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CARATTERIZZAZIONE DI NUOVE PROPRIETA’ STRUTTURALI DELLA SUBUNITA' UMANA OSCP DI ATP SINTASI E ANALISI BIOFISICA DELLE SUE INTERAZIONI CON L’INIBITORE MITOCONDRIALE IF1-1 E CON LA CICLOFILINA D, CORRELATE A SITUAZIONI FISIOPATOLOGICHE CONNESSE CON LA REGOLAZIONE DEL m-PTP COME L'INVECCHIAMENTO, IL CANCRO E LA MALATTIA DI ALZHEIMER
No full textIn questa tesi di dottorato, viene presentata, per la prima volta, una caratterizzazione a livello molecolare del dominio C-terminale della proteina umana OSCP in soluzione, tramite l’impiego sinergico di diverse tecniche strutturali avanzate come l’NMR, il Bio-SAXS e l’ITC. Successivamente, grazie alle informazioni raccolte anche per il dominio umano N-terminale, è stato possibile porre le basi per una prima indagine biofisica dell’intera subunità in soluzione, comprendendone nuove ed inaspettate proprietà strutturali che aiuteranno a fare luce sugli emergenti ruoli terapeutici di OSCP, anche in qualità di subunità regolatrice del m-PTP. Le nuove informazioni raccolte su OSCP hanno poi permesso di effettuare i primi studi a livello molecolare su due processi di interazione proteina-proteina connessi con la regolazione del m-PTP e con le patologie cellulari ad esso correlate. Il primo riguarda un nuovo legame fra OSCP e la prima isoforma dell’inibitore mitocondriale dell’ATP sintasi IF1-1, ad oggi noto solamente per l’azione inibitrice dell’attività idrolasica del complesso V in condizioni ipossiche, attraverso il legame con la porzione catalitica F1. La nuova interazione tra OSCP e IF1-1 è stata trovata in alcuni modelli cellulari tumorali, nei quali l’inibitore risulta essere particolarmente sovra-espresso, in condizioni in cui l’enzima sintetizza ATP (fosforilazione ossidativa). Questo nuovo legame non influenza l’attività enzimatica dell’ATP sintasi ma inibisce l’apoptosi cellulare, nello specifico attraverso un silenziamento del m-PTP, aiutando così la cellula tumorale a raggiungere una condizione di apoptosi-resistenza e immortalità. Il secondo processo, invece, coinvolge la nota interazione fra OSCP e CypD, finora mai studiata a livello molecolare. La nostra indagine ha permesso di portare alla luce nuove, seppur preliminari, informazioni riguardo il meccanismo strutturale di questo legame proteina-proteina che resta, ad oggi, uno dei pochi processi molecolari riconosciuti dal mondo scientifico come direttamente coinvolto nell’apertura del m-PTP.
Viene inoltre riportata la caratterizzazione strutturale dell’interazione fra la chinasi umana CK2α (subunità catalitica) e un nuovo poliossometallato a base di rutenio. Attraverso i risultati sperimentali ottenuti, è stato proposto un interessante meccanismo di inibizione dell’enzima, la cui attività catalitica è particolarmente importante per i meccanismi di proliferazione sfruttati delle cellule tumorali.In this PhD work, it is provided the first characterization at a molecular level of the C-terminal domain of the human OSCP in solution, by a synergic use of different and advanced structural techniques as NMR, Bio-SAXS and ITC. Thanks to the information collected also for the human N-terminal domain, it has been possible to perform a preliminary investigation of the whole subunit in solution, understanding new and unexpected structural proprieties that will help to elucidate the emerging therapeutic roles of the OSCP, even as a regulatory subunit of the m-PTP. The new information about the OSCP allowed us to study for the first time two protein-protein interaction processes at a molecular level, which are involved in the m-PTP regulation and its related pathologies. The first one is the novel binding event between the OSCP and the first isoform of the ATPase inhibitor IF1-1 that is known for its inhibitory action against the complex V hydrolytic activity in anoxia, through the binding within the F1 catalytic module. The new interaction between the OSCP and IF1-1 was detected in some tumor cell models, where the inhibitor is overexpressed, during mitochondrial respiration and ATP synthesis (oxidative phosphorylation). This novel binding event does not affect the ATP synthase catalysis but it prevents the apoptosis, specifically through the m-PTP desensitization, helping in this way the cancer cell to achieve an apoptosis-resistant phenotype and immortality. The second binding process regards the well known interaction between the OSCP and CypD which has never been studied at a molecular level. Our investigation allowed to elucidate new, despite preliminary, aspects about the structural mechanisms of this protein-protein binding event that remains, nowadays, one of the few molecular processes accepted form the scientific community as directly involved into the m-PTP sensitization.
It is also reported, as a side project, the structural characterization of the interaction between human protein kinase CK2α (catalytic subunit) and a novel ruthenium-based polyoxometalate, which provides an interesting inhibition mechanism against the cancer-related CK2, at a molecular level
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Get PDFWe 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
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
HT-SuMD: making molecular dynamics simulations suitable for fragment-based screening. A comparative study with NMR
Get PDFFragment-based lead discovery (FBLD) is one of the most efficient methods to develop new drugs. We present here a new computational protocol called High-Throughput Supervised Molecular Dynamics (HT-SuMD), which makes it possible to automatically screen up to thousands of fragments, representing therefore a new valuable resource to prioritise fragments in FBLD campaigns. The protocol was applied to Bcl-XL, an oncological protein target involved in the regulation of apoptosis through protein-protein interactions. Initially, HT-SuMD performances were validated against a robust NMR-based screening, using the same set of 100 fragments. These independent results showed a remarkable agreement between the two methods. Then, a virtual screening on a larger library of additional 300 fragments was carried out and the best hits were validated by NMR. Remarkably, all the in silico selected fragments were confirmed as Bcl-XL binders. This represents, to date, the largest computational fragments screening entirely based on MD
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
The OSCP Subunit of ATP Synthase is a Dimer in Solution: Strategy to Induce the Monomeric Protein as a New Tool for Drug Discovery
No full textThe F-ATP synthase subunit Oligomycin Sensitivity Conferral Protein (OSCP) contributes to the structural and functional coupling between the F1 catalytic and the FO transmembrane domains of the synthase. A body of literature suggests that OSCP is also a potential pharmacological target, as it mediates interactions with both proteins and small molecules capable of influencing ATP synthase activity and mitochondrial Permeability Transition (PT). However, the possibility of further exploring this aspect has been so far hindered by the poor properties of the isolated protein in solution, as reported by previous studies. Here, we use an integrated approach based on Nuclear Magnetic Resonance (NMR), Small Angle X-ray Scattering (SAXS) and Mass Spectrometry under native conditions (nMS) to demonstrate that the isolated OSCP subunit is not affected by nonspecific aggregation, but it is involved in a dimerization equilibrium. By the analysis of the anchoring region between the OSCP subunit and the b subunit of the ATP synthase, we identified a peptide mimicking the sequence of the C-terminal helix of the b subunit (b-CT) and capable to interfere with the OSCP dimerization equilibrium, stabilizing the monomeric state of the protein. This study offers valuable insights on the structural properties of the isolated OSCP subunit in solution, opening new avenues for future pharmacological studies and a more thorough comprehension of the emerging therapeutic potential of this protein
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