Teses / dissertações sobre o tema "153.9/3/09"

Introduction: Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by an aberrant interstitial deposition of collagen in the lung parenchyma, leading to progressive lung function impairment. Repeated injuries to the alveolar epithelium are believed to lead to an imbalance of the extracellular matrix (ECM) turnover supported by metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The kinase GSK-3 is a mediator of the cytokine homeostasis and favors the production of pro-inflammatory mediators. Its specific inhibitor, SB216763, displayed anti-inflammatory and anti-fibrotic properties of in a mouse model of Bleomycine (BLM)-induced pulmonary fibrosis. Furthermore, GSK-3 seems to be involved in the epithelial to mesenchimal transition (EMT). The purpose of this study was to assess the expression of MMP9, TIMP1, MMP2 and and TIMP2 in the early inflammatory response and in the late fibrosis induced by BLM in a mouse model of pulmonary fibrosis. Next goal was to elucidate whether the in vivo treatment with SB216763 could modulate MMPs activity and their balance with TIMPs. Moreover, in vitro experiments were aimed to identify the signal pathways through which GSK-3 can modulate MMPs and TIMPs. Methods: Cohorts of C57BL6 mice (12 weeks old) were randomized to receive intratracheal instillation of either saline (PBS), or BLM alone, or BLM in association with SB216763. The inhibitor was administered intraperitoneally the day after BLM and every 48 hours up to the sacrifice on day 7 (inflammatory phase) or 28 (fibrotic phase), when bronchoalveolar lavage (BAL) was performed and lungs harvested. MMP9 and MMP2 activity was measured by gel zymography in BAL fluid (BALF). MMPs and TIMPs expression levels were evaluated by Real Time-PCR and Western Blotting. Their tissue localization was evaluated by IHC analysis. In vitro experiments were performed using A549 cells as epithelial model, assessing expression and activity of MMPs downstream of TNFα, with and without GSK-3 silencing by siRNA. Results: MMP9 and MMP2 levels were elevated in BALF from BLM-treated mice at day 7. At this time point, the inflammatory cells recruited in the intra-alveolar spaces were alveolar macrophages (AMs), lymphocytes and neutrophils. Furthermore, the lung IHC staining indicated a strong positivity for MMP9, MMP2, TIMP1 and TIMP2 in interstitial AM (iAM) and, specifically, in injured and cuboidalized epithelium. BLM-treated mice sacrificed at day 28 presented high levels of MMP2 and TIMP1 and low levels of MMP9 in the BALF. Interestingly, in vivo GSK-3 inhibition reduced MMP9, MMP2 and TIMP1 levels in the BALF and their positivity in iAMs and in epithelium. Moreover, in vitro experiments performed with A549 cells, as epithelial model, demonstrated that GSK-3α and GSK-3β silencing increased MMP9 expression after stimulation with the pro-inflammatory cytokine TNFα. This effect was achieved by the interaction of GSK-3 with NF-kB and ERK signalling transduction pathway. Conclusions: Our mouse model of BLM-induced pulmonary fibrosis clearly showed an imbalance between MMPs and TIMPs during inflammation and fibrosis. Importantly, we proposed GSK-3 as a crucial mediator of their expression in AMs and in damaged epithelium. Next, in vitro results with A549 cells suggested that GSK-3 silencing induce MMP-9 secretion, downstream of TNFα, in a p65-mediated and ERK1,2 dependent manner.
