Cristiana Pires - Academia.edu (original) (raw)

Papers by Cristiana Pires

Regular and Young Investigator Award Abstracts

Background Natural killer (NK) cells are innate cytotoxic lymphocytes capable of killing virally ... more Background Natural killer (NK) cells are innate cytotoxic lymphocytes capable of killing virally infected cells and malignant tumors. Unlike T cells, NK cells are human leukocyte antigen (HLA)-agnostic and cause little to no Graft vs. Host Disease in allogenic transfusions, making them excellent candidates for off-the-shelf therapies. However, current techniques to insert a chimeric antigen receptor (CAR) gene into NK cells primarily employ viral vectors. Viral methods can give rise to complications such as insertional mutagenesis, which can lead to gene silencing or oncogene activation. 1 Here we have developed a CRISPR genome editing strategy to modify primary NK cells and report the targeted virus-free CAR integration within peripheral blood NK cells. Methods In our strategy, the transgene (e.g., CAR) is encoded in a linear double stranded DNA (dsDNA) template and produced by polymerase chain reaction (PCR). The template includes homology to the intended target for insertion that is defined by nuclease mediated double strand break formation by a Cas9-single guide RNA (gRNA) ribonucleoprotein complex (figure 1A). 2 Results We show efficient (>80%) CRISPR knockout of the inhibitory NKG2A receptor encoded by the KLRC1 gene within primary NK cells. Optimization of electroporation timing showed that delivery of 3 mg of donor DNA on day 4 of ex vivo expansion results in gene knock-in rates of up to 24% for the transgene (figure 1B). Lastly, we report that the use of K562-mbIL15-41BBL feeder cells improves expansion of CRISPR edited cells by approximately twenty-fold. Conclusions These data provide a proof-of-principle for on-target integration of long transgenes, including CAR, without viral vectors within primary NK cells via CRISPR-Cas9 genome editing. Overall, this virus-free manufacturing strategy could enable the development of novel genetically-programmed NK immunotherapies. Ethics Approval The study was approved by the IRB at University of Wisconsin-Madison, approval number 2018-0103. Abstract 1231 Figure 1 Knock-in via CRISPR-Cas9 at the KLRC1 locus (A) Schematic showing 2.5kb transgene encoding mCherry fluorescent protein and genome editing strategy. (B) Flow cytometry results showing 23.8% knock-in of a transgene into the KLRC1 locus in human primary NK cells 14 days after nucleofection of CRISPR reagents. RNP: ribonucleoprotein. NKG2A is encoded by the KLRC1 locus

Foods

Absorption at the intestinal epithelium is a major determinant of cholesterol levels in the organ... more Absorption at the intestinal epithelium is a major determinant of cholesterol levels in the organism, influencing the entry of dietary cholesterol and the excretion of endogenous cholesterol. Several strategies are currently being followed to reduce cholesterol absorption, using both pharmacological agents or food ingredients with hypocholesterolemic properties. Coffee has recently been shown to affect cholesterol bioaccessibility, although it has not been shown if this translates into a decrease on cholesterol bioavailability. In this work, coffee obtained with different commercial roasting (light and dark) and grinding (finer and coarser) was evaluated regarding their effect on cholesterol absorption through Caco-2 monolayers, mimicking the intestinal epithelium. The fluorescent dehydroergosterol was used as a sterol model, which was shown to permeate Caco-2 monolayers with a low-to-moderate permeability coefficient depending on its concentration. In the presence of coffee extract...

ex aequo - Revista da Associação Portuguesa de Estudos sobre as Mulheres

Resumo Este artigo apresenta uma análise sobre o assédio sexual em ambientes de lazer noturno a p... more Resumo Este artigo apresenta uma análise sobre o assédio sexual em ambientes de lazer noturno a partir das perceções de bystanders (observadores/as) e da avaliação do processo de implementação da certificação Sexism Free Night no Porto. Através de um questionário online (N=546) foi possível verificar que, de acordo com os/as bystanders, o assédio sexual é muito prevalecente em ambientes de lazer noturno. Esses dados influenciaram a implementação de um modelo de certificação assente no envolvimento da gerência de estabelecimentos de lazer noturno (n=7) e na formação ao staff dos estabelecimentos aderentes (n=46). Os resultados demonstram que os ambientes de lazer noturno são contextos estratégicos para a prevenção e intervenção em situações de assédio sexual.

