37 research outputs found
Hyperbranched {DNA} clusters
No full textTaking advantage of the base-pairing specificity and tunability of DNA interactions, we investigate the spontaneous formation of hyperbranched clusters starting from purposely designed DNA tetravalent nanostar monomers, encoding in their four sticky ends the desired binding rules. Specifically, we combine molecular dynamics simulations and Dynamic Light Scattering experiments to follow the aggregation process of DNA nanostars at different concentrations and temperatures. At odds with the Flory– Stockmayer predictions, we find that, even when all possible bonds are formed, the system does not reach percolation due to the presence of intracluster bonds. We present an extension of the Flory– Stockmayer theory that properly describes the numerical and experimental results
Analysis & optimization of an innovative method for fetal cells enrichment from maternal blood
No full textLAUREA MAGISTRALEI difetti cromosomici alla nascita colpiscono 1 su ogni 150 gravidanze. Questi difetti possono essere diagnosticati con metodi invasivi, che comportano il rischio di perdita fetale, oppure possono essere sottoposti a screening con metodi non invasivi senza una determinata diagnosi. Per ottenere un test diagnostico e non invasivo per tali malattie, nel 2016, Lattuada ha introdotto un metodo che utilizza un sistema di frazionamento in campo-flusso gravitazionale (GrFFF), per l'arricchimento delle cellule fetali da un campione di sangue materno. Questo sistema mira a separare eritroblasti fetali dalle restanti cellule del campione, in base alle loro proprietà fisiche. In questa tesi, tale sistema viene studiato a livello computazionale per comprendere i comportamenti delle cellule nelle diverse fasi dell'applicazione, ed in particolare ci si è focalizzati in tre sezioni: sedimentazione delle cellule, ottimizzazione dell'ingresso, per una migliore distribuzione delle cellule dopo la sedimentazione, e le proprietà del flusso, in termini di forze effettive e movimento delle cellule. Dopo il completamento di queste attività, vengono proposte varie modifiche di progettazione per un'ottimizzazione della procedura di separazione.Chromosomal birth defects effect 1 of every 150 pregnancies. These defects can either be diagnosed by invasive methods, that bear the risk of miscarriage, or screened by non-invasive methods without a certain diagnosis. To obtain a method that is both non-invasive and diagnostic for such diseases, in 2016, a method that utilizes a gravitational field-flow fractionation (GrFFF) system was introduced by Lattuada, for the enrichment of fetal cells from a sample drawn from circulating maternal blood. This system targets to separate mainly fetal nucleated red blood cells (nRBC) from the remaining cells in the sample, according to their physical properties. In this dissertation, that system is investigated computationally to comprehend the behaviours of the cells in different stages of the application. The operation is handled in three sections: sedimentation of the cells, inlet optimization for a better distribution of the cells after sedimentation and the flow properties, in terms of effective forces and movement of the cells with respect to those. After the completion of the aforementioned, various design modifications are proposed for an optimization of the overall separation procedure
Analisi computazionale dell'interazione cellula-fluido in tecniche di frazionamento fluido in campo gravitazionale
No full textLAUREA MAGISTRALELa scoperta delle cellule fetali nucleate circolanti nel sangue periferico materno ha segnato un punto di svolta per la diagnosi prenatale: l’isolamento e l’analisi del DNA costituisce un metodo di indagine sicuro, in grado di effettuare diagnosi complete senza interferire con l’ambiente fetale. Il metodo di separazione oggetto dello studio proposto è il frazionamento fluido in campo gravitazionale (GrFFF) applicato al canale Lattuada, il quale, grazie all’azione combinata della forza gravitazionale e del flusso all’interno del canale, permette la separazione delle cellule d’interesse senza indurre alterazioni. L’obiettivo del lavoro consiste nella definizione di un modello computazionale a volumi finiti per lo studio del comportamento della cellula e la possibile deformazione. Si è implementato e validato un modello a due fasi fluide, medium e cellula, e mediante analisi di sensitività, è stato ottimizzato in termini di dimensione della mesh ed intervallo temporale. Inoltre, è stata valutata l’influenza dei parametri in ingresso sulla soluzione, per garantire l’affidabilità dei risultati. L’effetto della viscosità è risultato determinante, pertanto, si è ritenuto cruciale definirne il valore, applicando un problema inverso così da ottenere risultati attendibili. Lo studio proposto ha dato dei risultati soddisfacenti, ponendo le basi per ulteriori sviluppi futuri volti al miglioramento del modello stesso.The discovery of nucleated fetal cells circulating in maternal peripheral blood has marked a turning point in the prenatal diagnosis field: the isolation and subsequent analysis of DNA constitutes a safe method to perform diagnoses. The proposed study focuses on the Gravitational Field-Flow Fractionation (GrFFF) separation method applied to the Lattuada channel. The GrFFF system consists of a combination of gravitational force and a flow inside a microfluidic channel