Introduzione: La fibrosi polmonare idiopatica (IPF) è un'interstiziopatia caratterizzata da un decorso fatalmente progressivo, che conduce alla completa compromissione della funzione polmonare. Pur essendo la sua patogenesi non pienamente compresa, il primum movens sembra essere un danno epiteliale ripetuto che induce l'aberrante deposizione di matrice extracellulare (ECM), il cui turnover non viene più garantito da metalloproteasi (MMPs) e loro inibitori tissutali (TIMPs). La chinasi GSK-3 è nota per il suo ruolo cardine nella modulazione di citochine pro-infiammatorie ed in diverse vie del segnale e processi cellulari verosimilmente coinvolti nella patogenesi della IPF. La sua inibizione in vivo, in un modello murino di fibrosi polmonare indotta da bleomicina (BLM), si è dimostrata in grado di modulare sia la risposta infiammatoria precoce che la fase fibrotica tardiva. Il ruolo della chinasi nella modulazione dell'equilibrio fra MMPs e TIMPs nella fibrogenesi polmonare non è ad oggi chiarito. Metodi: Diverse coorti di topi C57BL6 sono state sottoposte all'instillazione endotracheale di BLM o PBS e trattate o meno, per via intraperitoneale, con l'inibitore di GSK-3 SB216763. Per misurare attività, espressione proteica e genica delle MMPs sono stati utilizzati, rispettivamente, zimografia, WB analysis e RT-PCR del liquido di lavaggio bronco alveolare (BALF) di topi sacrificati a 7 o 28 giorni dalla somministrazione di BLM. L'espressione proteica e genica delle TIMPs è stata valutata con WB e RT-PCR. E' stata inoltre eseguita l'analisi istologica ed immunoistochimica dei polmoni, ai due intervalli di tempo. L'effetto in vitro del silenziamento di GSK-3 è stato infine studiato su una linea di cellule epiteliali alveolari umane (A549). Risultati: Il trattamento con BLM ha dimostrato di aumentare la cellularità ed i livelli di MMP2 e MMP9 nel BALF al giorno +7. Si è inoltre registrato un significativo aumento dell'espressione di MMP2, MMP9, TIMP1 e TIMP2, all'analisi immunoistochimica, a livello di macrofagi alveolari (iAMs) e cellule epiteliali alveolari cuboidalizzate (CEACs). I topi sacrificati al giorno +28 hanno mostrato un incremento dei livelli di MMP2 e TIMP1, ma non di MMP9, nel BALF. L'inibizione in vivo di GSK-3 mediante SB216763 si è dimostra in grado di ridurre il reclutamento di cellule infiammatorie e la secrezione di MMPs al giorno +7, a livello del BALF; ha inoltre ridotto i livelli di MMP2 al giorno +28. Anche l'espressione di MMPs e TIMPs è stata modulata dall'inibizione di GSK-3, a livello di iAMs e CEACs, ad entrambi gli intervalli di tempo. Gli esperimenti in vitro su A549 hanno invece dimostrato che il silenziamento di GSK-3 è in grado di potenziare la secrezione di MMP9 indotta dallo stimolo proinflammatorio con TNFa, che attiva la via di NF-kB. Conclusioni: Il nostro lavoro dimostra un ruolo di GSK-3 nella modulazione dell'espressione di MMPs e TIMPs in un modello murino di fibrosi polmonare indotta da BLM, che interessa sia la fase infiammatoria precoce che quella fibrotica tardiva. L'effetto dell'inibizione di GSK-3 sembra esplicarsi, in particolare, a livello di iAMs e CEACs. I dati in vitro, pur preliminari, indicano un effetto opposto del silenziamento di GSK-3 sulla secrezione di MMP9 in una linea di cellule epiteliali alveolari (A549); il silenziamento di GSK-3 potenzia l’effetto di TNFα sulla secrezione di MMP-9, tramite un meccanismo che coinvolge p65 ed è ERK1,2-dipendente

The identification of new molecular insights always drove the research. Data from genetic, molecular biology and biochemistry suggest new directions, open new fields and lead to new discoveries. The significance of these data is, in certain situation, difficult to envision. In this view, physiology could represent one of the possible read-out of the overall complex modifications inside the cell. In particular, electrophysiological analysis shed light on both physiological and pathological conditions in excitable and non-excitable cells. In excitable cell, ion channels, cellular microenvironment, transcription factors and accessory proteins shape the electric profile of the cell. In my PhD, I mainly used this approach to test the effect of mutations associated with arrhythmic diseases (in both cardiac arrhythmia and epilepsy) and of physiopathological remodeling in response to endurance training. In particular, I performed the following projects concerning: - Characterization of the biophysical properties of the hHCN1 L157V mutation found in a patient affected by idiopathic generalized epilepsy. This mutation resulted to decrease the current density and leading to an increased excitability in single neonatal rat cortical neurons. - Analysis of the cardiac endurance training-associated microRNAs (miRNAs) in a trained mouse model and of the role of the muscle-specific miRNAs in modulating membrane excitability in the neonatal rat ventricular cardiomyocytes. These results highlights new miRNAs potentially involved in the cardiac electrical remodeling associated with endurance training. - Characterization of the impact of the T78M cav-3 variant found in a cohort of arrhythmic patients. This variant induces modification of several ionic currents leading to a pro-arrhythmogenic profile. The leitmotiv of these projects is the identification of the causes underlying the pathophysiological modification of excitable cells by ion channels, membrane proteins and post-transcriptional molecules.