Contemporary Drug Problems

This essay is based in a self-reflexive collective process and intends to present the chemsex sce... more This essay is based in a self-reflexive collective process and intends to present the chemsex scene in Lisbon and harm reduction responses implemented to address the needs of chemsex practitioners. The analysis considered professional experiences, participant observation, literature review of the relevant data in Portugal and autoethnographic data from a chemsex practitioner and peer educator. This essay aims to present the community-led creation of a transdisciplinary collaborative network able to assess and respond to chemsex-related risks in Lisbon. Specifically, we aim to: (i) present the chemsex scenes in Lisbon; (ii) discuss the setting up and preliminary results of a collaborative network and harm reduction responses targeting chemsex practitioners. The work implemented in Lisbon demonstrates that chemsex is a global trend with localized idiosyncrasies that must be addressed when designing local tailored interventions. It also reiterated that harm reduction organizations are ...

Science Immunology, 2022

Type 1 conventional dendritic cells (cDC1s) are rare immune cells critical for the induction of a... more Type 1 conventional dendritic cells (cDC1s) are rare immune cells critical for the induction of antigen-specific cytotoxic CD8 + T cells, although the genetic program driving human cDC1 specification remains largely unexplored. We previously identified PU.1, IRF8, and BATF3 transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts, but reprogramming of human somatic cells was limited by low efficiency. Here, we investigated single-cell transcriptional dynamics during human cDC1 reprogramming. Human induced cDC1s (hiDC1s) generated from embryonic fibroblasts gradually acquired a global cDC1 transcriptional profile and expressed antigen presentation signatures, whereas other DC subsets were not induced at the single-cell level during the reprogramming process. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the cDC1-inducing transcription factor network as key drivers of the process. Combining IFN-γ, IFN-β...

Basically, there are 2 main needs to tackle: the first is to rapidly identify and assess NPS, by ... more Basically, there are 2 main needs to tackle: the first is to rapidly identify and assess NPS, by using specific screening instruments and then transmit the results to the National and European Early Warning Systems; the second is to improve awareness and knowledge about NPS among partygoers, professionals and policy makers. Promoting drug checking in nightlife contexts in the EU countries linked to the EWS and informing local service providers both on NPS and the results of drug checking seem to be the best way to meet the needs.

From the 1990s until the pre-COVID-19 outbreak, commercial nightlife has become central to the ur... more From the 1990s until the pre-COVID-19 outbreak, commercial nightlife has become central to the urban branding, and tourism marketing strategies of many European cities. However, the night-time leisure industry has stalled in Europe as a consequence of the COVID-19 pandemic and the unprecedented lock-down and physical distancing measures imposed by several national governments. This short paper aims to picture the uncertain scenario that the nightlife industry of Europe, and Portugal, might have to face in post-pandemic times.

Volume 4: Policy and Planning, 2021

Journal of Alcohol and Drug Education, 2018

Pharmaceutics, 2021

Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The r... more Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). In this work, we characterize variations in the tightness of the cell monolayer over the stable time interval and evaluate the conditions required for their re-use in permeability assays. The monolayer integrity was assessed through TEER measurements and permeability of the paracellular marker Lucifer Yellow (LY), complemented with nuclei and ZO-1 staining for morphological studies and the presence of tight junctions. Over 150 permeability assays were performed, which showed that manipulation of the cell monolayer in the permeability assay may contribute significantly to the flux of LY, leading to Papp values that are dependent on the sampling duration. The assay also leads to a small decr...

Food Chemistry, 2021

Edible wild plants are part of the ethnobotanical and gastronomic heritage of different geographi... more Edible wild plants are part of the ethnobotanical and gastronomic heritage of different geographical areas. Corema album (L.) D. Don is an endemic species of the dune systems of the Atlantic coast of the Iberian Peninsula. The aerial parts of Corema album are a source of nutrients and antioxidants. The Corema album white berry (Portuguese crowberry) is rich in calcium, iron, and zinc. The plant also shows high phenolic content and antioxidant capacity associated with the leaves, fruit, and flowers. The presence of organic acids, namely phenolic acids, such as hydroxycinnamic acids, and long chain polyunsaturated fatty acids (PUFAs) omega-3 and omega-6 has also been confirmed. Toxicity studies evaluated by cell viability tests with human intestinal epithelium model cells (Caco-2) have shown that, at low concentrations, plant extracts may present beneficial effects.

