Daniela Freitas

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90–120 nm; small exosome vesicles, Exo-S, 60–80 nm) and discovered an abundant population of non-membranous nanoparticles termed ‘exomeres’ (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.Lyden and colleagues use asymmetric flow field-flow fractionation to classify nanoparticles derived from cell lines and human samples, including previously uncharacterized large, Exo-L and small, Exo-S, exosome subsets.

Helicobacter pylori chronic infection and mucosal inflammation switches the human gastric glycosylation pathways.

Helicobacter pylori exploits host glycoconjugates to colonize the gastric niche. Infection can persist for decades promoting chronic inflammation, and in a subset of individuals lesions can silently progress to cancer. This study shows that H. pylori chronic infection and gastric tissue inflammation result in a remodeling of the gastric glycophenotype with increased expression of sialyl-Lewis a/x antigens due to transcriptional up-regulation of the B3GNT5, B3GALT5, and FUT3 genes. We observed that H. pylori infected individuals present a marked gastric local pro-inflammatory signature with significantly higher TNF-α levels and demonstrated that TNF-induced activation of the NF-kappaB pathway results in B3GNT5 transcriptional up-regulation. Furthermore, we show that this gastric glycosylation shift, characterized by increased sialylation patterns, favors SabA-mediated H. pylori attachment to human inflamed gastric mucosa. This study provides novel clinically relevant insights into the regulatory mechanisms underlying H. pylori modulation of host glycosylation machinery, and phenotypic alterations crucial for life-long infection. Moreover, the biosynthetic pathways here identified as responsible for gastric mucosa increased sialylation, in response to H. pylori infection, can be exploited as drug targets for hindering bacteria adhesion and counteract the infection chronicity.

Medical Management of Pancreatic Adenocarcinoma

Pancreatic cancer is the fourth leading cause of cancer death in the United States. In 2008, an estimated 34,290 people died from pancreatic cancer and 37,680 new cases were diagnosed. Despite modern treatment, 90% of patients die within 1 year of diagnosis. Pancreatectomy is still the only potentially curative approach, but most patients have incurable disease by the time they are diagnosed, and fewer than 20% are candidates for surgery. In the present paper the English-language literature addressing the medical management in pancreatic cancer was reviewed. Based on these data we will discuss the role of currently used chemotherapy and target therapy in pancreatic cancer, as well as perspectives of the emerging strategies that are arising in order to improve the outcomes of this complex disease.

Downstaging of a rectal gastrointestinal stromal tumor by neoadjuvant imatinib therapy allowing for a conservative surgical approach

Gastrointestinal stromal tumor (GIST) is a recently recognized pathology that originates from the interstitial cells of Cajal1 and is the most common mesenchymal neoplasm of the gastrointestinal tract. Although surgery is the only treatment available, it is only possible in approximately half of the cases2. This malignancy is characterized by a high risk of metastatic relapse, specifically in the liver and peritoneum. Imatinib mesylate, a tyrosine kinase inhibitor that inhibits c-kit signal transduction, is often used as a treatment for metastatic GIST. Neoadjuvant therapy is defined as a treatment administered prior to the main (usually surgical) treatment for a particular neoplasm. It is used not only to eliminate circulating tumor cells but also to reduce the size of the tumor, thereby facilitating surgery and perhaps increasing the chance of organ preservation. Since imatinib results in significant tumor shrinkage in at least 50% of treated patients,3,4 its use as a neoadjuvant treatment is logical.

Discrimination and Mental Health Among Gay, Lesbian, and Bisexual Youths in Portugal: The Moderating Role of Family Relationships and Optimism

ABSTRACT This study investigates associations between mental health, perception of discrimination (unfair treatment and personal rejection), and family environment dimensions—satisfaction, intimacy, admiration, and conflict in the relationships with the mother and the father, and familial optimism—in a sample of 84 gay, lesbian, and bisexual (GLB) Portuguese adolescents. Results show that participants with higher levels of mental health experience less discrimination, feel more respected by their fathers, have fewer conflicts with both parents, and belong to families with higher levels of optimism. No significant differences were found with regard to satisfaction and intimacy in the relationships with both parents and in the perception of admiration by the mothers. Conflict with parents moderated the relation between discrimination and mental health. Having frequent experiences of conflict with parents was found to exacerbate the negative impact of occasional acts of discrimination on mental health. Results highlight the relevance of a positive family environment as a protection mechanism in the face of discrimination, and the need to lower the levels of homophobia and heterosexism in social contexts in order to promote the well-being of GLB adolescents.

Resilience in the face of peer victimisation and discrimination: The who, when and why in five patterns of adjustment

Victimisation has a negative effect on psychosocial functioning. Based on the resilience theory, and with a sample of 2975 Portuguese students, the present study aims to: i) identify patterns of adjustment in the face of peer victimisation and perceptions of discrimination; ii) explore the association between the patterns of adjustment and the characteristics of participants (the who) and of the victimisation (the when and why). Cluster analysis revealed five patterns of adjustment: Unchallenged; Externally Maladjusted; Internally Maladjusted; Resilient, and At-Risk. The results suggest that there is no complete resilience in the face of social victimisation. Group differences were found regarding: i) gender, type of course, sexual orientation, ethnicity, nationality, parental educational level and religious beliefs; ii) the age at which peer victimisation was more frequent, and; iii) the motives underlying discrimination. Globally considered, peer victimisation is representative of the wider cultural environment and interventions should also target social prejudices.

