Alterations at the blood-brain barrier and brain parenchyma along brain metastasization of breast cancer

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2016Despite the restricted permeability of the blood-brain barrier (BBB), the brain is a privileged organ regarding the appearance of metastases, particularly from breast cancer. Patients with brain metastases from breast cancer have a severe prognosis, rendering this issue a serious oncologic problem that deserves further attention. Therefore, additional studies are required to establish when breast cancer cells cross the brain endothelium and what are the routes used for the transendothelial migration, to understand what is their precise phenotype along the processes of transmigration and establishment of brain metastases, to determine the alterations occurring in brain endothelium, to study how endothelial cells communicate with malignant ones to promote the attraction of malignant cells into the brain vasculature and tumour-associated vascular development. Based on this, we aimed at establishing the temporal profile of breast cancer metastasization to the brain and characterize the metastasizing cells phenotype, as well as, to investigate the vascular events and BBB properties along the process of metastasization to this target organ. In addition, we aimed to assess signalling mechanisms involved in attraction of carcinoma cells into the brain and proliferation in the nervous tissue. To establish the temporal evolution of the players involved in such processes, we used cerebella, cranial hippocampi, and striata of female mice inoculated with 4T1 breast cancer cells sacrificed at 5 hours, 3 days, 7 days or 10 days, and of female mice injected with vehicle (control) sacrificed at 5 hours. Our results showed the presence of brain metastasis of breast cancer at 7-days after inoculation, which increased thereafter. The malignant cells crossed the BBB as mesenchymal cells and, once inside the brain, these cells underwent a complete or partial mesenchymal-epithelial transition to acquire the epithelial characteristics that allow the establishment of new tumours. In addition, the process of brain metastasization of BC contributed to the downregulation of the tight junction protein claudin-5 of brain microvascular endothelial cells, as well as to the entrance of the blood-borne component thrombin in brain parenchyma. On the other hand, hypervascularization in cranial hippocampus appeared to be associated to the process of brain colonization by breast cancer cells. Regarding the role of platelet-derived growth factor B signalling along the process of brain metastasization, we found that this growth factor was expressed by tumour cells and its expression increased during the formation of brain metastasis. Interestingly, a continuous entrance of cysteine-X amino acid-cysteine receptor 4 (CXCR4)-positive cells into the brain parenchyma appeared to occur along the process of brain metastasization of breast cancer. In sum, this study contributes to clarify the time-course and interdependence of the signalling events, BBB breach and phenotypic transition of malignant cells along endothelial transposition and brain metastases establishment by breast cancer cells. Moreover, the demonstration of early cellular and molecular events points to novel targets for modulation in order to prevent metastasis formation and development.Apesar da barreira hematoencefálica (BHE) ter uma permeabilidade restrita, o cérebro é um órgão preferencialmente afetado pelo aparecimento de metástases, particularmente de cancro mama. Pacientes com metástases cerebrais provenientes do cancro da mama têm um prognóstico severo, tornando a metastização num sério problema oncológico que merece toda a atenção. Por este motivo, novos estudos são necessários para estabelecer quando é que as células cancerígenas da mama atravessam o endotélio cerebral e quais são as vias que utilizam para migrarem através do endotélio, para se perceber qual o fenótipo que têm ao longo dos processos de migração para dentro do encéfalo e durante o estabelecimento de metástases, para determinar as alterações que ocorrem no endotélio cerebral, para estudar como as células endoteliais comunicam com as células malignas para promover a atracão das células cancerígenas da mama para a vasculatura do encéfalo e o desenvolvimento vascular associado ao tumor. Com base nisto, tivemos com objetivos estabelecer o perfil temporal da metastização do cancro da mama para o encéfalo e caracterizar o fenótipo destas mesmas células, assim como, estudar as alterações vasculares e as propriedades da BHE ao longo do processo de metastização para este órgão secundário. Para além disso, também pretendíamos avaliar os mecanismos de sinalização envolvidos na atracão das células tumorais para o encéfalo e na proliferação no tecido nervoso. Para estabelecer a evolução temporal dos intervenientes envolvidos em tais processos, utilizámos cerebelos, hipocampos craniais e estriados de ratinhos fêmea inoculados com células cancerígenas da mama 4T1 sacrificados às 5 horas, 3 dias, 7 dias, ou 10 dias, e de ratinhos fêmea injetados com veículo (controlo) sacrificados às 5 horas. Os nossos resultados mostraram a presença de metástases cerebrais do cancro mama 7 dias após a inoculação, aumentando ao longo do tempo. As células malignas atravessaram a BHE como células mesenquimais e, uma vez dentro do encéfalo, estas células sofreram uma transição completa ou parcial de fenótipo mesenquimal para epitelial para adquirirem as características epiteliais necessárias para o estabelecimento de novos tumores no encéfalo. Além disso, o processo de metastização cerebral do cancro da mama contribuiu para a diminuição da expressão da proteína das junções de oclusão claudina-5 nas células endoteliais microvasculares cerebrais, assim como para a entrada do componente sanguíneo trombina no parênquima cerebral. Por outro lado, o aumento de vascularização no hipocampo cranial aparentou estar associado ao processo de colonização do encéfalo pelas células cancerígenas da mama. Relativamente ao papel da sinalização do fator de crescimento B derivado de plaquetas ao longo do processo de metastização cerebral, descobrimos que as células tumorais expressavam este fator de crescimento e que a sua expressão aumentou durante a formação de metástases no encéfalo. Curiosamente, a entrada contínua de células que expressam o recetor CXCR4 para dentro do parênquima cerebral aparentou ocorrer ao longo do processo de metastização cerebral do cancro da mama. Deste modo, este estudo contribui para clarificar o curso temporal e a interdependência de vias de sinalização, a quebra da BHE e a transição fenotípica das células malignas ao longo na transposição do endotélio e estabelecimento de metástases cerebrais pelas células cancerígenas da mama. Além disso, a demonstração dos eventos celulares e moleculares iniciais aponta para novos alvos para modulação de modo a prevenir a formação e desenvolvimento de metástases.This study was supported by Fundação para a Ciência e Tecnologia (FCT – UID/DTP/04138/2013), Portugal, and by National Research, Development and Innovation/Hungarian Scientific Research Fund (NKFIH/OTKA – K-100807 and K-116158), Hungary

