Multiple pulmonary nodules presenting a difficult diagnostic challenge

We describe the case of a 56 years-old man with a subacute onset of symptoms mimicking a granulomatosis with polyangiitis. He was admitted to our hospital with acute respiratory failure requiring oxygen therapy, fever and crusted rhinitis. Despite initial improvement in radiological and clinical features with a steroids therapy, his condition worsened rapidly and he was re admitted to our department with ARDS. Despite antibiotic, antiviral and antifungal therapy, an endotracheal intubation was necessary and ultimately the patient passed away. Only a histological examination on autopsy had shown the presence of a diffuse Anaplastic Large Cell Lymphoma (ALCL), a rare type of non-Hodgkin lymphoma (NHL) originated from mature post-thymic T cells. It represents 1–3% of NHL. Different subtypes have been described: Kinase (ALK)-negative ALCL, ALK-positive ALCL and breast implantassociated ALCL. ALK-negative ALCL affects mainly old males and has the worst prognosis

The Opa1-Dependent Mitochondrial Cristae Remodeling Pathway Controls Atrophic, Apoptotic and Ischemic Tissue Damage

SummaryMitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo

Auxiliary selection: the change from early-middle to modern English and the comparison to Romance languages.

reservedL’elaborato affronta la tematica della selezione degli ausiliari in lingue romanze e germaniche. In particolare, lo scopo è quello di ripercorrere l’evoluzione da inglese antico-medio a moderno e operare un confronto con le principali lingue romanze. La prima sezione prevede un’introduzione al tema e un’attenzione particolare alla distinzione tra verbi intransitivi, considerando tre distinte classificazioni dei suddetti verbi, utili per comprendere la modalità attraverso cui avviene la scelta dell’ausiliare. Specificatamente, vengono analizzate le distinzioni proposte da Enciclopedia Treccani, alla voce curata da E. Jezek, il paper Gradients in Auxiliary Selection with Intransitive Verbs, Antonella Sorace, e On triple auxiliation in Romance, Michele Loporcaro. Il secondo capitolo è focalizzato sul fenomeno di cambiamento tra inglese antico-medio e moderno nella distinzione tra gli ausiliari have e be, esaminando i dati forniti dal paper Perfects, Resultatives, and Auxiliaries in Earlier English, Thomas McFadden; Artemis Alexiadou. Infine, nella terza ed ultima sezione, l’elaborato riprende più approfonditamente i dati analizzati da Sorace e propone un confronto tra inglese e lingue romanze.The paper deals with auxiliary selection in Romance and Germanic languages. In particular, the aim is to analyse the development from early-middle to modern English and compare it to Romance languages. The first section presents the topic and focuses on the distinction of intransitives verbs, taking into account three different classifications of these verbs, which are useful to deeply understand how the auxiliary is chosen. I will analyse the distinctions proposed by Enciclopedia Treccani by E. Jezek, the paper Gradients in Auxiliary Selection with Intransitive Verbs, Antonella Sorace, e On triple auxiliation in Romance, Michele Loporcaro. The second chapter is focused on the phenomenon of change from early-middle and modern English in the distinction between auxiliaries have and be, examining the data presented in the paper Perfects, Resultatives, and Auxiliaries in Earlier English, Thomas McFadden; Artemis Alexiadou. In the third section, the paper examines in depth Sorace’s data and proposes a comparison between English and Romance languages

FORMAZIONE DI COMPLESSI DELLA PROTEINA MITOCONDRIALE OPA 1 NEL MIOCARDIO ESPOSTO A STRESS OSSIDATIVO E CARATTERIZZAZIONE DI MUTANTI IN RESIDUI CISTEINICI

