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Frezzetti D.,IRGS Biogem S.c.ar.l. | Frezzetti D.,University of Naples Federico II | De Menna M.,SEMM | De Menna M.,Centro Of Ingegneria Genetica E Biotecnologie Avanzate | And 17 more authors.
Oncogene | Year: 2011

miR-21 is a microRNA (miRNA) frequently overexpressed in human cancers. Here we show that miR-21 is upregulated both in vitro and in vivo by oncogenic Ras, thus linking this miRNA to one of the most frequently activated oncogenes in human cancers. Ras regulation of miR-21 occurs with a delayed kinetic and requires at least two Ras downstream pathways. A screen of human thyroid cancers and non-small-cell lung cancers for the expression of miR-21 reveals that it is overexpressed mainly in anaplastic thyroid carcinomas, the most aggressive form of thyroid cancer, whereas in lung its overexpression appears to be inversely correlated with tumor progression. We also show that a LNA directed against miR-21 slows down tumor growth in mice. Consistently, a search for mRNAs downregulated by miR-21 shows an enrichment for mRNAs encoding cell cycle checkpoints regulators, suggesting an important role for miR-21 in oncogenic Ras-induced cell proliferation. © 2011 Macmillan Publishers Limited.


Leccia F.,SEMM | Nardone A.,University of Naples Federico II | Corvigno S.,University of Naples Federico II | Vecchio L.D.,CEINGE Biotecnologie Avanzate | And 4 more authors.
Cytometry Part A | Year: 2012

To determine whether cell cultures maintain the cellular heterogeneity of primary tissues and may therefore be used for in vitro modeling of breast cancer subtypes, we evaluated the expression of a cell surface marker panel in breast cancer cell cultures derived from various subtypes of human breast carcinoma. We used a four-color flow cytometry strategy to immunophenotype seven human breast cancer cell cultures and four reference breast cancer cell lines. We analyzed 28 surface markers selected based on their potential to distinguish epithelial or mesenchymal lineage, to identify stem cell populations, and to mediate cell adhesion and migration. We determined their ability to form mammospheres and analyzed luminal cytokeratins CK18, CK19, and myoepithelial/basal CK5, SMA (alpha-smooth muscle actin), and vimentin expression by western blot. All cell surface markers showed a unimodal profile. Ten/28 markers were homogenously expressed. Four (CD66b, CD66c, CD165, CD324) displayed negative/low expression. Six (CD29, CD55, CD59, CD81, CD151, CD166) displayed homogenous high expression. Eighteen (CD9, CD10, CD24, CD26, CD44, CD47, CD49b, CD49f, CD54, CD61, CD90, CD105, CD133, CD164, CD184, CD200, CD227, CD326) were heterogeneously expressed. Spearman's rank test demonstrated a significant correlation (p< 0.001) between mesenchymal phenotype and breast cancer cell cultures. Breast cancer cell cultures, all CD44+, displayed concomitant high expression of only three antigens (CD10, CD54, CD90), and low expression of CD326; cell cultures formed mammospheres and expressed CK5, SMA and vimentin, and were weakly CK19-positive. We demonstrate that breast cancer cell cultures preserve inter-tumor heterogeneity and express stem/progenitor markers that can be identified, quantified and categorized by flow cytometry. Therefore, cell cultures can be used for in vitro modeling of breast cancer subtypes; immunophenotyping may mirror breast cancer heterogeneity and reveal molecular characteristics of individual tumors useful for testing target therapy. © 2012 International Society for Advancement of Cytometry.