Revista Portuguesa de Enfermagem de Saúde Mental, 2020

CONTEXTO: O uso nocivo de álcool é um dos determinantes de risco mais importantes na saúde da pop... more CONTEXTO: O uso nocivo de álcool é um dos determinantes de risco mais importantes na saúde da população, sendo o consumo esporádico excessivo (CEE) um indicador importante do mesmo. OBJECTIVO: O objetivo deste estudo é analisar a prevalência deste padrão de consumo (i.e., frequência, intensidade e duração) e explorar a sua relação com comportamentos de risco numa amostra de jovens e jovens adultos/as na cidade do Porto. MÉTODOS: Foram administrados um total de 403 questionários num período de 1 mês na cidade do Porto a jovens entre os 18 e os 29 anos (M = 22,76, DP = 3,57). O estudo enquadra-se no âmbito da epidemiologia social, dado procurar problematizar o CEE numa perspectiva biopsicossocial. RESULTADOS: Dos participantes que ingeriram bebidas alcoólicas nos últimos 12 meses, 67,7% reportaram ter feito consumos esporádicos excessivos, a maioria entre 1 a 2 vezes por semana, num período entre 3 a 4 horas. O número de bebidas por CEE foi em média de 5 para mulheres e de mais de 10 para homens. A disparidade na prevalência de consumo e a associação entre intensidade de consumo, género e comportamentos de risco, foram resultados particularmente relevantes para uma tentativa de operacionalizar uma definição compreensiva de CEE. CONCLUSÕES: Os resultados foram discutidos problematizando os critérios associados à definição de CEE. No sentido de potenciar respostas mais ajustadas às reais necessidades dos/as jovens que exibem este padrão de consumo, as políticas de saúde pública devem ser capazes de integrar a frequência, duração e intensidade do consumo na avaliação de risco.

BIO-PROTOCOL, 2020

Ectopic expression of transcription factor combinations has been recently demonstrated to reprogr... more Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on antigen cross-presentation, a critical step for inducing CD8 + T cell cytotoxic responses. The rarity of naturally occurring cDC1s and lack of in vitro methodologies for the generation of pure cDC1 populations strongly hinders the study of cDC1 lineage specification and function. Here, we describe a protocol for the generation of induced DCs (iDCs) by lentiviral-mediated expression of the transcription factors PU.1, IRF8 and BATF3 in mouse embryonic fibroblasts. iDCs acquire DC morphology, cDC1 phenotype and transcriptional signatures within 9 days. iDCs generated with this protocol acquire functional ability to respond to inflammatory stimuli, engulf dead cells, process and cross-present antigens to CD8 + T cells. DC reprogramming provides a simple and tractable system to generate high numbers of cDC1-like cells for high content screening, opening new avenues to better understand cDC1 specification and function. In the future, faithful induction of cDC1 fate in fibroblasts may lead to the generation of patient-specific DCs for vaccination.

Frontiers in Immunology, 2019

Cell reprogramming concepts have been classically developed in the fields of developmental and st... more Cell reprogramming concepts have been classically developed in the fields of developmental and stem cell biology and are currently being explored for regenerative medicine, given its potential to generate desired cell types for replacement therapy. Cell fate can be experimentally reversed or modified by enforced expression of lineage specific transcription factors leading to pluripotency or attainment of another somatic cell type identity. The possibility to reprogram fibroblasts into induced dendritic cells (DC) competent for antigen presentation creates a paradigm shift for understanding and modulating the immune system with direct cell reprogramming. PU.1, IRF8, and BATF3 were identified as sufficient and necessary to impose DC fate in unrelated cell types, taking advantage of Clec9a, a C-type lectin receptor with restricted expression in conventional DC type 1. The identification of such minimal gene regulatory networks helps to elucidate the molecular mechanisms governing development and lineage heterogeneity along the hematopoietic hierarchy. Furthermore, the generation of patient-tailored reprogrammed immune cells provides new and exciting tools for the expanding field of cancer immunotherapy. Here, we summarize cell reprogramming concepts and experimental approaches, review current knowledge at the intersection of cell reprogramming with hematopoiesis, and propose how cell fate engineering can be merged to immunology, opening new opportunities to understand the immune system in health and disease.

ex aequo - Revista da Associação Portuguesa de Estudos sobre as Mulheres, 2018

Neste artigo, analisa-se a tríade sexo feminino, consumo de substâncias psicoativas em contextos ... more Neste artigo, analisa-se a tríade sexo feminino, consumo de substâncias psicoativas em contextos festivos e a sua relação com situações de violência sexual. Através da revisão bibliográfica e de dois grupos de discussão realizados com frequentadoras de contextos festivos e utilizadoras recreativas de substâncias psicoativas (n=12), com idades compreendidas entre os 16 e os 46 anos e de classe média, foram identificadas várias desigualdades de género que emergem em ambientes de diversão noturna. A normalização de situações de violência sexual nestes contextos e a reprodução de mitos da violação justificam a implementação de ações educativas informais que os previnam. Nesse sentido, analisam-se também propostas de intervenção que se têm focado nesse fenómeno sublinhando-se as boas práticas.