The heart is especially vulnerable to mitochondrial derangements. In fact, many cardiomyopathies are caused by anomalous mitochondrial metabolism or alterations of mitochondrial bioenergetics. The involvement of mitochondria in cardiac diseases is likely to be extended to structural changes, since mitochondrial activity and cellular signaling are tightly linked with mitochondrial morphology. A major example of this link is provided by the relationship between mitochondrial fragmentation and the progression of apoptosis. Among the proteins involved in mitochondrial dynamics, the dynamin-like GTPase Optic Atrophy 1 (OPA 1) is required for mitochondrial inner membrane fusion and maintenance of normal cristae structure. The research activity of this thesis was aimed at both investigating whether OPA 1 is altered in hearts subjected to ischemia and reperfusion and elucidating the relationships between OPA 1 changes and alterations in mitochondrial morphology. Due to the established role of mitochondria in generating reactive oxygen species, oxidative stress was investigated as a crucial mechanism altering OPA 1 structure and function.The susceptibility of OPA 1 to oxidation was initially characterized in intact hearts, cardiomyoblasts and isolated mitochondria. In isolated rat hearts perfused with hydrogen peroxide (H2O2) or subjected to ischemia and reperfusion OPA 1 was found to undergo the formation of high molecular weight (HMW) bands that were barely detectable in normoxic hearts. Since these HMW bands were only observed in electrophoreses carried out under non-reducing conditions, their formation reflected the oxidation of vicinal cysteinyl residues resulting in the generation of disulphide cross-bridges. In mitochondria isolated from heart perfused with H2O2, Blue Native-PAGE (BN-PAGE) analysis displayed the aggregation of OPA 1 in various complexes with high molecular weight (ranging from xx to xx). These complexes were not present in mitochondria isolated from normoxic rat hearts. Then OPA 1 complex formation was analyzed both in HL-1, a proliferating atrial cardiomyocites derived from mouse AT-1 cells, and in mouse embryonic fibroblasts (MEFs), as a model cell line. Oxidative stress caused by incubation with H2O2 resulted in the formation of OPA 1 complexes that disappeared under reducing conditions, confirming the presence of intermolecular cross-bridges. Notably, under the same experimental condition causing OPA 1 aggregation (i.e., incubation with increasing concentration of H2O2) a fragmentation of mitochondrial network was observed in HL-1 cells suggesting that oxidation of cysteine residues might hamper the ability of OPA 1 to favour mitochondrial fusion. This hypothesis was validated by identifying which residues of cysteine were involved in the formation of disulfides cross-bridges. Since NMR and crystallographic structure of OPA 1 is not available, 3D structure information were obtained by means of homology modeling. In a collaborative effort with Prof. S. Moro (Faculty of Pharmacy, University of Study of Padova), analyses were carried out aimed at identifying the cysteine residues exposed on the protein surface that is also the region involved in OPA 1 dimerization. As a result of this study, cysteines 853, 856 and 874 were indicated as the residues that more likely contribute to the formation of OPA 1 complexes. This hypothesis was verified by performing site-directed mutagenesis experiments on the expression construct containing wild-type Opa1 isoform 1. By substituting the native cysteine residues with serine. This approach allowed us obtaining the following mutants: (i) the double mutant in C853-6S; (ii) the single mutants in C853S or in C874S. New clones have been sequenced to ensure fidelity. Lentiviral vectors containing the mutated cysteine plasmids were produced in order to transfect OPA 1-/- MEF cell line (kindly given by Prof. L. Scorrano). Both C853S and C853-6S expressed detectable level of OPA 1, while cells containing OPA 1 mutated in the single residue C874S were not obtained. Cells harbouring the OPA 1 mutants were assessed for mitochondrial morphology. Under control conditions C853-6S cells displayed punctuate mitochondria that is similar to the mitochondrial phenotype displayed by OPA 1-/- MEF cells; conversely, the expression of the C853S mutant restored the fusion morphology. Cells were incubated with H2O2 in order to elucidate the relationship between OPA 1 aggregation and disulphide crossbridge formation. OPA 1 complex formation was inhibited in both cell lines. To investigate the correlation between OPA 1 aggregation and mitochondrial fission, C853S cells were treated with H2O2. In C853S mutants the time required for completing the fission process was doubled with respect of MEF wild type cells. In conclusion these findings highlight the relevance of cysteine 853 in maintaining the role of OPA 1 in mitochondrial fusion, and especially in the derangement of this function caused by oxidative stress. In fact, when cells expressing the mutant C853S were exposed to a severe oxidative stress, OPA 1 aggregation was prevented along with a significant delay in the occurrence of mitochondrial fission.Il cuore è particolarmente