The nutritional role of dietary fatty acids is quite controversial and often the object of mediatic campaigns either as panacea against all diseases or detrimental for human health. The most recent data are instead showing that it should be considered, in an optimal dietary regimen, the balance among all dietary fatty acids based on the current physiological needs, applying what it is currently known as personalised nutrition. It has been paid particular attention to the ratio between highly polyunsaturated fatty acids (HPUFAs) n-6, mainly arachidonic acid (ARA, 20:4n-6;) and n-3 HPUFAs, mainly eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) in tissues, since an inverse correlation between circulating n-3 HPUFAs and several chronic pathological states has been observed, possibly mediated by modulation of the endocannabinoid system (ECS). However, to assess this balance and controlling it by dietary means remains a great challenge for nutritionists. In addition, other nutritional factors may influence their tissue concentration. It is therefore compelling to individuate the mechanism(s) of action and what are the most efficient nutritional conditions to maximize n-3 HPUFAs health-related beneficial activities. The aim of this thesis was to evaluate the effects of some nutritional factors, such as conjugated linoleic acid (CLA), naturally occurring in the diet or produced by the probiotic Bifidobacterium breve (B. breve), in influencing n-3 PUFA metabolism to optimize the n-3 HUFA score (a biomarker of n-3 status in tissues) and possible interactions with the ECS in rodents and humans. We evaluated whether CLA, from different dietary sources, was able to enhance DHA biosynthesis, from its precursor alpha linolenic (ALA), in experimental models and humans. We found that different dietary ALA/CLA ratios affected n-3 PUFA and EC profile with a graded decrease of ALA and EPA and corresponding increase of DHA, while the EC arachidonoylethanolamine (AEA) decreased parallel to ratio ALA/CLA, whereas dietary ALA, in absence of CLA, was not able to increase significantly circulating DHA levels. We also evaluated whether supplementation with B. Breve as probiotic, along with ALA, in mice, as a nutritional factor that may increase dietary CLA, modulated n-3 fatty acid metabolism. We found that dietary probiotics increased DHA biosynthesis in liver and epididymal adipose tissue. Actually, we showed an increased of DHA biosynthesis in liver, but not an increase of CLA, while the ratio CD 16:2/CLA, a biomarker of peroxisomal beta oxidation, increased significantly but not associated to a higher PPAR α gene expression. The observed parameters suggested that these effects may be related to a pro-inflammatory event possibly triggered by the activation of toll like receptors by B. breve. In fact, in an ancillary experiment on rats challenged with LPS, we obtained similar results on fatty acid metabolism. In addition, dietary probiotics decreased AEA and its congener palmitoylethanolamine (PEA) levels in liver, while in adipose tissue we found a significant increased levels of AEA, PEA and the other AEA congener oleoylethanolamide (OEA). Our studies strongly suggest that background diet may play an important role in modulating fatty acid metabolism and in particular in modulating n-3/n-6 fatty acid balance, thereby differently affecting the ECS. Therefore, the synergistic effects of different nutritional factors, as it occurs in daily life in humans are somewhat difficult to predict and may explain the contradictory results in the literature on the effects of dietary fatty acids, in particular when they are singularly considered without taking into account the whole dietary regimen. The discover that gut microbiota may directly interfere and modify fatty acid metabolism opens to novel nutritional strategies in shaping the optimal milieu for contrasting several metabolic disorders by modulating the ECS. Future studies in humans will be focus on evaluating possible synergistic effects of CLA-enriched products with B. breve in maintaining and/or re-equilibrating energy and lipid metabolism, by modulating EC and congeners tissue profile, acting on energy metabolism and EPA and DHA related molecules, and also to assess if there is an optimal level of dietary EPA and DHA to allow a synergistic activity in different physio-pathological conditions.