Staying under the radar - the multifunctional LANA Protein

Many viruses have developed numerous strategies to recruit and take advantage of cellular protein degradation pathways to evade the cellular viral immune system. One such virus is the Kaposi´s Sarcoma associated herpesvirus (KSHV), first discovered in Kaposi´s Sarcoma lesions found in AIDS patients. Latency-Associated Nuclear Antigen (LANA) is a KSHV multifunctional protein responsible for tethering viral DNA to the chromosome ensuring maintenance and segregation of the viral genome during cell division. Besides its main role of viral maintenance, LANA also physically interacts with several host proteins to modulate cell functions. One such function is to recruit the EC5S ubiquitin-ligase complex by interacting with Elongin BC complex and Cullin 5 protein, which in turn ubiquitinate substrates such as NF-κB and p53 to allow persistent viral infection. Like any other post-translation modifications, ubiquitination is reversible through deubiquitination enzymes (DUBs). LANA also interacts with ubiquitin specific protease 7 (USP7), a deubiquitination enzyme involved in regulation of several proteins including p53. Interaction with USP7 is made through a conserved peptide motif, which is also present in LANA. This work addresses the role of LANA in the recruitment and modulation of the ubiquitination and deubiquitination pathways. Despite the continued efforts in uncovering new LANA interacting partners to form a functional EC5S ubiquitin-ligase complex, only MHV-68 LANA interacted directly with Elongin BC, other interactions were not direct and may require a linker protein. On the other hand, LANA interaction with USP7 was able to be analysed by X-ray structure determination. In addition to a conserved P/AxxS motif, a novel Glutamine (Gln) residue from KSHV LANA was shown to make a specific interaction with USP7. This Gln residue is also present in other herpesvirus protein and hence it might be a conserved motif within herpesviruses