soggetto a disfunzioni mitocondriali e numerose cardiomiopatie sono associate ad un anomalo metabolismo mitocondriale o ad alterazioni bioenergetiche mitocondriali. Studi recenti hanno messo in evidenza come l’attività dei mitocondri e le vie di trasduzione del segnale cellulare influenzino la morfologia dei mitocondri, ad esempio, la frammentazione della rete mitocondriale è correlata al processo di apoptosi. La proteina OPA 1, GTPasi appartenente alla famiglia delle dinamine, è coinvolta nel processo di fusione della membrana interna mitocondriale e nel mantenimento della struttura delle cristae interne mitocondriali. Tuttavia sono necessari maggiori studi atti a contribuire ad una maggiore comprensione delle proteine e dei meccanismi alla base delle funzionalità di OPA 1. Nel presente lavoro di tesi è stato studiato l’effetto dello stress ossidativo sulla proteina mitocondriale OPA 1 nel cuore, dato che i mitocondri sono sede di produzione e bersaglio delle specie reattive dell’ossigeno (ROS). Inizialmente, utilizzando cuori di ratto isolati e perfusi ex-vivo, è stato valutato l’effetto di ischemia/riperfusione e perfusione con perossido di idrogeno (H2O2) sulla proteina OPA 1. Nei campioni trattati è stata osservata la formazione di complessi ad alto peso molecolare (high molecular weight, HMW) della proteina in esame. In condizioni riducenti, tali complessi vengono disgregati. Questi risultati indicano che in condizioni di forte stress ossidativo con H2O2 si ha l’aggregazione di OPA 1 e che nella formazione dell’aggregato è coinvolta la formazione di ponti disolfuro. In seguito, mediante Blue Native-PAGE (BN-PAGE), è stata eseguita un’analisi dei complessi proteici a partire da mitocondri isolati da cuore di ratto in condizioni normossiche e da cuori perfusi con H2O2. L’analisi ha dimostrato che OPA 1 aggrega in complessi di diverso peso molecolare in condizioni native. La formazione del complesso di OPA 1 è stata analizzata sia cellule HL-1, una linea di cardiomiociti da atrioma murino, che in fibroblasti embrionali murini (mouse embryonic fibroblasts, MEFs). Al fine di indurre stress ossidativo le cellule sono state incubate con 1 mM H2O2 per 2 ore. Il complesso di OPA 1 scompare in condizioni riducenti, confermando la formazione di ponti disolfuro intermolecolari. Inoltre, in cellule HL-1 incubate con concentrazioni crescenti di H2O2 è stata osservata frammentazione della rete mitocondriale. Ci siamo proposti di individuare quali fossero i residui cisteinici coinvolti nella formazione del ponte disolfuro. La struttura della proteina OPA 1 non è stata determinata né mediante NMR né mediante tecnica cristallografica. In collaborazione con il Prof. S. Moro (Facoltà di Farmacia, Università degli Studi di Padova) è stata ipotizzata la struttura 3D della proteina mediante homology modelling al fine di caratterizzare quali fossero i residui cisteinici esposti sulla superficie proteica. In questo modo le cisteine 853, 856 e 874 sono stati individuati come residui più probabilmente coinvolti nella formazione del complesso di OPA 1. Per confermare questa ipotesi, sono stati eseguiti degli esperimenti di mutagenesi sito-diretta a partire da un costrutto plasmidico contenente la sequenza genica per l’isoforma 1 umana del gene OpaI. Mediante questa tecnica sono stati ottenuti plasmidi mutati a livello delle singole cisteine 853 (C853S) e 874 (C874S), ed un doppio mutante cisteina 853-6 (C853-6S). I cloni sono stati sequenziati per confermare le avvenute mutazioni. Sono stati prodotti vettori lentivirali contenenti i plasmidi mutagenizzati al fine di esprimere stabilmente la proteina di interesse nella linea cellulare MEF OPA 1-/- (gentilmente fornita dal Prof. L. Scorrano). Sono state ottenute linee cellulari esprimenti la proteina OPA 1 mutata a livello del singolo residuo cisteinico 853 e dei residui 853-6, mentre non è stata ottenuta la linea esprimente la proteina mutata in C874S. È stata quindi valutata la morfologia mitocondriale nelle linee cellulari ottenute. Le cellule C853-6S presentano mitocondri disgregati con un fenotipo simile alla linea MEF OPA 1-/-; al contrario, nelle cellule esprimenti la proteina OPA 1 mutata a livello del residuo 853, si verifica il ripristino del fenotipo di fusione mitocondriale. Al fine di chiarire la relazione tra l’aggregazione di OPA 1 e la formazione dei ponti disolfuro, le cellule sono state incubate con H2O2. La formazione del complesso di OPA 1 risulta inibita in ambedue le linee cellulari contenenti la proteina mutata. Le cellule C853S sono state trattate con H2O2 per verificare la correlazione tra l’aggregazione della proteina in esame e il processo di fissione mitocondriale. I cloni C853S completano il processo di fissione mitocondriale in un tempo doppio rispetto alla linea MEF WT. Per concludere, in condizioni di elevato stress ossidativo la proteina OPA 1 è esposta ad ossidazione che ne modifica lo stato di aggregazione. Tale processo è correlato ad una diminuzione della fusione mitocondriale e, nelle cellule HL-1, ad un aumentato rilascio di citocromo c. Il meccanismo di aggregazione della proteina è stato stabilito mediante mutagenesi sito specifica che mette in luce il ruolo del residuo cisteinico 853

