1,721,046 research outputs found
Ferritin and Transferrin Receptor 1: cryo-EM structural studies and protein engineering
Full text linkThe principal aim of this thesis is the structural determination of the residues involved in the complex between the human Ferritin H-chain (HFt) and the human Transferrin Receptor 1 (hTfR1 or CD71) at near to atomic resolution.
HFt nanoparticles represent one of the most appropriate vectors for cellular delivery of molecules thanks to their internalization by CD71, a transmembrane receptor overexpressed in most cancer cell types. As such, literature is continuously enriched by successful biomedical applications of this interaction; nevertheless, the epitopes of their recognition are to date unknown. To this end, we exploited single-particle cryo-electron microscopy (cryo-EM): this technique is currently under intense technological development and can now be used as a convenient method to determine atomic structures of protein complexes. So, we took advantage of modern cryo-electron microscopes and GPU-equipped computer clusters for single particle analysis calculations. Through the CD71-targeted distribution of specific payloads, the cellular internalization property of HFt might be used for a plethora of purposes, ranging from anticancer therapy to image-enhancement for diagnostics: the side aims of this thesis deal with cryo-EM structural analysis of two different Ferritins chimeras whose constructs have been designed and optimized by our group
Structural bioinformatics: methods development and applications to biomedical problems
Full text linkIn this thesis, I have used a variety of structural bioinformatics methods to investigate different biological and biomedical problems in collaboration with experimental groups, and I have developed a program to help protein structure comparisons. I have combined structure analyses and virtual screenings of several compound libraries to investigate the ligand binding properties of the sigma-1 receptor, which is an attractive therapeutic target for its roles in neural protection and pain. These studies have allowed me to: i) Propose steroid-based compounds as likely physiological ligands sigma-1 receptor; and ii) identify six FDA-approved drugs that were experimentally demonstrated to bind the receptor and improve the growth of cell from Huntington’s disease patients and can be therefore repurposed for use in this disease. I also carried out structure analyses and virtual screening towards COX-2 and COX-1 to compare the predicted binding of compounds with anti-inflammatory potential, obtained from olive oil with eco-sustainable procedures, with classic COX inhibitors. I performed sequence and structure analyses to identify the structural determinants of the biological activity of several Arabidopsis thaliana proteins, in comparison with their human homologues: i) the antiproliferative activity of the two sirtuins; and ii) the substrate specificity of N-acetyltransferase 2. Finally, I have implemented an automated pipeline that compares protein structure alignments produced by multiple programmes and identifies the consensus regions in their output
Novel insights into fungal degradation of lignin: role of a flavoenzyme in assisting laccase-mediated oxidations
Full text linkLignin is the third most abundant biopolymer on earth, after cellulose and chitin, and accounts for up to 30% of plant biomass. It could be an inestimable source of aromatics of interest for organic syntheses, and probably the primary one after petrol runs out. However, its heterogeneous, randomly assembled and extremely complex chemical structure (fundamental for its protective role in the plant cell wall) makes its valorisation and exploitation scarce. A solution to this problem can be found in nature. In fact, filamentous fungi stood out as efficient degraders of lignin owing to a synergistic action of redox proteins, secreted by the fungus, that perform an enzymatic combustion. These enzymes are mostly annotated within the “Auxiliary Activities” (AA) class defined in the Carbohydrate Active enZymes (CAZy) database 1 . AA encompass enzymes with different cofactors, such as heme (peroxidases, CAZy family AA2), copper (laccases, AA1) and many others. Attempts to simulate laccase-mediated lignin degradation in vitro showed that highly reactive radicals
produced by laccase oxidation are prone to repolymerization into higher molecular weight intermediates, that are even more recalcitrant to further enzymatic treatment. During in vivo degradation of lignin, fungi can prevent the massive repolymerization observed in vitro, leading to the hypothesis that they possess mechanisms to control or redirect the pool of radicals produced during oxidative attack of lignin. A few studies have pointed out the possible role of a few enzymes and their interplay in this event
Neural stem cells differentiated from human induced pluripotent stem cells (iPSCs) as a novel in vitro model to study developmental pathways in Huntington Disease
Full text linkHuntington Disease (HD) is an autosomal dominant disorder characterized by motor, cognitive and behavioral features caused by a CAG expansion in the HTT (huntingtin) gene beyond 35 repeats. Since the discovery of the HTT mutation 24 years ago, more than 15,000 papers have been published on HD. However, both the role of the huntingtin (wtHTT) in healthy individuals and the molecular mechanisms by which the mutated huntingtin causes the disease remain unclear. The discovery of induced pluripotent stem cell (iPSC) technology offer the possibility to generate patient-specific iPS cells and to enable the development of in vitro HD models that more accurately reflect the human disease. The disease-specific iPSCs can be differentiated into relevant cell-types affected in HD, holding a great potential for disease modeling and drug screening. In the present study, we have obtained dermal fibroblasts from 16 HD patients and 6 healthy controls. Fibroblasts from a young adult healthy control, a young subject with 43 CAG repeats, in an early stage of the disease and two JHD (Juvenile Huntington disease) patients were reprogrammed into induced pluripotent stem cells. All iPS clones that show an uniform flat morphology are characterized for their stemness and pluripotency, both in vitro through embryoid bodies formation and in vivo