News Article | November 2, 2016
Site: www.nature.com

There's much to consider when you're trying to choose the university and programme for your science PhD. But the main reason for your selection must be that it suits you — not that you don't know what else to do, not the institution's or department's reputation, not that a star researcher in your field is a faculty member there. Getting a PhD is hard enough, says Bruce Horazdovsky, associate dean for the Mayo Graduate School in Rochester, Minnesota. You don't want to make it harder by being “miserable while you're doing it”, he says. “You have to be engaged and like what you are doing. The best programme in the country is the one that best fits you.” How do you find that best fit? Prospective doctoral students will need to consider several factors and compare programmes and schools. Deciding which universities to apply to means identifying programmes that match your research interests and personality. You will need to evaluate how the school approaches career and professional development for its graduate students, and how its alumni fare after achieving their PhDs. Ultimately, the school you select will be the launch pad for your scientific career. Before you look at schools, you should have a clear idea of your chosen subfield of study. “Even at this stage, students ought to be thinking about what sort of specialization they want to do,” says David Bogle, pro-vice-provost of the doctoral school at University College London. He notes that a physics programme, for instance, could be great for astrophysics and string-theory research but offer nothing on materials science. Although it's not necessary to narrow down fields too specifically, it is imperative to find a programme that has at least several faculty members who are doing research that excites you, says Bogle, who chairs the League of European Research Universities' doctoral studies community in Leuven, Belgium. He advises students to look, not for a single high-profile researcher, but rather for a strong research environment with several professors working in similar areas. To get started, applicants can generally find descriptions of a school's research programmes and faculty members on the institution's website. Sometimes, more information is available: the European School of Molecular Medicine (SEMM), a graduate programme shared between two universities and three research centres in Milan and Naples, Italy, publishes an annual list of faculty members who are taking new students in the coming year. Other institutions may publish similar material. Group websites can also give applicants a feel for the size and culture of a laboratory. Look for photos of lab outings or celebrations, for announcements of student achievements and publications, and for other evidence that graduate students drive much of the research in the group. Applicants should also look up a lab group's latest research publications to get an idea of its members' current interests and to see how well and how often students in the lab are publishing papers. “If the publications coming out of a lab are numerous and high quality, you can be pretty sure that you will get published by the end of your PhD” — which is essential for success after graduation, says Francesca Fiore, coordinator of the SEMM graduate office in Milan. Applicants should also seek advice and guidance from their undergraduate or master's advisers to generate a shortlist of potential programmes. “Come talk to me,” says Andreas Berlind, an astrophysicist at Vanderbilt University in Nashville, Tennessee. “Let me help you make that initial list — it will save you a lot of time.” Advisers, he says, have enough deep knowledge of their field and its subfields to know which programmes tie in with the subjects a student is passionate about; they should also know where other researchers in that subfield are doing good work. For extra connections with the research world, applicants should try to attend large scientific conferences in their subfield; these often have travel fellowships so that undergraduates can attend. Students should also check online resources such as the US National Research Mentoring Network (nrmnet.net) and Facebook sites such as Equity Einstein, a group dedicated to making physics and astronomy more inclusive. Such resources will help students to connect with established researchers who can offer advice on training. They should also contact current graduate students in their subfield to learn about programmes' reputations. Applicants should not be shy about doing this, says Berlind, who is also Vanderbilt's director of graduate studies in astrophysics. It is the best way to get honest answers about the culture and atmosphere in a programme, he adds (see 'The value of hindsight'). Fiore also encourages correspondence with current students, especially for applicants who are pondering studying abroad. “Find someone from your home country,” if possible, she says, so that you can discuss their experience in your native language. Prospective students should never pin their hopes on working with one particular professor, because that person may not be taking students, may move away or might be a terrible fit as a mentor. If several faculty members are working in a similar area, the student has a better chance of landing a spot in one of those labs. Identify and contact at least two researchers, and ideally more, whom you'd like to do a PhD with, counsels Pamela McLean, director of neurobiology at Mayo Graduate School in Jacksonville, Florida. When you e-mail them, you can let them know of your interest in their work and find out whether they are taking on doctoral students in the next year. “A lot of times it will also strengthen your application,” she says. “Those names are often forwarded to admissions directors, and someone who has taken the initiative gets bonus points.” More programmes are publishing data on their websites about their graduate students, including the average time taken to achieve a PhD. Students should pay particular attention to this: anything much more than five years for US programmes or three for UK programmes can indicate that students are languishing in labs as labourers. Some institutions provide data on their graduates' career choices — the University of California, San Francisco, posts outcome data for most of its graduate-division programmes. It's unusual for these data to be long-term enough to give a realistic picture of what all PhD holders are doing ten years after earning their degree, but it is still useful to scan such listings to see if doctoral graduates are ending up in careers that applicants consider desirable. “If they're not there, that's a bad sign that the department doesn't see it as a priority to advertise how well students are doing,” says Berlind. Applicants should also determine whether they want to work on fundamental questions or do applied research. Students interested in the latter should seek programmes with strong ties to high-tech companies, the aerospace industry or hospitals, if their passion lies in those areas. For example, the Mayo Graduate School is spread across three large medical campuses in Minnesota, Florida and Arizona. Students should also give some thought to the overall structure and organization of graduate programmes; these can be small and based in single departments or wide-ranging and interdisciplinary. Umbrella programmes (sometimes called structured programmes in the United Kingdom) pull in faculty members across several departments or campuses. These are in contrast to more conventional, single-department programmes, and in many cases they offer numerous labs and more options for cross-disciplinary studies. But what they make up for in quantity, they may lose in the quality of training or mentoring. Departmental programmes often produce more close-knit communities, with seminars, journal clubs or other events geared specifically to their graduate students. Another structure is the bridge programme, which offers US students the chance to apply to a master's programme that filters directly into a PhD programme on the same or a nearby campus. (The master's-to-PhD route is common in the United Kingdom.) Such programmes are often a sound choice for those who feel that they need more preparation for doctoral studies. LaNell Williams, a second-year biophysics student, found that the Fisk–Vanderbilt Bridge Program run by Fisk and Vanderbilt universities in Nashville, Tennessee, let her meet up with other students who were from groups that are under-represented in science. In contrast to her experience as the only woman of colour in her undergraduate physics studies, Williams says that after a year in the Fisk–Vanderbilt programme, she feels comfortable and has formed a community with fellow students. “I have been able to thrive,” she says, “and see myself as a physicist.” The doctoral application process is not too early to think about ultimate career goals, says Horazdovsky. “Those can change,” he says. “However, you need to make sure you will have tools or experiences to achieve your goal by the end of graduate school.” For example, students who think they want to work at a mainly undergraduate institution will want significant teaching experience. Students who aim for industry will need exposure to business, companies and the jobs that PhD holders occupy. Students should also find out whether their programme of choice hosts, or at least encourages students to attend, conferences and workshops that help them to build teaching, networking and communications skills. Many programmes include career-development components that give students real-world exposure to career tracks. These can be extremely helpful for students who are not aiming for an academic research position and can include university internships, external internships and other options. Students at the application stage need to stand out from the crowd to get accepted by their school of choice. David Charbonneau, director of graduate admissions for Harvard University's department of astronomy in Cambridge, Massachusetts, looks for students who have persevered in the face of obstacles. “Most of what we do in science leads to dead ends,” he says. He seeks students who are passionate and hard-working, and who have demonstrated new ways of tackling problems — for example, by working through solutions to an ambitious research problem for several years. These attributes should come across through concrete examples in their letters of recommendation, he says. McLean says applicants should personalize their application statements by including a paragraph explaining which faculty members within a programme they would like to work with, and why. If prospective PhD students are unsure whether graduate school is the right decision, they should take a year or two to work as a research assistant in an academic or industry lab before making the hefty commitment to doctoral studies. Taking that time is no longer viewed as a negative, says McLean, but instead shows that applicants have realistic expectations and are aware of what's ahead. Allatah Mekile was uncertain of her next steps after finishing college at East Stroudsburg University of Pennsylvania, so she moved home and took an entry-level position as a research associate at a supplier of nutritional products. There, she worked for two years on a metabolic-engineering project before applying to graduate programmes; she is now a second-year doctoral student in biochemistry, cellular and molecular biology at Johns Hopkins University in Baltimore, Maryland. She says that her experience in industry also helped her to explain in her application letter why and how certain programmes aligned with her career goals. By eschewing the conventional path of going immediately into a doctoral programme after earning a bachelor's degree, and gambling that she'd be better prepared, Mekile showed that she was ready for graduate studies, says Bogle. “The whole point of going to graduate school is to take a bit of a risk. If you want to play it safe all the way through, then maybe graduate school — or research — isn't for you.”


