronze Age events that witnessed the abrupt fall of the Harappan Civilisation and the arrival of Indo-European speakers. This study focuses on the phylogeographic patterns of mitochondrial haplogroups H2 and H13 in the Indian Subcontinent and incorporates evidence from recently released ancient genomes from Central and South Asia. It found signals of Neolithic arrivals from Iran and later movements in the Bronze Age from Central Asia that derived ultimately from the Steppe. This study shows how a detailed mtDNA phylogeographic approach, combining both modern and ancient variation, can provide evidence of population movements, even in a scenario of strong male bias such as in the case of the Bronze Age Steppe dispersals., M.S., P.J., S.R., G.O.-G., K.D., G.F., A.F., B.Y., M.P., and M.B.R. received support from the Leverhulme Trust Doctoral Scholarship programme. This work was partially supported by the Portuguese Foundation for Science and Technology (FCT), through the project PTDC/EPH-ARQ/4164/2014 partially funded by European Regional Development Fund (FEDER) (COMPETE 2020 project 016899). P.S. was supported by FCT, ESF and POPH through the FCT Investigator Programme (IF/01641/2013) and acknowledges FCT IP and ERDF (COMPETE2020 -POCI) for the CBMA strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569). T.R. is supported by an FCT grant (SFRH/BPD/108126/2015) and acknowledges the project [NORTE-01-0145-FEDER-000013], supported by NORTE 2020-Portugal 2020, through FEDER.

Aging is associated with cognitive decline. Herein, we studied a large cohort of old age and young adult male rats and confirmed that, as a group, old rats display poorer spatial learning and behavioral flexibility than younger adults. Surprisingly, when animals were clustered as good and bad performers, our data revealed that while in younger animals better cognitive performance was associated with longer dendritic trees and increased levels of synaptic markers in the hippocampus and prefrontal cortex, the opposite was found in the older group, in which better performance was associated with shorter dendrites and lower levels of synaptic markers. Additionally, in old, but not young individuals, worse performance correlated with increased levels of BDNF and the autophagy substrate p62, but decreased levels of the autophagy complex protein LC3. In summary, while for younger individuals "bigger is better", "smaller is better" is a more appropriate aphorism for older subjects., Portuguese Foundation for Science and Technology (FCT) with fellowships granted to: Cristina Mota (SFRH/BD/81881/2011), Susana Monteiro (SFRH/BD/69311/2010), Sofia Pereira das Neves and Sara Monteiro-Martins (PIC/IC/83213/2007); and by the European Commission within the 7th framework program, under the grant agreement: Health-F2-2010-259772 (Switchbox). In addition, this work was co-funded by the Northern Portugal Regional Operational Programme (ON.2 SR&TD Integrated Program – NORTE-07-0124-FEDER-000021), through the European Regional Development Fund (FEDER) and by national funds granted by FCT (PEst-C/SAU/LA0026/2013), and FEDER through the COMPETE (FCOMP-01-0124-FEDER-037298).

Cervical cancer is the fourth most common cancer in women. Although cure rates are high for early stage disease, clinical outcomes for advanced, metastatic, or recurrent disease remain poor. To change this panorama, a deeper understanding of cervical cancer biology and novel study models are needed. Immortalized human cancer cell lines such as HeLa constitute crucial scientific tools, but there are few other cervical cancer cell lines available, limiting our understanding of a disease known for its molecular heterogeneity. This study aimed to establish novel cervical cancer cell lines derived from Brazilian patients. We successfully established one (HCB-514) out of 35 cervical tumors biopsied. We confirmed the phenotype of HCB-514 by verifying its' epithelial and tumor origin through cytokeratins, EpCAM and p16 staining. It was also HPV-16 positive. Whole-exome sequencing (WES) showed relevant somatic mutations in several genes including BRCA2, TGFBR1 and IRX2. A copy number variation (CNV) analysis by nanostring and WES revealed amplification of genes mainly related to kinases proteins involved in proliferation, migration and cell differentiation, such as EGFR, PIK3CA, and MAPK7. Overexpression of EGFR was confirmed by phospho RTK-array and validated by western blot analysis. Furthermore, the HCB-514 cell line was sensitive to cisplatin. In summary, this novel Brazilian cervical cancer cell line exhibits relevant key molecular features and constitutes a new biological model for pre-clinical studies., Barretos Cancer Hospital Research Support Department (NAP) for sample collection, Barretos Cancer Hospital Biobank for sample processing, Dr. Flávia de Paula and Gabriela Fernandes for technical support of STRs and BRCA2 Sanger validation, respectively, and Dr. Laura Musselwhite (Duke University) for revising the manuscript. This study was supported by grants from the FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII- BIOPLAT - Process number 01.13.0469.00) and Barretos Cancer Hospital. PhD scholarship from FINEP (Grant numbers 384088/2014-7 and 380434/2015-6) and Barretos Cancer Hospital to MNR.

Essential thrombocytosis (ET) is a chronic myeloproliferative neoplasm characterized by the presence of thrombocytosis and it can be complicated by thrombotic and/or hemorrhagic events. Treatment options include low-dose aspirin and cytoreductive agents such as hydroxyurea. In cases of extreme thrombocytosis, therapeutic thrombocytapheresis can be a useful procedure. We present a case of a 61-year-old-man previously diagnosed with CALR-mutated ET, who develop acute myeloid leukemia. When recovering after induction chemotherapy, he developed an extreme thrombocytosis up to 2337 × 109 /L regardless hydroxyurea was started. Two therapeutic trombocytapheresis were performed and anagrelide was added to cytoreductive regimen. Platelet count stabilized around 570 × 109 /L. Both procedures were performed with the Spectra Optia Apheresis System version 11.3 (Terumo BCT) and we decided to use a higher collection preference and lower collection speed than manufacturer's recommendations. Platelet count decreased from 2380 × 109 /L to 1035 × 109 /L in the first procedure and from 1813 × 109 /L to 768 × 109 in the second procedure. Platelet collection efficiency was calculated to be 110.3% and 86.1% in the first and second thrombocytapheresis, respectively. Therapeutic thrombocytapheresis with Spectra Optia is a safe and efficient therapy to treat patients with primary thrombocytosis while effect of cytoreductive agents is attained. Platelet collection efficiency was calculated to be higher than previously reported. We suggest that changes in technical parameters such as a deeper aspiration point and/or lower collection speed may increase procedure's efficiency.