that allows the separation of the cells of interest without inducing alterations. The subject of the work concerns the definition of a finite volume computational model to study cell behaviour in terms of interaction and potential deformation inside the channel. The present study is based on a two-fluid phases model that has been implemented and validated by employing sensitivity analysis, thus optimised in terms of mesh size and time interval. An analysis of the input parameters was carried out to guarantee model reliability to establish which factors are particularly influential. The outcomes demonstrated that viscosity was a decisive parameter, so to optimize the model, we implemented an inverse problem to define those values that guarantee the achievement of reliable results. The proposed study gave satisfactory results, laying the foundations for future developments to improve the model itself
Treatment of kidney clear cell carcinoma, lung adenocarcinoma and glioblastoma cell lines with hydrogels made of DNA nanostars
No full textOvercoming the systemic administration of chemotherapy to reduce drug toxicity and the application of personalised medicine are two of the major challenges in the treatment of cancer. To this aim, efforts are focused on finding novel nanomaterials for the targeted administration of drugs and bioactive molecules in the tumor sites. DNA-based hydrogels are promising candidates for these applications. However, while such materials are fairly known from a structural and physical standpoint, their effects on cell cultures are far less investigated. Here, we studied the biological response of three different cell lines (clear cell renal cell carcinoma 786-O, lung adenocarcinoma H1975 and glioblastoma U87MG) to the treatment with DNA-GEL - a DNA-based hydrogel composed of interacting DNA nanostars. Additionally, we investigated the structural modification of DNA-GELs under cell culture conditions. The results we collected show a cell type specificity of the response, with interesting implications for future applications
Protective effects of vitamin D3 on fimbrial cells exposed to catalytic iron damage
No full textBACKGROUND:
Recently, vitamin D3 (1alpha, 25-dihydroxyvitamin D) has shown its capability to take part in many extraskeletal functions and its serum levels have been related to patient survival rate and malignancy of many types of neoplasms, including ovarian cancers. Catalytic iron is a free circulating form of iron that is able to generate reactive oxygen species and consequently to promote a number of cellular and tissutal dysfunctions including tumorigenesis. In fertile women an important source of catalytic iron is derived from retrograde menstruation. Epithelial secretory cells from fimbriae of fallopian tubes are greatly exposed to catalytic iron derived from menstrual reflux and so represent the site of origin for most serous ovarian cancers. The aim of this study was to assess whether vitamin D3 can play a role in counteracting catalytic iron-induced oxidative stress in cells from fimbriae of fallopian tubes.
METHODS:
The cells, isolated from women undergoing isteroannessiectomy, were treated with catalytic iron 50-75-100 mM and vitamin D3 at a concentration ranging from 0.01 to 10 nM to study cell viability, radical oxygen species production, p53, pan-Ras, Ki67 and c-Myc protein expressions through Western Blot, and immunocytochemistry or immunofluorescence analysis.
RESULTS:
The pre-treatment with vitamin D3 1 nM showed its beneficial effects that consists in a significant decrease in ROS production. In addition a novel finding is represented by the demonstration that pre-treatment with vitamin D3 is also able to significantly counteract tumoral biomarkers activation, such as p53, pan-Ras, Ki67 and c-Myc, and consequently the catalytic iron-induced cellular injury.
CONCLUSIONS:
This study demonstrates for the first time that vitamin D3 plays an important role in preventing catalytic iron-dependent oxidative stress in cultured fimbrial cells. These results support the hypothesis that vitamin D3 could counteract carcinogenic changes induced by catalytic iron
A tag-less method for direct isolation of human umbilical vein endothelial cells by gravitational field-flow fractionation
No full textThe analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedure
Mitochondrial DNA copy number in peripheral blood : a potential non-invasive biomarker for female subfertility
No full textPURPOSE:
Low mitochondrial DNA (mtDNA) content in oocytes and in cumulus cells is an indicator of poor oocyte quality. Moreover, initial evidence showed a correlation between mtDNA content in cumulus cells and mtDNA copy number in peripheral blood cells. On these bases, we deemed of interest investigating the correlation between mtDNA copy number in peripheral blood and natural fecundity.
METHODS:
This is a nested case-control study drawn from a prospective cohort of pregnant women referred for routine first trimester screening for aneuploidies (from 11 + 0 to 12 + 6 weeks of gestation) between January 2012 and March 2013 at the "Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico" of Milan, Italy. Cases were subfertile women who attempted to become pregnant for 12-24 months. Controls were the two subsequently age-matched women who became pregnant in less than 1 year. MtDNA was quantified using real-time PCR and normalized to nuclear DNA.