Measles - an old children's disease to remember

O sarampo é uma doença viral grave e altamente contagiosa. O vírus do sarampo é um vírus de ARN, cadeia simples, do gênero Morbillivirus, da família Paramyxoviridae, transmitido pelas vias aéreas, através de gotículas de aerossóis ou por contato direto com secreções respiratórias de indivíduos infectados. A infecção é adquirida pelo contato dessas gotículas com a mucosa do trato respiratório ou conjuntiva. Em 2005, a Organização Mundial de Saúde estabeleceu que o objetivo para a erradicação do sarampo na Região Europeia seria até 2010 mas, apesar das medidas adotadas pelos diversos países, o sarampo ressurgiu, com 10271 casos relatados somente em 2013 em 30 estados da União Europeia, com mais de 91% deles na Alemanha, Itália, Holanda e Reino Unido. No início de 2017, Portugal foi ameaçado por um surto de sarampo, relatando nos primeiros cinco meses do ano 31 casos confirmados, 20 (65%) em adultos (com idade igual ou superior a 18), dos quais 45% (13) foram em profissionais de saúde. Face a este surto de sarampo ter tantos casos em adultos, os autores decidiram fazer uma breve revisão, tentando relembrar uma infeção antiga, não tão conhecida pelos médicos mais jovens e que pode ser esquecida na abordagem de um doente adulto. Os autores salientam igualmente que o vírus do sarampo pode virtualmente ser erradicado, pois existe uma vacina eficaz e não existe na natureza nenhum reservatório para o vírus, além dos humanos.Measles is a serious, highly contagious viral disease. The measles virus is a single-stranded, RNA virus of the genus Morbillivirus within the family Paramyxoviridae, transmitted by air, through droplets of aerosols or by direct contact with respiratory secretions of infected individuals. The infection is acquired through the mucosa of respiratory tract or conjunctiva. In 2005, the World Health Organization established that measles eradication in the European Region should be achieved by 2010, but despite the measures adopted by the various countries, measles re-emerged, with 10271 cases reported only in 2013 in 30 states of European Union, with more than 91% of them in Germany, Italy, Netherland and United Kingdom. In the beginning of 2017, Portugal was threatened with a measles outbreak, reporting in the first five months of the year 31 confirmed cases, 20 (65%) of them in adults (18 or older), of which 45% (13) were in health professionals. Because this measles outbreak had so many cases in adults, the authors decided to make a brief review, trying to remember an old infection, not so well known by younger doctors, and that can be overlooked in the approach of the adult patients. The authors also point out that measles virus could virtually be eradicated as there is an effective vaccine and there is no reservoir in nature for the virus other than humans

Evaluation of the antibacterial activity and colouring capacity of two Hylocereus spp. Epicarps

Betalains are a group of secondary metabolites named chromoalkaloids that are synthesized from tyrosine. These compounds have gained some attention in the last few years mainly due to their interesting bioactive potential, namely antioxidant, antimicrobial, and other bioactive properties [1]. Their strong and vibrant colours are also one of the characteristics by which these compounds have gained visibility in the food and pharmaceutical industries [2]. Betalains can be divided in two groups regarding the colour range: betaxanthins in the orange to yellow range, and betacyanins in the purple to pink range. Thereby, these compounds can be used as natural colouring agents, providing alternatives to the massively used artificial counterparts [3]. Although there are already some natural options in the market, these are not enough to meet the needs of the food industry, due to the growing concern of consumers regarding what they eat.To FCT and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019); C. Lobo Roriz (SFRH/BD/117995/2016) and T.C.S. Pires (SFRH/BD/129551/2017) grants and National funding by FCT- Foundation for Science and Technology, P.I., through the institutional scientific employment program-contract for L. Barros contract. This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479:ValorNatural®.info:eu-repo/semantics/publishedVersio

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface-binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms

Enjoo decorrente da higienização dentária e condição periodontal de mulheres grávidas