Accepted manuscript, The process of generating new functional neurons in the adult mammalian brain occurs from the local neural stem and progenitor cells and requires tight control of the progenitor cell's activity. Several signaling pathways and intrinsic/extrinsic factors have been well studied over the last years, but recent attention has been given to the critical role of cellular metabolism in determining the functional properties of progenitor cells. Here, we review recent advances in the current understanding of when and how metabolism affects neural stem cell (NSC) behavior and subsequent neuronal differentiation and highlight the role of lipocalin-2 (LCN2), a protein involved in the control of oxidative stress, as a recently emerged regulator of NSC activity and neuronal differentiation., IF/00231/2013 of the Portuguese Foundation for Science and Technology (FCT, Portugal). This work was supported by the Foundation for Science and Technology (FCT) and COMPETE through the project EXPL/NEU-OSD/2196/2013. The work has been developed under the scope of the project NORTE-01-0145-FEDER-000013, which is supported by the Northern Portugal Regional Operational Programme (NORTE 2020) under the Portugal 2020 Partnership Agreement through the European Regional Development Fund (FEDER) and is funded by FEDER funds through the Competitiveness Factors Operational Programme (COMPETE) and by National funds through the Foundation for Science and Technology (FCT) under the scope of the project POCI-01-0145-FEDER-007038, info:eu-repo/semantics/publishedVersion

We investigated the spatial epidemiology of bovine tuberculosis (TB) in wildlife in a multihost system. We surveyed bovine TB in Portugal by serologic analysis of elutes of dried blood spots obtained from hunted wild boar. We modeled spatial disease risk by using areal generalized linear mixed models with conditional autoregressive priors. Antibodies against Mycobaterium bovis were detected in 2.4% (95% CI 1.5%-3.8%) of 678 wild boar in 2 geographic clusters, and the predicted risk fits well with independent reports of M. bovis culture. Results show that elutes are an almost perfect substitute for serum (Cohen unweighted κ = 0.818), indicating that serologic tests coupled with dried blood spots are an effective strategy for large-scale bovine TB surveys, using wild boar as sentinel species. Results also show that bovine TB is an emerging wildlife disease and stress the need to prevent further geographic spread and prevalence increase., Plan Nacional (grant CGL2017-89866 from the Ministry of Economy and Competitiveness, Spain, and Fondo Europeo de Desarollo Regional) and Programa Operacional Regional do Norte (grant ON.2 O Novo Norte), Quadro de Referência Estratégico Nacional through the Fundo Europeu de Desenvolvimento Regional. N.S. was supported by PhD grant SFRH/BD/69390/2010 from Fundação para a Ciência e Tecnologia, info:eu-repo/semantics/publishedVersion

Accepted manuscript, This report presents the methods and results of the Thoracic Auto-Segmentation Challenge organized at the 2017 Annual Meeting of American Association of Physicists in Medicine. The purpose of the challenge was to provide a benchmark dataset and platform for evaluating performance of autosegmentation methods of organs at risk (OARs) in thoracic CT images., The challenge organizers would like to thank Artem Mamonov and Andrew Beers from Harvard Medical School for providing valuable support in creating the challenge scoring system on the challenge website, Kirk Smith and Tracy Nolan from University of Arkansas for Medical Sciences for data curation to TCIA, Tim Lustberg from Maastro Clinic for data collection, and AAPM for sponsoring this challenge. Of the challenge participants, Brent van der Heyden would like to thank Frank Verhaegen and Mark Podesta for valuable discussions; Bruno Oliveira would like to thank Sandro Queirós, Pedro Morais, Helena R. Torres, Jaime C. Fonseca, João Gomes-Fonseca, and João L. Vilaça for all the contributions to his work; Leonid Zamdborg would like to acknowledge Thomas M Guerrero and Edward Castillo. This project was support in part by the CPRIT (Cancer Prevention Research Institute of Texas) grant No. RP110562-P2, the National Institutes of Health Cancer Center Support (Core) grant No. CA016672 to the University of Texas MD Anderson Cancer Center, and in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government, info:eu-repo/semantics/publishedVersion

Tese de doutoramento em Medicina, As doenças de foro respiratório são altamente prevalentes a nível mundial, encontrando-se no grupo das doenças mais fatais em países desenvolvidos. Epitélio pulmonar gerado in-vitro a partir de células estaminais pluripotentes humanas tem aplicações em medicina regenerativa, no estabelecimento de novos modelos de doença, ensaios farmacológicos pré-clínicos e estudo do desenvolvimento humano. Neste trabalho descrevemos uma estratégia para gerar células da via aérea e alveolares maturas a partir de células estaminais pluripotentes humanas. Seguimos os paradigmas do desenvolvimento para gerar progenitores pulmonares (PPs) a partir das células estaminais e descobrimos que a glycogen synthase kinase 3 (GSK3) desempenha um papel central no balanço entre expansão de progenitores e a sua maturação em multi-linhagem. É importante realçar que a maioria das células geradas através do nosso método exibia características similares a células pulmonares pós-natais. Para além disso, o nosso modelo permitiu inferir um novo mecanismo envolvido no desenvolvimento pulmonar humano. Demonstramos que o efeito da inibição da maturação pela GSK3 no seu estado inibido é em parte devido à regulação do ciclo celular. Em culturas libertadas da inibição da GSK3, a via de sinalização NOTCH favorece a maturação dos PPs em populações epiteliais distais, inibindo as mais proximais, enquanto que a via WNT promove a maturação de células de Clara, implicando desta forma ambas as vias de sinalização no processo de especificação proximo-distal do epitélio pulmonar. Finalmente, demonstramos que o modelo desenvolvido é passível de ser utilizado com plataforma para desenvolvimento de modelos de doença respiratória humana e que poderá ser uma ferramenta importante para estudos sobre a fisiologia e importância de populações epiteliais pulmonares raras., Respiratory diseases are highly prevalent worldwide and consistently rank within the group of the most fatal diseases in developed countries. Lung epithelium generated in-vitro from human pluripotent stem cells (hPSCs) has tremendous potential for applications in regenerative medicine, disease modeling, pre-clinical drug screenings and the study of human development. Here we describe a strategy to derive mature airway and alveolar lung epithelium from hPSCs. We followed developmental cues to derive hPSCs towards lung progenitors (LPs) and found that glycogen synthase kinase 3 (GSK3) plays a central role in the balance between progenitor expansion and multilineage maturation of LPs. Importantly, the majority of the cells generated by our method exhibited characteristics similar to those found in postnatal human lungs. Our model offers novel mechanistic insight into human lung development. We show that the maturation inhibiting effects of GSK3 inhibition (GSK3i) in our cultures is partly due to cell cycle regulation. In addition, upon release of GSK3i, NOTCH signaling induces a distal cell fate at the expense of proximal and ciliated cell fates, whereas WNT signaling promotes a proximal club cell fate, implicating both signaling pathways in proximodistal specification of the human lung. Finally, we demonstrate that the model we developed is suitable for modelling of human respiratory diseases and could be a valuable tool to study the physiology and relevance of rare lung epithelium populations., National Institutes of Health (HL120046 and 1U01HL134760), the Thomas R Kully IPF Research Fund and the Portuguese Science Foundation (FCT) (PD/BD/52320/2013).

