301 research outputs found
Endotelio corneale umano: dagli studi in vitro all’applicazione dell’ ingegneria tissutale
No full textL'endotelio corneale regola lo stato di idratazione stromale necessario per la trasparenza corneale. In età adulta, la densità cellulare endoteliale (ECD) diminuisce annualmente dello 0,6%. Poiché le cellule endoteliali corneali umane hanno ridotta capacità proliferativa in vivo, la loro perdita è compensata dalla migrazione e allargamento delle cellule vicine. Quando l' ECD scende al di sotto del valore soglia di 500 cellule/mm2, in seguito a invecchiamento o trauma o una condizione patologica, l'endotelio non è in grado di garantire una corretta idratazione corneale, causando edema, opacità corneale e disturbi visivi. Il trapianto corneale, con le relative limitazioni, ad oggi è l'unico trattamento efficiente per le malattie endoteliali corneali. Tuttavia, la carenza mondiale di cornee donatrici sta diventando sempre più un problema non trascurabile, con solo 1 cornea disponibile ogni 70 cornee richieste. Questo ha indotto a sviluppare strategie alternative per il trattamento di malattie endoteliali corneali, tra cui gli approcci di ingegneria tissutale.
L'ingegneria tissutale è un approccio terapeutico emergente che combina l'utilizzo di cellule endoteliali corneali con l’utilizzo di un appropriato biomateriale per la coltura ed il trapianto di queste cellule. Il nostro gruppo di ricerca ha precedentemente dimostrato che il legame di un decapeptide contenente il motivo peptidico RGD (Arg-Gly-Asp) allo scaffold di chitina garantisce il mantenimento del comportamento delle cellule epiteliali corneali umane. Le caratteristiche dello scaffold sono state ottimizzate per produrre un substrato con proprietà biomeccaniche simili allo stroma corneale umano per trasparenza, architettura, rigidità e resistenza meccanica.
In questo progetto di ricerca, il nostro obiettivo è quello di studiare l'utilizzo della chitina funzionalizzata con l’ RGD come potenziale substrato anche per l'adesione e l'espansione delle cellule corneali endoteliali umane. Gli esperimenti sono stati condotti al fine di ottenere un tessuto endoteliale ingegnerizzato e, in una prospettiva futura, una cornea umana tridimensionale con tutti i suoi strati (epitelio + stroma + endotelio).
Tuttavia, l'ingegneria tissutale dell’ endotelio corneale è una sfida complessa per diversi motivi: a) le cellule corneali endoteliali hanno una bassa capacità proliferativa che deve essere stimolata finemente in vitro con terreni di coltura appropriati; b) durante la coltura in vitro, le cellule corneali endoteliali vanno incontro a senescenza prematura (in particolare nelle colture cellulari derivate da donatori più anziani) e a una trasformazione fenotipica assumendo un fenotipo mesenchimale, la cosiddetta transizione endoteliale-mesenchimale; e) pochi marcatori molecolari specifici definiscono la qualità delle cellule corneali endoteliali, necessari per controllare le loro funzioni fisiologiche cellulari; d) infine, per l’approccio di ingegneria tissutale dell’ endotelio corneale, non è stato ancora sviluppato un biomateriale in grado di creare un microambiente favorevole all'attività delle cellule corneali endoteliali.