Indicators for managing and planning urban green spaces: a case study in Padova (Northern Italy)

Sustainable and integrated management of urban green spaces is fundamental for the planning of our cities and towns. Therefore, understanding current conditions and informing citizens on urban green spaces is critical for such sustainable and integrated view. Furthermore, this knowledge is essential to appreciate existing and potential ecosystem services and disservices derived from urban trees. Here we present the usage of simple indicators to analyze and present current conditions of urban green spaces and their management. The final aim is to highlight possible scenarios and models for future planning and management. A subarea of Padova (Northern Italy), the basso Isonzo, was used as a test area and as part of the Urban Green Belts (UGB), an Interreg Central Europe project aiming to \u201csmart integrated models for sustainable management of urban green spaces for creating healthier and liveable urban environments\u201d. A number of indicators have been used, among which the share of green space, number of public trees per inhabitant, number of tree species, and age distribution of trees. Results enabled to represent GIS based solutions with reference to inventory information, types of urban green spaces, and supply of different functions. Richness and composition in tree species in different urban areas highlighted diverse conditions and can be used to detect possible different influences on related ecosystem services or disservices. Future management and planning options are proposed aiming to sustainable and integrated green spaces. For example, management efforts should be concentrated on those urban areas with un-balanced diameter distributions with a particular attention towards lack of small diameters, indicating a need of increasing tree planting, or large diameter, indicating a difficulty in growing trees to maturity

A Novel Benchtop Device for Efficient and Simple Purification of Cytokines, Growth Factors and Stem Cells from Adipose Tissue

Lipoaspirates represent a source of adult stem cells, cytokines, and growth factors of adipocyte origin with immunomodulation and regenerative medicine potential. However, rapid and simple protocols for their purification using self-contained devices that can be deployed at the points of care are lacking. Here, we characterize and benchmark a straightforward mechanical dissociation procedure to collect mesenchymal stem cells (MSCs) and soluble fractions from lipoaspirates. IStemRewind, a benchtop self-contained cell purification device, allowed a one-procedure purification of cells and soluble material from lipoaspirates with minimal manipulation. The recovered cellular fraction contained CD73+, CD90+, CD105+, CD10+ and CD13+ MSCs. These markers were comparably expressed on MSCs isolated using IstemRewind or classic enzymatic dissociation procedures, apart from CD73+ MSCs, which were even more abundant in IStemRewind isolates. IstemRewind-purified MSCs retained viability and differentiation into adipocytes and osteocytes, even after a freezing-thawing cycle. Levels of IL4, IL10, bFGF and VEGF were higher compared to the pro-inflammatory cytokines TNFα, IL1β and IL6 in the IStemRewind-isolated liquid fraction. In sum, IStemRewind can be useful for straightforward, rapid, and efficient isolation of MSCs and immunomodulatory soluble factors from lipoaspirates, opening the possibility to directly isolate and employ them at the point-of-care

Mitochondrial injury and protection in ischemic pre- and postconditioning

: Mitochondrial damage is a determining factor in causing loss of cardiomyocyte function and viability, yet a mild degree of mitochondrial dysfunction appears to underlie cardioprotection against injury caused by postischemic reperfusion. This review is focused on two major mechanisms of mitochondrial dysfunction, namely, oxidative stress and opening of the mitochondrial permeability transition pore. The formation of reactive oxygen species in mitochondria will be analyzed with regard to factors controlling mitochondrial permeability transition pore opening. Finally, these mitochondrial processes are analyzed with respect to cardioprotection afforded by ischemic pre- and postconditioning

Differences Between Early- and Late-Onset Asthma: Role of Comorbidities in Symptom Control

Asthma can present in early childhood or de novo in adulthood. Our understanding of the burden of comorbidities in adult asthmatic patients stratified by age at onset is incomplete