RESULTS:
One hundred and four subfertile women and 208 controls were selected. The median (IQR) mtDNA copy number was 95 (73-124) and 145 (106-198), respectively (p < 0.001). The area under the ROC curve was 0.73 (95% CI 0.67-0.79) (p < 0.001). The Youden index was 105 mtDNA copy number. The crude OR for subfertility in women with mtDNA copy number below this threshold was 5.72 (95% CI 3.43-9.55). The accuracy of mtDNA copy number assessment in peripheral blood progressively decreased with increasing female age.
CONCLUSIONS:
Low mtDNA copy number in peripheral blood is associated with an increased risk of subfertility and may represent a biomarker of natural fecundity
Mitochondrial DNA Copy Number in Peripheral Blood in the First Trimester of Pregnancy and Different Preeclampsia Clinical Phenotypes Development: A Pilot Study
No full textInflammation and oxidative stress are intrinsically linked to early poor placentation, typical of pregnancies complicated by preeclampsia associated with intrauterine growth restriction (PE-IUGR). Low mitochondrial DNA copy number (mtDNAcn) in peripheral blood constitutes a good peripheral surrogate marker of inflammation and oxidative stress. On these basis, we explored a possible correlation between mtDNAcn in peripheral blood in the first trimester of pregnancy and the PE-IUGR onset. To shed light on this issue, we setup a nested case–control study from a prospective cohort of pregnant women undergoing first-trimester aneuploidies screening. Two groups of patients affected by PE classified according to the clinical phenotype were identified: (1) patients who developed PE-IUGR and (2) patients who developed PE associated with appropriate for gestational age intrauterine fetal growth (PE-AGAf). Controls were women with a physiologic pregnancy matched to cases on the basis of age (±6 months, ratio 2:1). Mitochondrial DNA copy number was quantified using real-time polymerase chain reaction and normalized to nuclear DNA. The median (interquartile range) mtDNAcn in peripheral blood in patients with PE-IUGR (n = 12) and in patients with PE-AGAf (n = 16) was 70 (44-97) and 108 (95-145), respectively (P =.004). Both these values were significantly lower than that detected in the control group (161[133-183], P <.001). The area under the receiver–operator curve for PE-IUGR and PE-AGAf were 0.94 (95% confidence interval [CI]: 0.88-1.00, P <.001) and 0.81 (95%CI: 0.70-0.91, P <.001), respectively. In conclusion, MtDNAcn in peripheral blood resulted significantly lower both in patients affected by PE-IUGR and in those affected by PE-AGAf when compared to controls. The accuracy of this biomarker resulted particularly good in predicting PE-IUGR
Maternal blood mitochondrial DNA content during normal and intrauterine growth restricted (IUGR) pregnancy
No full textOBJECTIVE: We investigated mitochondrial DNA (mtDNA) content in the maternal circulation of normal pregnancies of different gestational ages and in pregnancies complicated by intrauterine growth restriction (IUGR).
STUDY DESIGN: We examined 70 maternal blood samples: 13 nonpregnant women; 45 normal pregnancies, divided into the 3 trimesters;
and 12 pregnancies complicated by IUGR. MtDNA content was determined by real-time quantitative polymerase chain reaction, using a genomic control and a target gene.
RESULTS: A highly significant progressive reduction in circulating mtDNA was observed in pregnant women of first, second, and third trimesters and compared to nonpregnant women (mean value: 237, 188, 144, and 283, respectively; P .001). Moreover, mtDNA was significantly increased in women carrying IUGR fetuses compared to women with normal pregnancies (430 vs 144; P .001).
CONCLUSION: MtDNA could provide new insight into the mechanisms that occur during physiological gestation. Furthermore, mtDNA content may help recognize the IUGR disease in pregnancy
Breve nota sui criteri museologici per il nuovo ordinamento del Museo Diocesano di Palermo
No full textIl contributo mette a fuoco i criteri ordinativi per il nuovo assetto museologico del Museo Diocesano di Palermo, del quale l’autore è il curatore scientifico.
Si precisano gli elementi che hanno condotto alla ridefinizione dell’identità del museo, rispetto ai cinque allestimenti precedenti e in relazione all’apertura al pubblico del piano nobile del Palazzo Arcivescovile, sede museale, e degli arcivescovi nel corso dei secoli.
La missione che ne consegue viene esplicitata in relazione allo spirito che deve informare i musei ecclesiastici e sulla base di un principio di contemporaneità che esula dalla periodizzazione delle opere e le trascende.The contribution focuses on the ordering criteria for the new museological structure of the Diocesan Museum of Palermo, of which the author is the scientific curator.
The elements that led to the redefinition of the museum's identity are specified, with respect to the five previous installations and in relation to the opening to the public of the noble floor of the Archbishop's Palace, seat of the museum, and of the archbishops over the centuries.
The ensuing mission is made explicit in relation to the spirit that must inform ecclesiastical museums and on the basis of a principle of contemporaneity that goes beyond the periodization of the works and transcends them