Accepted Manuscript, BACKGROUND: Lateralized dysfunction has been suggested in obsessive-compulsive disorder (OCD). However, it is currently unclear whether OCD is characterized by abnormal patterns of brain structural asymmetry. Here we carried out what is by far the largest study of brain structural asymmetry in OCD.METHODS: We studied a collection of 16 pediatric datasets (501 patients with OCD and 439 healthy control subjects), as well as 30 adult datasets (1777 patients and 1654 control subjects) from the OCD Working Group within the ENIGMA (Enhancing Neuro Imaging Genetics through Meta Analysis) Consortium. Asymmetries of the volumes of subcortical structures, and of measures of regional cortical thickness and surface areas, were assessed based on T1-weighted magnetic resonance imaging scans, using harmonized image analysis and quality control protocols. We investigated possible alterations of brain asymmetry in patients with OCD. We also explored potential associations of asymmetry with specific aspects of the disorder and medication status.RESULTS: In the pediatric datasets, the largest case-control differences were observed for volume asymmetry of the thalamus (more leftward; Cohen's d = 0.19) and the pallidum (less leftward; d = -20.21). Additional analyses suggested putative links between these asymmetry patterns and medication status, OCD severity, or anxiety and depression comorbidities. No significant case-control differences were found in the adult datasets.CONCLUSIONS: The results suggest subtle changes of the average asymmetry of subcortical structures in pediatric OCD, which are not detectable in adults with the disorder. These findings may reflect altered neurodevelopmental processes in OCD., This research was funded by the Max Planck Society (Germany). Additional funding was from the Japan Society for the Promotion of Science (KAKENHI Grant No. 18K15523 [to YA], KAKENHI Grant No. 16K04344 [to YH], KAKENHI Grant Nos. 16K19778 and 18K07608 [to TNakam], and KAKENHI Grant No. 26461762 [to AN]); the Carlos III Health Institute (Grant No. PI14/00419 [to PA], Grant No. PI040829 cofunded by European Regional Development Fund [to LL], Grant No. FI17/00294 [to IM-Z], Grant No. PI16/00950 [to JMM], and Grant Nos. CPII16/00048, PI13/01958, and PI16/00889 cofunded by European Regional Development Funds [to CS-M]); the Ontario Mental Health Foundation (Research Training Fellowship [to SHA]); Alberta Innovates Translational Health Chair in Child and Youth Mental Health (to PDA), the Ontario Brain Institute (to PDA); the National Institute of Mental Health (Grant No. K23MH104515 [to JTB], Grant No. K23-MH092397 [to BPB], Grant No. K23MH082176 [to KDF), Grant No. R21MH101441 [to RM], Grant No. R01MH081864 [to JO and JP], and Grant No. R01MH085900 [to JO and JF], Grant No. R21MH093889 [to HBS]); Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant No. 2011/21357–9 [to MCB], Grant No. 2011/21357–9 [to GFB], Grant No. 2011/21357–9 [to MQH], and Grant No. 2011/21357–9 [to ECM]); the Swiss National Science Foundation (Grant No. 320030_130237 [to SB; principal investigator, Susanne Walitza]); the Hartmann Müller Foundation (Grant No. 1460 [to SB]); the David Judah Fund at the Massachusetts General Hospital (to BPB); EU FP7 Project TACTICS (Grant No. 278948 [to JB]); the National Natural Science Foundation of China (Grant No. 81560233 [to YC] and Grant No. 81371340 [to ZW]); the International OCD Foundation (Grant No. K23 MH115206 [to PG]); the Wellcome Sir Henry Dale Fellowship (Grant No. 211155/Z/18/Z [to TUH]); the Jacobs Foundation (to TUH); the Brain and Behavior Research Foundation (2018 NARSAD Young Investigator Grant No. 27023 [to TUH]); the Agency for Medical Research and Development (Grant No. JP18dm0307002 [to YH]); the Michael Smith Foundation for Health Research (to FJ-F); the Federal Ministry of Education and Research of Germany (Grant No. BMBF-01GW0724 [to NK]); the Deutsche Forschungsgemeinschaft (Grant No. KO 3744/7–1 [to KK]); the Helse Vest Health Authority (Grant Nos. 911754 and 911880 [to GK]); the Norwegian Research Council (Grant No. HELSEFORSK 243675 [to GK]); the Marató TV3 Foundation (Grant Nos. 01/2010 and 091710 [to LL]); the Agency for Management of University and Research Grants (Grant No. 2017 SGR 881 [to LL] and 2017 SGR 1247 from the Generalitat de Catalunya [to JMM]); Fundação para a Ciência e a Tecnologia (Grant No. PDE/BDE/113604/2015 from the PhD-iHES Program [to RM], Grant No. PDE/BDE/113601/2015 from the PhD-iHES Program [to PSM]); the Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant-in-Aid for Scientific Research (Grant Nos. 22591262, 25461732, and 16K10253 [to TNakao]); the Government of India Department of Science and Technology (DST INSPIRE Faculty Grant No. -IFA12-LSBM-26 [to JCN] and Grant No. SR/S0/HS/0016/2011 [to YCJR]); the Government of India Department of Biotechnology (Grant No. BT/06/IYBA/2012 [to JCN] and Grant No. BT/PR13334/Med/30/259/2009 [to YCJR]); the New York State Office of Mental Health (to HBS); the Italian Ministry of Health (Grant No. RC13-14-15-16A [to GS]); the National Center for Advancing Translational Sciences (Grant No. UL1TR000067/KL2TR00069 [to ERS]); the Canadian Institutes of Health Research (to SES); the Michael Smith Foundation for Health Research (to SES); the British Columbia Provincial Health Services Authority (to SES); the Netherlands Organization for Scientific Research (Grant No. NWO/ZonMW Vidi 917.15.318 [to GAvW]); the Wellcome-DBT India Alliance (Grant No. 500236/Z/11/Z [to GV]); the Shanghai Key Laboratory of Psychotic Disorders (Grant No. 13dz2260500 [to ZW]).