Adaptação da Escala de Discriminação Quotidiana para Jovens Portugueses

O objetivo deste estudo foi a adaptacao da Escala de Discriminacao Quotidiana para a populacao jovem portuguesa. Apos traducao, ajustes semânticos aos itens, inclusao de dois itens para aumento da validade de constructo e a afericao da validade facial, os itens da escala foram respondidos por 2996 jovens estudantes do ensino secundario em Portugal. A Analise Fatorial Exploratoria (AFE) revelou uma estrutura de dois fatores - Tratamento Injusto e Rejeicao Pessoal - com quatro itens cada (sete itens da escala original e um formulado neste estudo). A Analise Fatorial Confirmatoria (AFC) validou um modelo hierarquico, em que os dois fatores observados na AFE estao subordinados a um fator latente de segunda ordem, a Percepcao de Discriminacao. A escala permite diferenciar a discriminacao percepcionada por grupos alvo de preconceito social, observando-se diferencas significativas nos valores reportados entre pessoas negras e brancas e entre jovens lesbicas, gays, bissexuais e participantes heterossexuais. O instrumento explica parcialmente variacoes ao nivel de saude mental, observando-se uma correlacao negativa moderada entre a discriminacao percepcionada e a saude mental. Os resultados sugerem que a versao adaptada da escala apresenta validade de constructo, sendo bastante apropriada para avaliar e comparar a percepcao de discriminacao de jovens.

Resilience in LGB Youths: A Systematic Review of Protection Mechanisms

Considerando las disparidades observadas en varios indicadores de ajuste entre jovenes gais, lesbianas y bisexuales y jovenes heterosexuales, ha sido enfatizada en la literatura la necesidad de identificar los mecanismos que promueven la resiliencia entre las minorias sexuales. Esta revision sistematica trato de responder la pregunta: ?cuales son los mecanismos de proteccion contra la homofobia entre los jovenes LGB? De las 147 referencias localizadas en la busqueda sistematica, 13 estudios fueron analizados. Los mecanismos de proteccion identificados fueron ordenados segun dos criterios: especificos a la orientacion sexual o generales, y considerando su nivel de expresion, personales, relacionales o contextuales. Los resultados demuestran que la mayoria de estos mecanismos son exclusivamente promotores del ajuste positivo en la presencia del riesgo. Los mecanismos de proteccion generales se revelan mas a menudo compensatorios de los riesgos que los especificos.

Adaptação da Escala de Discriminação Quotidiana para Jovens Portugueses = Adaptation of the Everyday Discrimination Scale to Portuguese Youth

O objetivo deste estudo foi a adaptacao da Escala de Discriminacao Quotidiana para a populacao jovem portuguesa. Apos traducao, ajustes semânticos aos itens, inclusao de dois itens para aumento da validade de constructo e a afericao da validade facial, os itens da escala foram respondidos por 2996 jovens estudantes do ensino secundario em Portugal. A Analise Fatorial Exploratoria (AFE) revelou uma estrutura de dois fatores - Tratamento Injusto e Rejeicao Pessoal - com quatro itens cada (sete itens da escala original e um formulado neste estudo). A Analise Fatorial Confirmatoria (AFC) validou um modelo hierarquico, em que os dois fatores observados na AFE estao subordinados a um fator latente de segunda ordem, a Percepcao de Discriminacao. A escala permite diferenciar a discriminacao percepcionada por grupos alvo de preconceito social, observando-se diferencas significativas nos valores reportados entre pessoas negras e brancas e entre jovens lesbicas, gays, bissexuais e participantes heterossexuais. O instrumento explica parcialmente variacoes ao nivel de saude mental, observando-se uma correlacao negativa moderada entre a discriminacao percepcionada e a saude mental. Os resultados sugerem que a versao adaptada da escala apresenta validade de constructo, sendo bastante apropriada para avaliar e comparar a percepcao de discriminacao de jovens.

Molecular weight of surface immobilized hyaluronic acid influences CD44-mediated binding of gastric cancer cells

The physiological importance of the interactions between hyaluronic acid (HA) and its main membrane receptor, CD44, in pathological processes, e.g. cancer, is well recognized. However, these interactions are mainly studied in solution, whereas HA in the extracellular matrix (ECM) is partially immobilized via its interactions with other ECM components. We therefore, developed substrates in which HA is presented in an ECM-relevant manner. We immobilized HA with different molecular weights (Mw) in a Layer-by-Layer (LbL) fashion and studied the interactions of the substrates with CD44 and two human gastric cancer cell lines that overexpress this receptor, namely AGS and MKN45. We demonstrate that MKN45 cells are more sensitive to the LbL substrates as compared with AGS. This difference is due to different CD44 expression: while CD44 is detected mainly in the cytoplasm of AGS, MKN45 express CD44 predominantly at the cell membrane where it is involved in the recognition and binding of HA. The invasiveness of the studied cell lines was also evaluated as a function of HA Mw. Invasive profile characterized by low cell adhesion, high cell motility, high expression of cortactin, formation of invadopodia and cell clusters was observed for MKN45 cells when they are in contact with substrates presenting HA of high Mw.