Tocchio A.,SEMM | Tocchio A.,Fondazione Filarete | Tamplenizza M.,Fondazione Filarete | Martello F.,Fondazione Filarete | And 9 more authors.
Biomaterials | Year: 2015

Despite significant progresses were achieved in tissue engineering over the last 20 years, a number of unsolved problems still remain. One of the most relevant issues is the lack of a proper vascularization that is limiting the size of the engineered tissues to smaller than clinically relevant dimensions. Sacrificial molding holds great promise to engineered construct with perfusable vascular architectures, but there is still the need to develop more versatile approaches able to be independent of the nature and dimensions of the construct. In this work we developed a versatile sacrificial molding technique for fabricating bulk, cell-laden and porous scaffolds with embedded vascular fluidic networks. These branched fluidic architectures are created by highly resistant thermoplastic sacrificial templates, made of poly(vinyl alcohol), representing a remarkable progress in manufacturability and scalability. The obtained architecture, when perfused in bioreactor, has shown to prevent the formation of a necrotic core in thick cell-laden constructs and enabled the rapid fabrication of hierarchically branched endothelium. In conclusion we demonstrate a novel strategy towards the engineering of vascularized thick tissues through the integration of the PVA-based microfabrication sacrificial approach and perfusion bioreactors. This approach may be able to scale current engineered tissues to clinically relevant dimensions, opening the way to their widespread clinical applications. © 2014 Elsevier Ltd.