O objetivo deste livro é criar um “guião" para o estudante de medicina, para o ajudar a tornar-se proficiente na ciência e na arte da Semiologia Médica. O livro é composto por quatro capítulos. O capítulo 1, "A Arte da Entrevista Clínica” fornece ao leitor uma visão da herança da comunicação clínica e da relevância da relação médico-doente. No capítulo 2 “Exame Físico de Screening” e no capítulo 3 “Exame Físico Completo” o estudante recebe um guia prático e sistematizado para duas abordagens, distintas, mas complementares, ao exame físico: uma mais curta e mais frequentemente aplicada e outra mais clássica e detalhada. O capítulo 4 “Algoritmos de Sistematização” agrega uma abordagem decisiva ao processo mental que cada clínico utiliza no processo de decisão clínica. E, com isto, pretendemos ajudar cada estudante a transformar-se num Médico.

Background. Hemophilia is a rare disorder characterized by spontaneous bleeding, with pain being a critical aspect. However, a systematic assessment of hemophilia-related pain in Portugal has never been conducted. Objective. To understand the pain experience among Portuguese people with hemophilia (PWH) by describing its prevalence, characteristics, and impact and uncovering intervention needs in the realm of hemophilia-related pain care. Methods. A cross-sectional observational survey, with age-adapted versions of questions concerning pain, emotional distress, and quality of life, was answered by 104 adults, 21 children/teenagers (10-17 years), and 19 children (1-9 years). Results. Pain was reported by 82 (78.8%) adults, 16 (76.2%) children/teenagers, and 13 (68.4%) children, with 65 (62.5%), 13 (61.9%), and eight (42.1%) of them reporting pain lasting more than three months, respectively. The mean number of pain locations (SD) was 5.23 (3.95) for adults, 4.13 (3.48) for children/teenagers, and 3.15 (1.99) for children age 1-9 years, with lower limbs pain causing the greatest negative impact. More frequent pain-triggering factors were physical effort/movements (61, 74.4%) for adults and hemarthrosis for younger groups (children/teenagers: 14, 87.5%; children: 9, 69.2%). Bleeds yielded the highest mean pain intensity (adults: M [SD] = 5.67 [2.09]; children/teenagers: M [SD] = 5.69 [2.15]). Adults with pain revealed more anxiety (odds ratio [OR] =1.698, P= 0.003) and depression (OR = 1.961, P= 0.025) and lower quality of life (OR = 0.928, P= 0.001). Conclusions. The current findings highlight the high prevalence, duration, and frequency of pain at all ages, its potentially simultaneous acute and chronic nature, its likelihood to affect multiple locations concurrently, and its detrimental impact. Important insights concerning intervention needs are presented, ultimately contributing to the improvement of hemophilia-related pain management and patient care., This work was supported by the Novo Nordisk HERO Research Grant 2015. PRP is the recipient of a postdoctoral grant (SFRH/BPD/103529/2014) from the Portuguese Foundation of Science and Technology.

Background: The implementation of population screening for cervical lesions in rural areas has been particularly challenging for a variety of reasons, including geographic isolation, limited access to medical care, lack of awareness of cancer risks, lower education levels, and embarrassment about having the clinical examination. Methods: This study was designed to evaluate the use of self-sampling for human papillomavirus (HPV) screening in the rural communities served by our hospital. To develop the study group, we trained a local police officer who was already interacting with the rural community to explain cancer risks and the ease of self-sampling to the women. Results: We identified a study group of 386 women, of whom 369 (95.6%) agreed to participate in the study. To test for high-risk HPV, we performed the Cobas PCR test, and of 340 valid samples, 45 (12.2%) were positive for at least 1 of the high-risk HPV subtypes. All positively tested women were referred for colposcopy and Papanicolaou testing. The follow-up biopsies showed that 40% had low-grade intraepithelial neoplasia (CIN1), 6.6% had high-grade intraepithelial neoplasia (CIN2/3), and 4.4% had squamous cell carcinoma. Conclusions: Self-collection for HPV testing by women living in isolated communities is highly efficient and has potential for cost-effective screening by taking advantage of existing support networks, such as the local police service, (undefined)

Colorectal cancer (CRC) is a major cause of cancer-related death worldwide. CRC increased risk has been associated with alterations in the intestinal microbiota, with decreased production of short chain fatty acids (SCFAs). SCFAs produced in the human colon are the major products of bacterial fermentation of undigested dietary fiber and starch. While colonocytes use the three major SCFAs, namely acetate, propionate and butyrate, as energy sources, transformed CRC cells primarily undergo aerobic glycolysis. Compared to normal colonocytes, CRC cells exhibit increased sensitivity to SCFAs, thus indicating they play an important role in cell homeostasis. Manipulation of SCFA levels in the intestine, through changes in microbiota, has therefore emerged as a potential preventive/therapeutic strategy for CRC. Interest in understanding SCFAs mechanism of action in CRC cells has increased in the last years. Several SCFA transporters like SMCT-1, MCT-1 and aquaporins have been identified as the main transmembrane transporters in intestinal cells. Recently, it was shown that acetate promotes plasma membrane re-localization of MCT-1 and triggers changes in the glucose metabolism. SCFAs induce apoptotic cell death in CRC cells, and further mechanisms have been discovered, including the involvement of lysosomal membrane permeabilization, associated with mitochondria dysfunction and degradation. In this review, we will cover the current knowledge on the transport of SCFAs by CRC cells and their effects on CRC metabolism and survival. The impact of increasing SCFA production by manipulation of colon microbiota on the prevention/therapy of CRC will also be addressed., This article is a result of the project EcoAgriFood NORTE-01-0145-FEDER-00009, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)It counts also with the support of the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 -Programa Operacional Competitividade e Internacionalizacao (POCI).It has also been supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01- 0145-FEDER-006684)., info:eu-repo/semantics/acceptedVersion