Parkinson’s disease is the second most common neurodegenerative disorder after Alzheimer’s disease and affects about 1% of the population over 65 years old. This disorder can be both sporadic and familial and some genetic forms are due to mutations in SNCA gene, encoding for the protein alpha-synuclein (aS). PD pathological hallmarks are the prominent death of the dopaminergic neurons in the substantia nigra pars compacta and the presence of proteins and lipid inclusions, termed Lewy’s body (LBs), in the surviving neurons in parkinsonian brains. The main constituent of LBs is an aggregated fibrillar beta-sheet rich form of aS. aS aggregation process was widely studied in the past years: the protein is unfolded in its native state, but in pathological conditions it tends to aggregate forming oligomeric species. These oligomers constitute a heterogeneous and transient ensemble and rapidly convert into amyloid fibrils when they reach a critical concentration. Amyloid fibrils then deposit in LBs along with several other proteins and lipids. aS aggregation was mainly studied in vitro, but recently more efforts were put into the study of this process in cell and animal models, to identify not only aS aggregation intermediates, but also the associated toxic mechanism(s) that lead to neurons cell death in PD. In this thesis two main issues were faced: the study of aS aggregation in cells using unconventional methods and the characterization of the effects of the family of chaperone-like proteins 14-3-3, on aS aggregation. In the first part, two cellular models for the study of aS aggregation were set and characterized: the first one is obtained just overexpressing aS and allowed the characterization of an ensemble of heterogeneous oligomeric species (about 6±4 monomers per oligomer) using a new fluorescence microscopy method termed Number and Brightness analysis. These oligomeric species induced autophagic lysosomal pathway activation and mitochondrial fragmentation in this model. The second cellular model provides a method to study aS fibrils and larger aggregates in a physiological environment: aS was overexpressed in cells and aggregation was triggered by introducing in cell cytoplasm recombinant aS fibrils fragments, termed seeds. In both cases aS overexpression and aggregation cause cellular death, in good agreement with what was previously published by others groups. The characterization of aS aggregation in cells went further looking at the variation in cellular metabolism, possibly induced by mitochondrial damage. These changes were quantified measuring NADH fluorescence properties in the two models with respect to the control. These results showed that in cells presenting aS oligomer or aggregates, NADH fluorescence lifetime and emission spectra change, suggesting that these measurements may be used to detect aS aggregates in live cells and in vivo using a non-invasive dye-free method. The second part of the thesis concerns the ability of 14-3-3 chaperone-like proteins of interacting with aS and of interfering with aS aggregation process rescuing the induced toxicity in cells. Among the seven 14-3-3 isoforms, 14-3-3 eta can re-route aS amyloidogenic process in vitro, leading to the formation of curved objects rather than aS fibrils. These curved objects have diameters and curvatures that depend on 14-3-3 eta amount in the aggregation assays; moreover, 14-3-3 eta molecules were found in these aggregates, suggesting the formation of a stable complex between the two proteins. When aS amount is too large or seeds are used to trigger the aggregation process in vitro, 14-3-3 eta is not able any more to affect aS aggregation and is sequestered into aS fibrils. In cell models, 14-3-3 eta overexpression leads to a rescue when aS was only overexpressed, but not when aggregation in cell cytoplasm was triggered by seeds. Overexpressed 14-3-3 eta was found to interact with overexpressed aS using image correlation spectroscopy methods (cross raster image correlation spectroscopy and cross Number and Brightness analysis), mainly at plasma membrane. Moreover, 14-3-3 eta is sequestered into aggregates when aS aggregation is triggered by seeds, highlighting another possible toxic mechanism due to aS aggregation. All the results obtained in cells are in good agreement with the in vitro results previously reported, further suggesting that 14-3-3 proteins and eta isoform in particular are interesting in aS aggregation frame and may be used to interfere in the process to rescue its toxic effects.