Per questo motivo, in questo progetto di ricerca abbiamo affrontato alcune sfide che rendono difficile l’utilizzo delle cellule corneali endoteliali, in termini di (I) ottimizzazione della tecnica di coltura delle cellule corneali endoteliali umane (Capitolo I), (II) identificazione di marcatori funzionali specifici delle cellule corneali endoteliali (Capitolo II), (III) prevenzione della transizione endotelio-mesenchimale che induce ad un trans-differenziamento cellulare verso un fenotipo mio-fibroblastico che causa una perdita della funzione cellulare (Capitolo III) e (IV) analisi dello scaffold selezionato per coltivare le cellule corneali endoteliali (Capitolo IV).The corneal endothelium (CE) is the innermost layer of the cornea that regulates the stromal hydration state required to maintain corneal transparency. During adulthood, the endothelial cell density (ECD) decreases by 0.6% each year. As human corneal endothelial cells (hCECs) do not proliferate, the loss of aging-induced hCECs is compensated by migration and enlargement of neighbouring cells. When ECD falls below a threshold of 500 cells/mm2, by aging or trauma/disease, the endothelium does not have enough pumping power to guarantee a correct corneal hydration, leading to oedema, corneal opacity, and visual impairment. Corneal transplantation, with related problems, is the only efficient treatment for corneal endothelial diseases up to date. However, the worldwide donor corneas shortage is increasingly becoming a non-negligible issue, with only 1 cornea available for 70 needed. This has led to investigate alternative strategies for treating corneal endothelial diseases, such as tissue engineering approaches.
Corneal endothelial tissue engineering is an emerging therapeutic approach that involves the use of hCECs combined with a biomaterial to create tissue engineered grafts for transplantation. Our research group has previously demonstrated that proper binding of an RGD (Arg-Gly-Asp) peptide to the chitin scaffold guaranteed maintenance of human corneal epithelial cells behaviour. Scaffold characteristics were optimised to produce a substrate with biomechanical properties resembling the human corneal stroma for transparency, architecture, stiffness, and mechanical strength.
In this research project, our aim is to investigate the use of this functionalized biological scaffold as a potential substrate also for hCECs adhesion and expansion. The experiments were carried out in order to obtain a functional tissue engineered endothelial graft and, from a future perspective, a three-dimensional human cornea with all its layers (epithelium + stroma + endothelium). If successful, this elegant approach has the potential to increase access to corneal therapy by treating multiple patients.
However, CE tissue engineering is a major challenge for several reasons: a) the hCECs have a low natural proliferative capacity that must be finely stimulated in vitro with an appropriate mitogen-rich medium; b) during in vitro expansion, hCECs undergo premature senescence (particularly in cultures derived from older donors) and phenotypic transformation to a mesenchymal phenotype, so-called Endothelial-Mesenchymal Transition (EnMT), which must be prevented c) few specific molecular markers define the quality of cultured hCECs, which are needed to control their physiological cell functions; d) finally, to develop a tailored engineered corneal endothelium, a substrate material that is able to create a favourable microenvironment for hCECs activity has not been yet developed.
Thus, in this research project we analysed some challenges faced with hCECs in terms of (I) optimization of hCECs culture techniques (Chapter I), (II) identification of specific hCECs functional markers (Chapter II), (III) prevention of EnMT which leads to a cellular trans-differentiation towards a myofibroblastic phenotype causing a cellular loss of function (Chapter III), and (IV) analysis of the identified scaffold to make bioengineered corneal endothelial grafts (Chapter IV)
Impact of culture media on primary human corneal endothelial cells derived from old donors