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that results from the death of dopamine (DA) neurons. Over recent years, differentiated or undifferentiated neural stem cells (NSCs) transplantation has been widely used as a means of cell replacement therapy. However, compelling evidence has brought attention to the array of bioactive molecules produced by stem cells, defined as secretome. As described in the literature, other cell populations have a high-neurotrophic activity, but little is known about NSCs. Moreover, the exploration of the stem cell secretome is only in its initial stages, particularly as applied to neurodegenerative diseases. Thus, we have characterized the secretome of human neural progenitor cells (hNPCs) through proteomic analysis and investigated its effects in a 6-hydroxidopamine (6-OHDA) rat model of PD in comparison with undifferentiated hNPCs transplantation. Results revealed that the injection of hNPCs secretome potentiated the histological recovery of DA neurons when compared to the untreated group 6-OHDA and those transplanted with cells (hNPCs), thereby supporting the functional motor amelioration of 6-OHDA PD animals. Additionally, hNPCs secretome proteomic characterization has revealed that these cells have the capacity to secrete a wide range of important molecules with neuroregulatory actions, which are most likely support the effects observed. Overall, we have concluded that the use of hNPCs secretome partially modulate DA neurons cell survival and ameliorate PD animals' motor deficits, disclosing improved results when compared to cell transplantation approaches, indicating that the secretome itself could represent a route for new therapeutic options for PD regenerative medicine. Stem Cells Translational Medicine 2018;7:829-838., Portuguese Foundation for Science and Technology: Ciência 2007 Program and IF Development Grant (IF/00111/2013) to A.J.S., Ph.D. scholarships to S.I.A. (SFRH/BD/81495/ 2011); Canada Research Chair in Biomedical Engineering (LAB). This article has been developed under the scope of the project NORTE‐01‐0145‐FEDER‐000013 and NORTE‐01‐0145‐FEDER‐000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the projects POCI‐01‐0145‐FEDER‐007038, PTDC/NEU‐NMC/0205/2012, UID/NEU/04539/2013, and POCI‐01‐0145‐FEDER‐007440 cofunded by the Programa Operacional Factores de Competitividade, QREN, the European Union (FEDER), and by the National Mass Spectrometry Network under the contract REDE/1506/REM/2005, info:eu-repo/semantics/publishedVersion

The methylation profile of genes in precursor lesions in cervical cancer was characterized to improve screening techniques for high-grade intraepithelial neoplasia., Objectives The methylation profile of genes in precursor lesions in cervical cancer was characterized to improve screening techniques for high-grade intraepithelial neoplasia. Methods A total of 447 cervical cytology samples obtained from women who underwent colposcopy were examined. The cases were distributed as follows: (1) cervices without cervical intraepithelial neoplasia (CIN; n = 152); (2) cervices with a CIN grade of 1 (CIN 1; n = 147); and (3) cervices with a CIN grade of 2 or 3 (CIN 2/3; n = 148). The methylation pattern for a panel of 15 genes was analysed by quantitative methylation-specific PCR (qMSP) and compared between the groups (≤CIN 1 vs. CIN 2+). Results In the validation set, seven genes presented significantly different methylation profiles according to diagnosis, namely, DAPK1 (p = 0.001), EPB41L3 (p = 0.001), HIC1 (p = 0.028), hsa-miR-124-2 (p = 0.001), LMX1A (p = 0.001), SOX1 (p = 0.001), and TERT (p = 0.001). Six genes showed a significant increase in the frequency of methylation in the presence of hr-HPV, namely, DAPK1 (p = 0.001), EPB41L3 (p = 0.001), hsa-miR-124-2 (p = 0.001), LMX1A (p = 0.001), SOX1 (p = 0.001), and TERT (p = 0.001). The methylation of the hsa-miR-124 gene showed sensitivity and specificity (86.7% and 61.3%, respectively) similar to that of the HPV test (91.3% and 50.0%, respectively). The independent factors associated with the diagnosis of CIN 2+ and the methylation of the hsa-miR-124-2 (OR = 5.1), SOX1 (OR = 2.8), TERT (OR = 2.2), and LMX1A (OR = 2.0) genes were a positive test for hr-HPV (odds ratio [OR] = 5.5). Conclusions Hypermethylation of the hsa-miR-124-2, SOX1, TERT, and LMX1A genes may be a promising biomarker for precursor lesions in cervical cancer regardless of the hr-HPV status., São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo–FAPESP) with the cooperation of the Coordination for the Improvement of Higher Education Personnel (Conselho Nacional de Desenvolvimento Científico e Tecnológico–CNPq-PPSUS) (Protocol Nos. 12/51221-4, 14/50014-0, 14/24415-8, and 15/15065-6), the National Institute of Science and Technology (Instituto Nacional de Ciência e Tecnologia–INCT-HPV) (FAPESP Proc. No. 2008/578891 and CNPq Proc. No. 573799/2008-3), and CAPES (PROSUP/CAPES Protocol No. 1571815), info:eu-repo/semantics/publishedVersion