PubMed | SEMM, Fondazione Filarete and Wuhan University of Technology
Type: | Journal: Biomaterials | Year: 2015

Despite significant progresses were achieved in tissue engineering over the last 20 years, a number of unsolved problems still remain. One of the most relevant issues is the lack of a proper vascularization that is limiting the size of the engineered tissues to smaller than clinically relevant dimensions. Sacrificial molding holds great promise to engineered construct with perfusable vascular architectures, but there is still the need to develop more versatile approaches able to be independent of the nature and dimensions of the construct. In this work we developed a versatile sacrificial molding technique for fabricating bulk, cell-laden and porous scaffolds with embedded vascular fluidic networks. These branched fluidic architectures are created by highly resistant thermoplastic sacrificial templates, made of poly(vinyl alcohol), representing a remarkable progress in manufacturability and scalability. The obtained architecture, when perfused in bioreactor, has shown to prevent the formation of a necrotic core in thick cell-laden constructs and enabled the rapid fabrication of hierarchically branched endothelium. In conclusion we demonstrate a novel strategy towards the engineering of vascularized thick tissues through the integration of the PVA-based microfabrication sacrificial approach and perfusion bioreactors. This approach may be able to scale current engineered tissues to clinically relevant dimensions, opening the way to their widespread clinical applications.


PubMed | Keele University, SEMM, University of Manchester, Fondazione Filarete and Loughborough University
Type: Journal Article | Journal: Macromolecular bioscience | Year: 2016

This study presents a custom-made in situ gelling polymeric precursor for cell encapsulation. Composed of poly((2-hydroxyethyl)methacrylate-co-(3-aminopropyl)methacrylamide) (P(HEMA-co-APM) mother backbone and RGD-mimicking poly(amidoamine) (PAA) moiteis, the comb-like structured polymeric precursor is tailored to gather the advantages of the two families of synthetic polymers, i.e., the good mechanical integrity of PHEMA-based polymers and the biocompatibility and biodegradability of PAAs. The role of P(HEMA-co-APM) in the regulation of the chemico-physical properties of P(HEMA-co-APM)/PAA hydrogels is thoroughly investigated. On the basis of obtained results, namely the capability of maintaining vital NIH3T3 cell line in vitro for 2 d in a 3D cell culture, the in vivo biocompatibility in murine model for 16 d, and the ability of finely tuning mechanical properties and degradation kinetics, it can be assessed that P(HEMA-co-APM)/PAAs offer a cost-effective valid alternative to the so far studied natural polymer-based systems for cell encapsulation.


Martello F.,Fondazione Filarete | Tocchio A.,SEMM | Tamplenizza M.,Fondazione Filarete | Gerges I.,Fondazione Filarete | And 9 more authors.
Acta Biomaterialia | Year: 2014

Poly(amido-amine) (PAA) hydrogels containing the 2,2-bisacrylamidoacetic acid-4-amminobutyl guanidine monomeric unit have a known ability to enhance cellular adhesion by interacting with the arginin-glycin-aspartic acid (RGD)-binding αVβ3 integrin, expressed by a wide number of cell types. Scientific interest in this class of materials has traditionally been hampered by their poor mechanical properties and restricted range of degradation rate. Here we present the design of novel biocompatible, RGD-mimic PAA-based hydrogels with wide and tunable degradation rates as well as improved mechanical and biological properties for biomedical applications. This is achieved by radical polymerization of acrylamide-terminated PAA oligomers in both the presence and absence of 2-hydroxyethylmethacrylate. The degradation rate is found to be precisely tunable by adjusting the PAA oligomer molecular weight and acrylic co-monomer concentration in the starting reaction mixture. Cell adhesion and proliferation tests on Madin-Darby canine kidney epithelial cells show that PAA-based hydrogels have the capacity to promote cell adhesion up to 200% compared to the control. Mechanical tests show higher compressive strength of acrylic chain containing hydrogels compared to traditional PAA hydrogels. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.