No full text: Corneal endothelial dysfunction is a major indication for corneal transplantation. However, a global shortage of donor corneal tissues and risks associated with corneal surgeries have prompted exploration of alternative options, including tissue-engineered grafts or cell injection therapy. Nonetheless, these approaches require a controlled culture of primary human corneal endothelial cells (HCEnCs). Although HCEnCs established from young donors are generally more proliferative and maintain a better phenotype, corneas from old donors are more frequently accessible from eye banks due to a lower corneal endothelial cell count than the necessary threshold required for transplantation. In this study, we investigated various culture media to evaluate which one is the most appropriate for stimulating the proliferation while maintaining cell morphology and function of HCEnCs derived from old donors (age >65 years). All experiments were performed on paired research-grade donor corneas, divided for the conditions under investigation in order to minimize the inter-donor variability. Cell morphology as well as expression of specific markers were assessed at both mRNA (CD166, SLC4A11, ATP1A1, COL8A1, α-SMA, CD44, COL1A1, CDKN2A, LAP2A and LAP2B) and protein (ZO-1, α-SMA, Ki67 and LAP2) levels. Results obtained showed how the Dual Media formulation maintained the hexagonal phenotype more efficiently than Single Medium, but cell size gradually increased with passages. In contrast, the Single Medium provided a higher proliferation rate and a prolonged in vitro expansion but acquired an elongated morphology. To summarize, Single medium and Dual media preserve morphology and functional phenotype of HCEnCs from old donor corneas at low passages while maintenance of the same cell features at high passages remains an active area of research. The new insights revealed within this work become particularly relevant considering that the elderly population a) is the main target of corneal endothelial therapy, b) represents the majority of corneal donors. Therefore, the proper expansion of HCEnCs from old donors is essential to develop novel personalised therapeutic strategies and reduce requirement of human corneal tissues globally.Corneal endothelial dysfunction is a major indication for corneal transplantation. However, a global shortage of donor corneal tissues and risks associated with corneal surgeries have prompted exploration of alternative options, including tissue-engineered grafts or cell injection therapy. Nonetheless, these approaches require a controlled culture of primary human corneal endothelial cells (HCEnCs). Although HCEnCs established from young donors are generally more proliferative and maintain a better phenotype, corneas from old donors are more frequently accessible from eye banks due to a lower corneal endothelial cell count than the necessary threshold required for transplantation. In this study, we investigated various culture media to evaluate which one is the most appropriate for stimulating the proliferation while maintaining cell morphology and function of HCEnCs derived from old donors (age >65 years). All experiments were performed on paired research-grade donor corneas, divided for the conditions under investigation in order to minimize the inter-donor variability. Cell morphology as well as expression of specific markers were assessed at both mRNA (CD166, SLC4A11, ATP1A1, COL8A1, α-SMA, CD44, COL1A1, CDKN2A, LAP2A and LAP2B) and protein (ZO-1, α-SMA, Ki67 and LAP2) levels. Results obtained showed how the Dual Media formulation maintained the hexagonal phenotype more efficiently than Single Medium, but cell size gradually increased with passages. In contrast, the Single Medium provided a higher proliferation rate and a prolonged in vitro expansion but acquired an elongated morphology. To summarize, Single medium and Dual media preserve morphology and functional phenotype of HCEnCs from old donor corneas at low passages while maintenance of the same cell features at high passages remains an active area of research. The new insights revealed within this work become particularly relevant considering that the elderly population a) is the main target of corneal endothelial therapy, b) represents the majority of corneal donors. Therefore, the proper expansion of HCEnCs from old donors is essential to develop novel personalised therapeutic strategies and reduce requirement of human corneal tissues globally
Economic factors affecting obesity: an application in Italy
No full textThe World Health Organization has stated that obesity is spreading around the world like a “global epidemic”. In 2004 the percentage of obese people in the Italian population was 9%, but the trend s increasing in recent years. Focusing on this country, the purpose of the paper is to analyze the socio-economic variables affecting obesity by means of a survey conducted in a consumer sample. Our analysis is based on a survey conducted in Italy, and the sample was composed of 999 consumers. We used a binary logit model and the dependent variable is body mass index (BMI), expressed in a dichotomic way (seriously overweight and obese, value 1, and normal weight, value 0). The results show that the condition of the seriously overweight and obese increases with age, especially in people over 65 of age. Also gender is correlated with the pathology: being seriously overweight and obese is far more likely for men than for women. An inverse relation was shown between obesity and education, and between obesity and the level of food knowledge. The results highlight that disadvantaged social categories are more susceptible to the problem of overweight and obesity. A policy implication of the analysis, to limit the spread of obesity, could lie in programs aimed at improving health and food awareness and focused on these minority groups.economics of obesity, BMI and consumer, logit model, Food Consumption/Nutrition/Food Safety, Health Economics and Policy,