Glioblastoma (GBM) is a universally fatal brain cancer, for which novel therapies targeting specific underlying oncogenic events are urgently needed. While the WNT pathway has been shown to be frequently activated in GBM, constituting a potential therapeutic target, the relevance of WNT6, an activator of this pathway, remains unknown. Methods: WNT6 protein and mRNA levels were evaluated in GBM. WNT6 levels were silenced or overexpressed in GBM cells to assess functional effects in vitro and in vivo. Phospho-kinase arrays and TCF/LEF reporter assays were used to identify WNT6-signaling pathways, and significant associations with stem cell features and cancer-related pathways were validated in patients. Survival analyses were performed with Cox regression and Log-rank tests. Meta-analyses were used to calculate the estimated pooled effect. Results: We show that WNT6 is significantly overexpressed in GBMs, as compared to lower-grade gliomas and normal brain, at mRNA and protein levels. Functionally, WNT6 increases typical oncogenic activities in GBM cells, including viability, proliferation, glioma stem cell capacity, invasion, migration, and resistance to temozolomide chemotherapy. Concordantly, in in vivo orthotopic GBM mice models, using both overexpressing and silencing models, WNT6 expression was associated with shorter overall survival, and increased features of tumor aggressiveness. Mechanistically, WNT6 contributes to activate typical oncogenic pathways, including Src and STAT, which intertwined with the WNT pathway may be critical effectors of WNT6-associated aggressiveness in GBM. Clinically, we establish WNT6 as an independent prognostic biomarker of shorter survival in GBM patients from several independent cohorts. Conclusion: Our findings establish WNT6 as a novel oncogene in GBM, opening opportunities to develop more rational therapies to treat this highly aggressive tumor., FCT - Foundation for Science and Technology (PTDC/SAU-GMG/113795/2009 and IF/00601/2012 to B.M.C.; SFRH/BD/92786/2013 to C.S.G.; SFRH/BD/88121/2012 to J.V.C.; SFRH/BD/81042/2011 to M.P.; SFRH/BD/93443/2013 to S.Q.) and Fundação Calouste Gulbenkian (B.M.C.), by FEDER funds through the Operational Programme Competitiveness Factors - COMPETE and National Funds through FCT under the project POCI-01-0145-FEDER-007038; by the project NORTE-01-0145-FEDER-000013 and NORTE-01-0246-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); and by the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), info:eu-repo/semantics/publishedVersion

Diagnosis of prostate cancer via PCA3 biomarker detection is promising to be much more efficient than with the prostatic specific antigens currently used. In this study, we present the first electrochemical and impedance-based biosensors that are capable of detecting PCA3 down to 0.128 nmol/L. The biosensors were made with a layer of PCA3-complementary single-stranded DNA (ssDNA) probe, immobilized on a layer-by-layer (LbL) film of chitosan (CHT) and carbon nanotubes (MWCNT). They are highly selective to PCA3, which was confirmed in impedance measurements and with polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Using information visualization methods, we could also distinguish between cell lines expressing the endogenous PCA3 long noncoding RNA (lncRNA) from cells that did not contain detectable levels of this biomarker. Since the methods involved in fabrication the biosensors are potentially low cost, one may hope to deploy PCA3 tests in any laboratory of clinical analyses and even for point-of-care diagnostics., This work had financial support from CNPq (Grant #164356/2015-0 and #113757/2018-2), FAPESP (Grant #2013/14262-7) and Barretos Cancer Hospital (Brazil). Andrey Soares thanks to Grant #2018/18953-8, São Paulo Research Foundation (FAPESP). The authors are also thankful to Maria Helena de Oliveira Piazzetta and Angelo Luiz Gobbi for the use of the Microfabrication Laboratory (LMF/LNNano/CNPEM) to manufacture interdigitated electrodes.

Upper urinary tract urothelial carcinomas are usually managed by radical nephroureterectomy (RNU), often followed by intravesical chemotherapy to minimize recurrence. Open surgery is the gold standard procedure for RNU, but it associates with high morbidity, and it has been increasingly replaced by minimally invasive strategies, such as laparoscopy and endoscopy. Although effective, endoscopic ureteral excision leaves the bladder unsutured, increasing the risk of tumor spillage, and precluding the immediate administration of intravesical chemotherapy. Here we describe a new method to close the bladder wall after ureteral excision, using barbed sutures via the endoscopic access. Our results in 8 female pigs demonstrate that this method is effective to close the bladder wall. The procedure was completed in a median time of 24 min, and no adverse events were registered in the follow-up or at the three-week necropsy. This technique improves a previous approach described by our group because the device is more flexible and allows to tie the knots inside the bladder. Barbed sutures have been used in the clinical practice for other types of surgeries, and therefore this method can further be adapted to human patients with no safety concerns. Its use may allow to administer intravesical chemotherapy, which reduces tumor recurrence and improves patient outcomes., The authors acknowledge Ana Goios for support in manuscript writing and technical editing, and for producing original illustrations for Figure 3. Doctoral grant of CUF Jose de Mello Saude.

Introduction Two developing forces have achieved prominence in medical education: the advent of competency-based assessments and a growing commitment to expand access to medicine for a broader range of learners with a wider array of preparation. Remediation is intended to support all learners to achieve sufficient competence. Therefore, it is timely to provide practical guidelines for remediation in medical education that clarify best practices, practices to avoid, and areas requiring further research, in order to guide work with both individual struggling learners and development of training program policies. Methods Collectively, we generated an initial list of Do’s, Don’ts, and Don’t Knows for remediation in medical education, which was then iteratively refined through discussions and additional evidence-gathering. The final guidelines were then graded for the strength of the evidence by consensus. Results We present 26 guidelines: two groupings of Do’s (systems-level interventions and recommendations for individual learners), along with short lists of Don’ts and Don’t Knows, and our interpretation of the strength of current evidence for each guideline. Conclusions Remediation is a high-stakes, highly complex process involving learners, faculty, systems, and societal factors. Our synthesis resulted in a list of guidelines that summarize the current state of educational theory and empirical evidence that can improve remediation processes at individual and institutional levels. Important unanswered questions remain; ongoing research can further improve remediation practices to ensure the appropriate support for learners, institutions, and society.