through teratoma formation assay. iPS cells from a JHD (85CAG) and healthy control, were differentiated into neurospheres of neural precursors (NPCs) by a new protocol optimized for differentiation of iPSCs derived embryoid bodies expressing all the three germ layers (ectoderm, mesoderm and endoderm) in neurospheres of Neural Stem Cells (NSCs). Differentiation assay confirmed that they possessed the potentiality, to differentiate into subtypes of neuronal and glial cells. Gene expression profiling was performed on iPSC HD-derived neurospheres using GeneChip Human Transcriptome Array 2.0. iPSC HD-derived neurospheres exhibit an impaired brain development processes when compared with iPSC-derived neurospheres from healthy individuals. This study aimed to produce a valid model of the Huntington disease. This novel in vitro model will permit us to have a closer view to neuronal development networks by morphologically and physiological studies on the cell types obtained from the neuropheres of NPC
Strategie, sistemi e sperimentazioni per architetture e città adattive. Strategie, sistemi, sperimentazioni per architetture e città adattive ai fenomeni di isola di calore e di ondate di calore, in regime di cambiamenti climatici e in un quadro di green economy
No full textI cambiamenti climatici e gli eventi meteorologici estremi sempre più frequenti che interessano il pianeta ormai da tempo, ci spingono a riflettere e a indagare su quanto le città riescano ad adattarsi a ciò che accade quotidianamente. Le aree urbane densamente costruite si trovano in una condizione aggravata da fenomeni quali l’effetto “isola di calore” e cambi di temperatura con picchi improvvisi, che comportano un aumento del discomfort ambientale in queste aree rispetto alle zone non urbanizzate. Tutto questo espone la popolazione a rischi considerevoli, soprattutto le fasce deboli, e genera vulnerabilità nell’”ecosistema urbano”.
La presente tesi di dottorato indaga tali fenomeni - ondate di calore e isole di calore urbano - e le possibili risposte che il mondo dell’architettura e della tecnologia possono fornire, in termini di adattività e flessibilità delle soluzioni, per il miglioramento della situazione a scala locale e urbana. In ambito europeo molti sono gli esempi di strategie e azioni messe in atto a tale scopo, tanto da consentire un’analisi sistemica e uno studio approfondito di un numero significativo di casi di studio. Questo ha portato alla costruzione di un set di indicatori quali-quantitativi che tengono conto della dimensione sociale e fisica, tramite i quali è stato possibile effettuare una valutazione delle azioni e degli interventi in oggetto e capire la loro vera efficacia contro gli effetti dei cambiamenti climatici. Scopo ultimo della tesi è quello di ottenere un quadro di analisi significativo, facilmente implementabile in futuro, attraverso cui costruire una matrice di efficacia delle azioni, che architetti, progettisti e pianificatori possano adottare come strumento progettuale già in fase decisionale e fino all’esecuzione.Climate changes and the extreme meteorological events are always more frequent, and they affected the planet since for some time; they inspire us to reflect and to investigate how cities can be adapted to what usually happens. The densely built urban areas are in an aggravated condition by phenomena such as the UHI (Urban Heat Island) and by the temperature changes with sudden peaks, that increase the environmental discomfort in these areas compared to the not-urbanized ones. All these circumstances expose the population to considerable risks, especially the vulnerable groups and produce vulnerability in the “urban ecosystem”.
This PHD thesis investigates these phenomena (heat waves and urban heat islands) and the possible answers that the architecture and the technology field can provide, in order to improve the situation at urban and local scale, in terms of adaptability and flexibility of the solutions. In the European context, there are many examples of strategies and actions applied for this aim, so much so to allow a systemic analysis and an in-depth study of a significant number of case study. This, led to the construction of a qualitative and quantitative indicators set that considers the social and physical dimension, through which it was possible to evaluate the actions and interventions in question; it also allowed to understand their true efficacy against the effects of the climate changes. The last thesis aim is to obtain a meaningful analysis framework - easily implementable in the future - which is possible to build an availability source, useful for architects, designers and planners in a decision-making phase and till the realization of it, as a design tool
Identification of a preferential substrate in glucose dehydrogenase from Pycnoporus cinnabarinus: a "sugar sweet" structural approach
Full text linkFungus-derived glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline (GMC) oxidoreductase superfamily. GDHs are the most popular enzymes used in glucose sensor strips manufactured for glycemic control by diabetic patients. These enzymes are classified in the “Auxiliary activities” of the Carbohydrate-Active Enzymes (CAZy) database, since they act together with laccases in the process of lignocellulose degradation. Among fungi, the Basidiomycota Pycnoporus cinnabarinus is able to completely degrade lignin. We solved the crystal structure of GDH from P. cinnabarinus in the ligand-free form and in complex with glucose. The glucose-bound structure revealed two glucose molecules in close proximity to the FAD cofactor. Their orientation guided us to investigate the activity of the enzyme towards the disaccharide laminaribiose. Structural data on the laminaribiose-bound GDH showed a well-defined orientation of the disaccharide in the active site. Remarkably, the functional analysis revealed that the preferential substrate of this enzyme is indeed laminaribiose, with a 90% higher activity compared to glucose. Since beta-1,3 glucans are products of lignocellulose degradation, our structure-function analysis is consistent with the hypothesis of a fine tuning of the gene regulation and expression to adapt fungal organisms to the diversity of their natural substrates
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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