Author response
No full textDetecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price (McKean and Lazzaro, 2011, Kominsky et al., 2010), and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues (Abbas et al., 2014), but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. Here we show that Drosophila melanogaster display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. We found the expression of the chemosensory cation channel dTRPA1 in these cells to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. Our findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism.sponsorship: Vlaams Instituut voor Biotechnologie Alessia Soldano Luis Franco Guangda Liu Natalia Mora Emre Yaksi Bassem A Hassanr Fonds Wetenschappelijk Onderzoek G.0702.12 Alessia Soldano Yeranddy A Alpizar Brett Boonen Alejandro Lopez-Requena Natalia Mora Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar Fonds Wetenschappelijk Onderzoek G.0077.15 Alessia Soldano Yeranddy A Alpizar Brett Boonen Alejandro Lopez-Requena Natalia Mora Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar Fonds Wetenschappelijk Onderzoek G.0680.10 Alessia Soldano Yeranddy A Alpizar Brett Boonen Alejandro Lopez-Requena Natalia Mora Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar Fonds Wetenschappelijk Onderzoek G.0681.10 Alessia Soldano Yeranddy A Alpizar Brett Boonen Alejandro Lopez-Requena Natalia Mora Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar Fonds Wetenschappelijk Onderzoek G.0503.12 Alessia Soldano Yeranddy A Alpizar Brett Boonen Alejandro Lopez-Requena Natalia Mora Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar Fonds Wetenschappelijk Onderzoek G.0654.15 Alessia Soldano Yeranddy A Alpizar Brett Boonen Alejandro Lopez-Requena Natalia Mora Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar Fonds Wetenschappelijk Onderzoek G.0761.10N Alessia Soldano Yeranddy A Alpizar Brett Boonen Alejandro Lopez-Requena Natalia Mora Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar Fonds Wetenschappelijk Onderzoek G.0596.12 Alessia Soldano Yeranddy A Alpizar Brett Boonen Alejandro Lopez-Requena Natalia Mora Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar Fonds Wetenschappelijk Onderzoek G.0565.07 Alessia Soldano Yeranddy A Alpizar Brett Boonen Alejandro Lopez-Requena Natalia Mora Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar KU Leuven GOA/14/011 Alessia Soldano Yeranddy A Alpizar Brett Boonen Luis Franco Alejandro Lopez-Requena Guangda Liu Natalia Mora Emre Yaksi Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar European Commission IUAP P7/13 Alessia Soldano Yeranddy A Alpizar Brett Boonen Luis Franco Alejandro Lopez-Requena Guangda Liu Natalia Mora Emre Yaksi Thomas Voets Rudi Vennekensr KU Leuven OT/12/091 Alessia Soldano Yeranddy A Alpizar Brett Boonen Luis Franco Alejandro Lopez-Requena Guangda Liu Natalia Mora Emre Yaksi Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talaverar KU Leuven PF-TRPLe Alessia Soldano Yeranddy A Alpizar Brett Boonen Luis Franco Alejandro Lopez-Requena Guangda Liu Natalia Mora Emre Yaksi Thomas Voets Rudi Vennekens Bassem A Hassan Karel Talavera (Vlaams Instituut voor Biotechnologie, Fonds Wetenschappelijk Onderzoek|G.0702.12, Fonds Wetenschappelijk Onderzoek|G.0077.15, Fonds Wetenschappelijk Onderzoek|G.0680.10, Fonds Wetenschappelijk Onderzoek|G.0681.10, Fonds Wetenschappelijk Onderzoek|G.0503.12, Fonds Wetenschappelijk Onderzoek|G.0654.15, Fonds Wetenschappelijk Onderzoek|G.0761.10N, Fonds Wetenschappelijk Onderzoek|G.0596.12, KU Leuven|GOA/14/011, KU Leuven|OT/12/091, European Commission|IUAP P7/13, KU Leuven PF-TRPLe)status: Publishe
Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium
No full textNanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle‐mediated RNA interference therapy aimed at enhancing its proliferative capacity, necessary for tissue regeneration. This work develops a strategy for siRNA nanoninjection to the human corneal endothelium. Nanoneedles can deliver p16‐targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is nontoxic ex vivo, and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNA interference therapy for the treatment of endothelial corneal dysfunction
Social Network to analyse the relationship between ‘victim-author’ and ‘motivation’ of violence against women in Italy.