Breast cancer (BrC) remains the leading cause of cancer-related death in women, mainly due to recurrent and/or metastatic events, entailing the need for biomarkers predictive of progression to advanced disease. MicroRNAs hold promise as noninvasive cancer biomarkers due to their inherent stability and resilience in tissues and bodily fluids. There is increasing evidence that specific microRNAs play a functional role at different steps of the metastatic cascade, behaving as signaling mediators to enable the colonization of a specific organ. Herein, we aimed to evaluate the biomarker performance of microRNAs previously reported as associated with prognosis for predicting BrC progression in liquid biopsies. Background Breast cancer (BrC) remains the leading cause of cancer-related death in women, mainly due to recurrent and/or metastatic events, entailing the need for biomarkers predictive of progression to advanced disease. MicroRNAs hold promise as noninvasive cancer biomarkers due to their inherent stability and resilience in tissues and bodily fluids. There is increasing evidence that specific microRNAs play a functional role at different steps of the metastatic cascade, behaving as signaling mediators to enable the colonization of a specific organ. Herein, we aimed to evaluate the biomarker performance of microRNAs previously reported as associated with prognosis for predicting BrC progression in liquid biopsies. Methods Selected microRNAs were assessed using a quantitative reverse transcription-polymerase chain reaction in a testing cohort of formalin-fixed paraffin-embedded primary (n = 16) and metastatic BrC tissues (n = 22). Then, miR-30b-5p and miR-200b-3p were assessed in a validation cohort #1 of formalin-fixed paraffin-embedded primary (n = 82) and metastatic BrC tissues (n = 93), whereas only miR-30b-5p was validated on a validation cohort #2 of liquid biopsies from BrC patients with localized (n = 20) and advanced (n = 25) disease. ROC curve was constructed to evaluate prognostic performance. Results MiR-30b-5p was differentially expressed in primary tumors and paired metastatic lesions, with bone metastases displaying significantly higher miR-30b-5p expression levels, paralleling the corresponding primary tumors. Interestingly, patients with advanced disease disclosed increased circulating miR-30b-5p expression compared to patients with localized BrC. Conclusions MiR-30b-5p might identify BrC patients at higher risk of disease progression, thus, providing a useful clinical tool for patients’ monitoring, entailing earlier and more effective treatment. Nonetheless, validation in larger multicentric cohorts is mandatory to confirm these findings., Research Center of Portuguese Oncology Institute of Porto (PI 74-CI-IPOP-19-2016). JL and CSG are supported by a PhD fellowship from FCT - Fundação para a Ciência e Tecnologia (SFRH/ BD/132751/2017 and SFRH/BD/92786/2013, respectively). SS is supported by a PhD fellowship IPO/ESTIMA-1 NORTE-01-0145-FEDER-000027. BMC is funded by FCT-Fundação para a Ciência e a Tecnologia (IF/00601/2012)

The authors report the case of a classic phenotype of Fabry disease in a 60-year-old male patient presenting with left ventricular hypertrophy and stroke. Genetic analysis revealed 2 GLA-gene variants, i.e., p.R356Q and p.G360R. This clinical case highlights that the finding of 2 or more GLA gene variants in a Fabry patient should lead to a careful evaluation in order to determine their exact role in the condition. This case also provides the first clinical evidence that the p.G360R mutation is pathogenic and responsible for a classic phenotype of Fabry disease. The clinical improvement following the initiation of enzyme replacement therapy reinforces the importance of Fabry disease awareness and diagnosis in patients exhibiting red flags, such as left ventricular hypertrophy and stroke.

Alzheimer disease (AD) is a neurodegenerative disorder characterized pathologically by the accumulation of amyloid-beta (Aβ) plaques and tau neurofibrillary tangles (NFTs). Recently, primary age-related tauopathy (PART) has been described as a new anatomopathological disorder where NFTs are the main feature in the absence of neuritic plaques. However, since PART has mainly been studied in post-mortem patient brains, not much is known about the clinical or neuroimaging characteristics of PART. Here, we studied the clinical brain imaging characteristics of PART focusing on neuroanatomical vulnerability by applying a previously validated multiregion visual atrophy scale. We analysed 26 cases with confirmed PART with paired clinical magnetic resonance imaging (MRI) acquisitions. In this selected cohort we found that upon correcting for the effect of age, there is increased atrophy in the medial temporal region with increasing Braak staging (r = 0.3937, p = 0.0466). Upon controlling for Braak staging effect, predominantly two regions, anterior temporal (r = 0.3638, p = 0.0677) and medial temporal (r = 0.3836, p = 0.053), show a trend for increased atrophy with increasing age. Moreover, anterior temporal lobe atrophy was associated with decreased semantic memory/language (r = - 0.5823, p = 0.0056; and r = - 0.6371, p = 0.0019, respectively), as was medial temporal lobe atrophy (r = - 0.4445, p = 0.0435). Overall, these findings support that PART is associated with medial temporal lobe atrophy and predominantly affects semantic memory/language. These findings highlight that other factors associated with aging and beyond NFTs could be involved in PART pathophysiology., NACC database is funded by NIA/NIH Grant U01 AG016976. NACC data are contributed by the NIA-funded ADCs: P30 AG019610 (PI Eric Reiman, MD), P30 AG013846 (PI Neil Kowall, MD), P30 AG062428–01 (PI James Leverenz, MD) P50 AG008702 (PI Scott Small, MD), P50 AG025688 (PI Allan Levey, MD, PhD), P50 AG047266 (PI Todd Golde, MD, PhD), P30 AG010133 (PI Andrew Saykin, PsyD), P50 AG005146 (PI Marilyn Albert, PhD), P30 AG062421–01 (PI Bradley Hyman, MD, PhD), P30 AG062422–01 (PI Ronald Petersen, MD, PhD), P50 AG005138 (PI Mary Sano, PhD), P30 AG008051 (PI Thomas Wisniewski, MD), P30 AG013854 (PI Robert Vassar, PhD), P30 AG008017 (PI Jeffrey Kaye, MD), P30 AG010161 (PI David Bennett, MD), P50 AG047366 (PI Victor Henderson, MD, MS), P30 AG010129 (PI Charles DeCarli, MD), P50 AG016573 (PI Frank LaFerla, PhD), P30 AG062429–01(PI James Brewer, MD, PhD), P50 AG023501 (PI Bruce Miller, MD), P30 AG035982 (PI Russell Swerdlow, MD), P30 AG028383 (PI Linda Van Eldik, PhD), P30 AG053760 (PI Henry Paulson, MD, PhD), P30 AG010124 (PI John Trojanowski, MD, PhD), P50 AG005133 (PI Oscar Lopez, MD), P50 AG005142 (PI Helena Chui, MD), P30 AG012300 (PI Roger Rosenberg, MD), P30 AG049638 (PI Suzanne Craft, PhD), P50 AG005136 (PI Thomas Grabowski, MD), P30 AG062715–01 (PI Sanjay Asthana, MD, FRCP), P50 AG005681 (PI John Morris, MD), P50 AG047270 (PI Stephen Strittmatter, MD, PhD). NIH grants to JFC (R01AG054008, R01NS095252, R01AG062348, RF1AG060961), the Tau Consortium, and Alzheimer’s Association (NIRG- 469 15-363188)