International Journal of Drug Policy, 2019

Background: Drug checking services are being implemented in recreational settings across the worl... more Background: Drug checking services are being implemented in recreational settings across the world, however these projects are frequently accused of a lack of evidence concerning their impact on people who use drugs. This paper describes the implementation of a drug checking service at the Boom Festival 2016 and explores the impact of this service on its users' behavioural intentions. Methods: 753 drug samples were submitted to the drug checking service for chemical analysis. All drug checking users were invited to fill a pre-analysis and a post-analysis questionnaire. 310 pre-and post-analysis questionnaires answered by users of the service were successfully matched. Results: When the test results were "unexpected" (N = 86), 94.3% of the service users reported that they would not to take the drug. When the test result indicated the sample contained "the expected substance plus adulterants" (N = 41) 32% of users stated they would not take it. When the test result was "only the expected substance" (N = 370), as anticipated, 98% of the participants reported they would take it. There was a statistically significant association between users' behavioural intentions and drug-checking result (χ 2 (2) = 350,042, p < .001). Conclusions: These results support the hypothesis that providing drug checking services in large-scale festivals helps users to better manage their drug use and deal with drug adulteration. The data corroborates the supposition that when provided with objective information about the content of their drugs, some users consider health protecting behaviours. Additionally, these results can contribute to the design of tailored harm reduction interventions that take into consideration clients' characteristics, profiles and motivations.

Colloids and Surfaces B: Biointerfaces, 2019

Biological systems are the result of the interactions established among their many distinct molec... more Biological systems are the result of the interactions established among their many distinct molecules and molecular assemblies. The high concentration of small molecules dissolved in the aqueous media alter the water properties with important consequences in the interactions established. In this work, the effects of high concentrations of the disaccharide trehalose on the solubility of a homologous series of fluorescent amphiphiles (NBD-Cn, n=4-16) and on their interaction with a lipid bilayer and a serum protein is quantitatively characterized. Both kinetic and equilibrium aspects are reported for a better understanding of the effects observed. The aqueous solubility of the most hydrophobic amphiphiles (n ³ 8) is strongly increased by 1 M trehalose, while no significant effect is observed for the most polar amphiphile (n=4). This results from a decrease in the magnitude of the hydrophobic effect at conditions of molecular crowding. A small decrease is observed on the equilibrium association with serum albumin. This is most significant for amphiphiles with longer alkyl chains, in agreement with their increased solubility in the aqueous media containing trehalose. The effects on the association of the amphiphiles with lipid bilayers are influenced by both equilibrium and kinetic aspects. On one hand, the decreased magnitude of the hydrophobic effect leads to a decrease in the affinity of the amphiphiles towards the membrane. However, this tendency may be overbalanced by the effects on the kinetics of the interaction (insertion/desorption) due to the increase in the viscosity of the aqueous media. It is shown that the distribution of amphiphilic drugs in the crowded biological media is significantly different from that predicted from studies in dilute solutions and that the effects are dependent on solute's hydrophobicity.

Science Immunology, 2018

Ectopic expression of transcription factors PU.1, IRF8, and BATF3 reprograms fibroblasts to dendr... more Ectopic expression of transcription factors PU.1, IRF8, and BATF3 reprograms fibroblasts to dendritic cells.

Experimental Hematology, 2017

Down syndrome (DS)/trisomy 21 children have a high risk of developing hematopoietic malignancies,... more Down syndrome (DS)/trisomy 21 children have a high risk of developing hematopoietic malignancies, including the characteristic perinatal transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both TMD and AMKL are associated with somatic mutations in GATA1, which result in the production of a truncated protein termed GA-TA1s. To date, the cell of origin that acquires the GATA1s mutation in TMD and AMKL remains unidentified. Analysis of the microRNAs (miRNAs) signature of adult myeloid leukemia stem cells identified miR-99a, miR-125b, miR-155, all encoded by chromosome 21. When engrafted into NSG recipients, CD34 + CD38cord blood cells overexpressing these 3 miRNAs (OE) initiate a lethal disease with characteristics of DS-AMKL, including splenomegaly and liver infiltration, and generated a high proportion of CD41 + megakaryocytes. Proteomic analysis revealed an increase in the naturally occurring GATA1 short splice form, suggesting that OE induces a rapid change in the splicing pattern of GATA1 or enriches for a population of cells preferentially expressing GATA1s. The perinatal onset of TMD and AMKL suggests an early developmental origin of the progenitors responsible for the disease. Using human pluripotent stem cell (hPSC) differentiation to recapitulate embryonic development, we found that cells sharing characteristics of yolk sac (YS)-derived erythro-myeloid progenitors (EMP), but not YS-derived primitive progenitors, are sensitive to the 3 miRNAs expression. OE in the EMP-like population led to an in vitro expansion of megakaryocytic progenitors that failed to undergo terminal differentiation and endowed them with engraftment capacity, as CD41 + CD61 + CD42 + xenografts were detected in NSG recipients. Taken together, our studies provide unique and previously unrecognized insights into the progenitor target population and the mechanism responsible for DS-associated leukemogenesis.