No full textThe paper aims to analyse the phenomenon of Violence against women in the Italian context during 2020. It proposes to study the relationship between ‘victim-author’ and ‘motivation’ in femicides committed in domestic environment. By means of the properties of the Social Network Analysis on bimodal data, the study detected main actors and motivations that generated the homicides with female victims. At the same time, the structural relationships allowed to investigate the existence of motivations that better characterized the action of the various actors. The bipartite graph visualization and centrality scores calculated have demonstrated the effectiveness of the methodology for the pursued objectives
Fluctuations in Corneal Endothelial LAP2 Expression Levels Correlate with Passage Dependent Declines in Their Cell Proliferative Activity
No full textThe corneal endothelium is the inner corneal mono-layered epithelium, fundamental for preserving corneal hydration and transparency. However, molecular mechanisms that regulate corneal endothelial cells (CEnCs), in particular regarding their proliferative capacity, have been only partially elucidated. CEnCs are quiescent in vivo and they easily undergo endothelial to mesenchymal transition (EnMT) in vitro. This study aims to analyze CEnCs behavior and expression in vitro, either in sub-confluent growing (S) or confluent (C) CEnCs cultures. Primary rabbit and human CEnCs were cultured and used for RT-PCR, immunofluorescence or western blot analysis. These methods allowed identifying a novel molecular marker, LAP2, that is upregulated in S while downregulated in C human or rabbit CEnCs. Those results were observed for several subsequent passages in culture and this, together with the correlation between ki67 and LAP2 expression, suggested LAP2 as a novel possible indicator for culture ageing. Finally, treatment with FGF and TGF beta in rCEnCs highlighted how LAP2 can vary as the cells regulate their proliferative state. In conclusion, we have identified a novel marker for CEnCs, LAP2, that regulates its expression depending on the cells sub/confluent state and that correlates with CEnCs proliferation
Louis-Philippe Dalembert, «vagabond jusqu’au bout de la fatigue»
No full textThe Haitian novelist and poet Louis-Philippe Dalembert (Port-au-Prince, 1962) has developed in his works of fiction the concept of vagabondage as a literary projection of his biographical wandering through multiple spaces. The aim of this essay is to study the presence of vagabondage and its distinctive features in those novels written by Dalembert that reflect the writer’s perpetual motion: Le Crayon du bon Dieu n’a pas de gomme (1996), L’Autre face de la mer (1998), L’Île du bout des rêves (2003), Les dieux voyagent la nuit (2006). The main characters are constantly moving, they are cosmopolitan wanderers who belong to many places at the same time, just like Dalembert himself. By analyzing the representation of movement in these fictions, we will show that the notion of vagabondage is depicted by the author as a positive and meaningful opportunity for the vagabond who travels across countries, languages and cultures
Una ridiscussione dei concetti di home e identity nell’Asia globalizzata: il caso di These Foolish Things (2004) e How to Get Filthy Rich in Rising Asia (2013)
No full textThe evolution of postcolonial studies in the last thirty years and the development of a transnational approach in literary studies (Jay 2010) have led to a renewed interest towards the subaltern voices, especially in relation to the phenomena of migration and diaspora and their global effects. In this light, the idea of 'home' is characterized by a sort of porosity and by a new geographical and emotional conceptualization which inevitably influences the personal and collective identities of migrant communities.
The aim of the paper is to analyze these topics from a cultural and literary standpoint through the examination of the two different kinds of migrant flows and postcolonial scenarios depicted in These Foolish Things (2004) by the English author Deborah Moggach, and How to Get Filthy Rich in Rising Asia (2013) by the Pakistan novelist Mohsin Hamid. In these novels, the chaotic Indian framework – the former margin of the British empire – is torn between its colonial past and the current effects of the permeability of its borders. It is, therefore, a perfect global context, wherein the experiences and the feelings of the modern Indian identities are reinterpreted by the two authors
- …