Nonsurgical treatment of hemorrhoidal disease (HD) includes medical and instrumental techniques. We aimed to compare the efficacy of the most frequently used nonsurgical strategies, either alone or in combination, applied in an ambulatory setting. Purpose: Nonsurgical treatment of hemorrhoidal disease (HD) includes medical and instrumental techniques. We aimed to compare the efficacy of the most frequently used nonsurgical strategies, either alone or in combination, applied in an ambulatory setting. Methods: Patients who received nonsurgical treatment for HD by proctology appointment at the Gastroenterology Department of Braga Hospital were evaluated. Isolated rubber band ligation (RBL) and a combination of RBL with a micronized purified flavonoid fraction (MPFF) were the 2 most frequently used strategies. Symptoms of HD (bleeding, pruritus, pain at rest, pain at defecation and prolapse) were assessed at days 0, 7, and 28 by using a severity grading scale (0 to 4/5). A Global Symptom score was constructed to assess the overall severity and compare the overall improvements of the HD symptoms between the 2 most frequently used strategies. Results: Nineteen patients underwent the combined treatment (RBL + MPFF group) and 25 the RBL treatment (RBL group). A comparison of the 2 treatment groups showed significant improvements in the combined treatment group in terms of bleeding at days 7 (P = 0.001) and 28 (P = 0.002) and in the pruritus intensity during the first week (P < 0.001). A trend toward clinical benefit was also verified in the combined treatment group for all other HD symptoms (pain at rest, pain at defecation and prolapse). Conclusion: A combined treatment approach with MPFF and RBL significantly reduced the intensity of bleeding during the first month and the pruritus during the first week

Acute myeloid leukaemia (AML) comprises a heterogeneous group of hematologic neoplasms characterized by diverse combinations of genetic, phenotypic and clinical features representing a major challenge for the development of targeted therapies. Metabolic reprogramming, mainly driven by deregulation of the nutrient-sensing pathways as AMPK, mTOR and PI3K/AKT, has been associated with cancer cells, including AML cells, survival and proliferation. Nevertheless, the role of these metabolic adaptations on the AML pathogenesis is still controversial. In this work, the metabolic status and the respective metabolic networks operating in different AML cells (NB-4, HL-60 and KG-1) and their impact on autophagy and survival was characterized. Data show that whereas KG-1 cells exhibited preferential mitochondrial oxidative phosphorylation metabolism with constitutive co-activation of AMPK and mTORC1 associated with increased autophagy, NB-4 and HL-60 cells displayed a dependent glycolytic profile mainly associated with AKT/mTORC1 activation and low autophagy flux. Inhibition of AKT is disclosed as a promising therapeutical target in some scenarios while inhibition of AMPK and mTORC1 has no major impact on KG-1 cells' survival. The results highlight an exclusive metabolic profile for each tested AML cells and its impact on determination of the anti-leukaemia efficacy and on personalized combinatory therapy with conventional and targeted agents., This work was developed under the scope of the project NORTE‐01‐0145‐FEDER‐000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and by FEDER, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI‐01‐0145‐FEDER‐007038. OP and BSM are supported by fellowships from the Fundação para a Ciência e Tecnologia (FCT, Portugal) (SFRH/BD/52292/2013 and SFRH/BPD/90533/2012, respectively), info:eu-repo/semantics/publishedVersion

Plant-based compounds are an option to explore and perhaps overcome the limitations of current antitumor treatments. Annona coriacea Mart. is a plant with a broad spectrum of biological activities, but its antitumor activity is still unclear. The purpose of our study was to determine the effects of A. coriacea fractions on a panel of cervical cancer cell lines and a normal keratinocyte cell line. The antitumor effect was investigated in vitro by viability assays, cell cycle, apoptosis, migration, and invasion assays. Intracellular signaling was assessed by Western blot, and major compounds were identified by mass spectrometry. All fractions exhibited a cytotoxic effect on cisplatin-resistant cell lines, SiHa and HeLa. C3 and C5 were significantly more cytotoxic and selective than cisplatin in SiHa and Hela cells. However, in CaSki, a cisplatin-sensitive cell line, the compounds did not demonstrate higher cytotoxicity when compared with cisplatin. Alkaloids and acetogenins were the main compounds identified in the fractions. These fractions also markedly decreased cell proliferation with p21 increase and cell cycle arrest in G2/M. These effects were accompanied by an increase of H2AX phosphorylation levels and DNA damage index. In addition, fractions C3 and C5 promoted p62 accumulation and decrease of LC3II, as well as acid vesicle levels, indicating the inhibition of autophagic flow. These findings suggest that A. coriacea fractions may become effective antineoplastic drugs and highlight the autophagy inhibition properties of these fractions in sensitizing cervical cancer cells to treatment., e FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/ 2013—FP XII-BIOPLAT), Barretos Cancer Hospital, CAPES, CNPq, FAPEMIG, UFSJ. RMR is a recipient of CNPq Productivity Grant

Background: Pan-intestinal capsule endoscopy (Pan-CE) has been used to assess both the small and large bowel inflammation in Crohn's disease (CD) patients in a single examination. The capsule endoscopy Crohn's disease activity index (CECDAI) was initially developed to measure mucosal disease activity in the small bowel, although in 2018, it was extended to the colon for standardization of inflammatory activity (CECDAIic). The aim of this study was to apply the CECDAIic in a cohort of CD patients that underwent Pan-CE to evaluate the inter-observer agreement and the correlation between this score and inflammatory parameters.Methods: The videos were read and scored using the CECDAIic by three independent experienced operators, blinded to the results of the standard workup. Statistical analysis was performed with SPSS®, using Kendall's coefficient to evaluate the inter-observer agreement. Spearman correlation (rs) was used to access the correlation between the score and inflammatory biomarkers.Results: Included 22 patients, 59.1% males with mean age of 30.7 ± 11.1 years. The median CECDAIic score was 9.17 (0-37). The overall CECDAIic score Kendall coefficient was 0.94, demonstrating a statistically significant (p