Objective: To verify the relationship among nausea, gingival bleeding and oral hygiene habits and in pregnant women.Material and methods: A retrospective study of documentary analysis was carried out on 1272 medical records of pregnant women entering the Dental Care Program for Pregnant Women, in a public university, in the period from 2000 to 2018. Incorrectly filled out medical records were excluded. The variables were analyzed: nausea prevalence, periodontal condition and oral hygiene habits. The data were processed and analyzed at a significance level of 5%. Results: The mean age of pregnant women was 27.49 years, 54.64% (n=695) white, 75.39% (n=959) were married and 44.43% (n=565) had low schooling until high school. Of the total 40.09% (n=510) had nausea during tooth brushing and was founded association between nausea and gestation period (p<0.0001); gingival bleeding and Oral Hygiene Index (p<0.0001); and between periodontal condition and flossing (p=0.000).Conclusion: The results suggest that nausea during tooth brushing was associated with the gestational period. There was an association between the presence of gingival bleeding on probing and the Simplified Oral Hygiene Index. Changes in periodontal conditions were observed, but at reversible levels.Objetivo: Analisar a relação existente entre enjoo, sangramento gengival à sondagem e práticas de higiene bucal, em gestantes.Materiais e métodos: Foi realizado um estudo, retrospectivo, de análise documental, em 1272 prontuários de gestantes ingressantes em um programa de atenção pré-natal, no período de 2000 a 2018. Foram excluídos os prontuários preenchidos incorretamente e selecionados dados sobre enjoo, condição periodontal e analisadas as práticas de higiene relacionadas. Foram calculadas as distribuições das frequências e realizados testes de associação ao nível de significância de 5%.Resultados: A idade média das gestantes foi de 27,49 anos (DP=10,47), 54,64% (n=695) eram brancas, 75,39% (n=959) casadas, e 44,43% (n=565) possuíam até ensino médio incompleto. Do total, 40,09%% (n=510) tiveram enjoo ao escovar os dentes e foram encontradas associação estatística entre enjoo e período gestacional (p<0,00001); sangramento gengival à sondagem e Índice de Higiene Oral Simplificado (p<0,0001), bem como condição periodontal e uso de fio dental (p=0,0007) e Índice de Higiene Oral Simplificado (p<0,0001).Conclusão: Conclui-se que houve associação entre enjoo e período gestacional; sangramento gengival à sondagem e Índice de Higiene Oral Simplificado; Índice Periodontal Comunitário, uso de fio dental e Índice de Higiene Oral Simplificado

A dor é total e não apenas um sintoma físico - acerca de um caso clínico em cuidados paliativos

info:eu-repo/semantics/publishedVersio

MLL1 is regulated by KSHV LANA and is important for virus latency

Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi's sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-A crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.info:eu-repo/semantics/publishedVersio

Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 (hACE2) receptor, a critical step in infection, and is the preferential target for spikeneutralizing antibodies. Post-translational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37°C, respectively. Deamidation is significantly slowed at 4°C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the hACE2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.Fil: Lorenzo Lopez, Juan Ramiro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Defelipe, Lucas Alfredo. European Molecular Biology Laboratory; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Aliperti Car, Lucio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Niebling, Stephan. European Molecular Biology Laboratory; Alemania. Centre for Structural Systems Biology; AlemaniaFil: Custódio, Tânia F.. European Molecular Biology Laboratory; Alemania. Centre for Structural Systems Biology; AlemaniaFil: Löw, Christian. European Molecular Biology Laboratory; Alemania. Centre for Structural Systems Biology; AlemaniaFil: Schwarz, Jennifer J.. European Molecular Biology Laboratory; AlemaniaFil: Remans, Kim. European Molecular Biology Laboratory; AlemaniaFil: Craig, Patricio Oliver. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: García Alai, María. European Molecular Biology Laboratory; Alemania. Centre for Structural Systems Biology; AlemaniaFil: Sánchez Miguel, Ignacio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Alonso, Leonardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface–binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection.Peer reviewe