PubMed | SEMM and Fondazione Filarete
Type: | Journal: Colloids and surfaces. B, Biointerfaces | Year: 2016

This study presents an innovative method for the synthesis of polymeric nanoparticles (NPs) for central nervous system (CNS) targeting. The method is based on Ultraviolet light (UV)-induced crosslinking of diacrylamide-terminated oligomers of poly(amidoamine)s (PAAs), a widely used class of synthetic polymers in biomedical field research, especially in drug delivery thanks to their excellent biocompatibility and controlled biodegradability. Previous attempts aiming at preparing PAA-based NPs by self-assembly were challenged by lack of structural stability and consequently their early degradation and premature drug release. Here, the UV-induced crosslinked PAA NPs demonstrated to overcome main disadvantages of the self-assembled ones, as they showed improved stability and controlled release properties. Besides the remarkable efficiency to produce monodisperse and stable PAA NPs, the UV-induced crosslinking method is featured by great versatility and low environmental impact, since it does not require use of organic solvents and multiple purification steps. The capability of PAA NPs to encapsulate a fluorescently labelled model protein was experimentally demonstrated in this study. Cell culture experiments showed that PAA NPs were biocompatible and highly permeable across an in vitro blood-brain barrier model, thus highlighting their great potential as drug delivery vectors for CNS delivery.


PubMed | SEMM, Fondazione Filarete and University of Milan
Type: Journal Article | Journal: Acta biomaterialia | Year: 2014

Poly(amido-amine) (PAA) hydrogels containing the 2,2-bisacrylamidoacetic acid-4-amminobutyl guanidine monomeric unit have a known ability to enhance cellular adhesion by interacting with the arginin-glycin-aspartic acid (RGD)-binding V3 integrin, expressed by a wide number of cell types. Scientific interest in this class of materials has traditionally been hampered by their poor mechanical properties and restricted range of degradation rate. Here we present the design of novel biocompatible, RGD-mimic PAA-based hydrogels with wide and tunable degradation rates as well as improved mechanical and biological properties for biomedical applications. This is achieved by radical polymerization of acrylamide-terminated PAA oligomers in both the presence and absence of 2-hydroxyethylmethacrylate. The degradation rate is found to be precisely tunable by adjusting the PAA oligomer molecular weight and acrylic co-monomer concentration in the starting reaction mixture. Cell adhesion and proliferation tests on Madin-Darby canine kidney epithelial cells show that PAA-based hydrogels have the capacity to promote cell adhesion up to 200% compared to the control. Mechanical tests show higher compressive strength of acrylic chain containing hydrogels compared to traditional PAA hydrogels.


PubMed | SEMM and Fondazione Filarete
Type: | Journal: Acta biomaterialia | Year: 2015

The potential of the 3D cell culture approach for creating in vitro models for drug screening and cellular studies, has led to the development of hydrogels that are able to mimic the in vivo 3D cellular milieu. To this aim, synthetic polymer-based hydrogels, with which it is possible to fine-tune the chemical and biophysical properties of the cell microenvironment, are becoming more and more acclaimed. Of all synthetic materials, poly(amidoamine)s (PAAs) hydrogels are known to have promising properties. In particular, PAAs hydrogels containing the 2,2-bisacrylamidoacetic acid-agmatine monomeric unit are capable of enhancing cellular adhesion by interacting with the RGD-binding V3 integrin. The synthesis of a new photocrosslinkable, biomimetic PAA-Jeffamine-PAA triblock copolymer (PJP) hydrogel is reported in this paper with the aim of improving the optical, biocompatibility and cell-adhesion properties of previously studied PAA hydrogels and providing an inexpensive alternative to the RGD peptide based hydrogels. The physicochemical properties of PJP hydrogels are extensively discussed and the behavior of 2D and 3D cell cultures was analyzed in depth with different cell types. Moreover, cell-laden PJP hydrogels were patterned with perfusable microchannels and seeded with endothelial cells, in order to investigate the possibility of using PJP hydrogels for fabricating cell laden tissue-like micro constructs and microfluidic devices. Overall the data obtained suggest that PJP could ultimately become a useful tool for fabricating improved in vitro models in order to potentially enhance the effectiveness of drug screening and clinical treatments.

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