La malattina di Parkinson è la seconda malattia neurodegenerative più comune dopo il morbo di Alzheimer e colpisce circa l’1% delle popolazione sopra i 65 anni di età. Questa malattia può essere sia sporadica che familiare e alcune forme genetiche sono dovute a mutazioni nel gene SNCA che codifica per la proteina alfa-sinucleina. Le caratteristiche patologiche principali della malattia di Parkinson sono la morte prevalentemente dei neuroni dopaminergici della substantia nigra pars compacta e la presentza di inclusioni proteiche e lipidiche, dette corpi di Lewy, nei neuroni che sopravvivono nei cervelli dei pazienti affetti dalla malattia. Il componente principale dei corpi di Lewy è una forma di alfa-sinucleina aggregata, fibrillare e ricca di foglietti beta. Il processo di aggregazione di alfa-sinucleina è stato ampiamente studiato negli anni passati: la proteina è non strutturata nella sua forma nativa, ma in condizioni patologiche tende ad aggregare formando specie oligomeriche. Questi oligomeri costituiscono un insieme etereogeneo e transiente e si convertono rapidamente in fibrille amiloidi quando raggiungono una concentrazione critica. Le fibrille amiloidi di alfa-sinucleina si depositano poi nei corpi di Lewy assieme ad altre proteine e lipidi. L’aggregazione di alfa-sinucleina è stata principalmente studiata in vitro, anche se più recentemente maggiori sforzi sono stati effettuati per caratterizzare il processo in modelli cellulari ed animali, per identificare non soltanto i diversi prodotti dell’aggregazione, ma anche i meccanismi tossici ad essi associati, che causano la morte dei neuroni nei pazienti affetti dalla malattia di Parkinson. In questa tesi due questioni principali sono state affrontate: lo studio dell’aggregazione di alfa-sinucleina in cellule utilizzando metodi non convenzionali di microscopia in fluorescenza e la caratterizzazione degli effetti di una famiglia di proteine chaperoniche, le 14-3-3, sul processo di aggregazione. Nella prima parte, due modelli cellulari per lo studio dell’aggregazione di alfa-sinucleina sono stati approntati e caratterizzati: il primo viene ottenuto sovraesprimento soltanto alfa-sinucleina e ha permesso la caratterizzazione di un ensemble di oligomeri eterogenei in cellule vive (circa 6±4 monomeri per oligomero) utilizzando un nuovo metodo di microscopia in fluorescenza chiamato Number and Brightness analysis. Queste specie oligomeriche inducono l’attivazione del sistema autofagico-lisosomiale e la frammentazione dei mitocondri in questo modello cellulare. Il secondo modello cellulare fornisce un metodo per lo studio delle fibrille di alfa-sinucleina e di aggregati più grandi in un ambiente di rilevanza fisiologica: alfa-sinucleina è stata sovrespressa in cellule e l’aggregazione è stata promossa introducendo nel citoplasma delle cellule frammenti di fibrille ottenute da alfa-sinucleina ricombinante, detti seeds. In entrambi i casi la sovraespressione e l’aggregazione di alfa-sinucleina hanno causato morte cellulare, in buon accordo con quello che è stato riportato in precedenza da altri gruppi di ricerca. La caratterizzazione dell’aggregazione di alfa-sinucleina in cellule è continuata osservando la variazione nel metabolismo cellulare, potenzialmente indotta da danni ai mitocondri. Queste variazione sono state quantificate misurando le proprietà della fluorescenza del NADH nei due modelli, rispetto al controllo. Questi risultati hanno mostrato che in cellule che presentano oligomeri o aggregati di alfa-sinucleina, il tempo di vita della fluorescenza del NADH e il suo spettro di emissione cambiano. Quindi, queste misure potrebbero essere ottimizzare per rilevare la presenza di aggregati di alfa-sinucleina in cellule e in vivo, utilizzando un metodo di indagine non invasivo e dye-free. La seconda parte della tesi riguarda l’abilità delle proteine chaperoniche 14-3-3 di interagire con alfa-sinucleina e di interferire con il suo processo di aggregazione, riducendone la tossicità in cellule. Tra le sette isoforme della famiglia di 14-3-3, la 14-3-3 eta può revertire il processo di fibrillazione di alfa-sinucleina in vitro, portando alla formazione di oggetti curvi invece che di fibrille canoniche. Questi oggetti curvi hanno diametri e curvature che dipendono dalla quantità di 14-3-3 eta nel saggio di aggregazione: inoltre, molecole di 14-3-3 eta sono state trovate in questi aggregati, suggerendo la formazione di un complesso stabile costituito dalle due proteine. Quanto la quantità di alfa-sinucleina è troppo grande o i seeds vengono utilizzati per promuovere il processo di aggregazione in vitro, la 14-3-3 eta non è più in grado di interferire con il processo di aggregazione di alfa-sinucleina e viene sequestrata nelle fibrille. Nei modelli cellulari, la sovraespressione di 14-3-3 eta riduce la tossicità indotta da alfa-sinucleina quando quest’ultima è soltato sovraespressa e oligomerizza, ma non quando l’aggregazione in cellule viene promossa dai seeds. È stato mostrato, utilizzando tecniche di image correlation spectroscopy (cross raster image correlation spectroscopy e cross Number and Brightness analysis) che la 14-3-3 eta sovraespressa può interagire con alfa-sinucleina sovraespressa, principalmente alla membrana plasmatica. Inoltre, la 14-3-3 eta viene sequestrata negli aggregati quando il processo di aggregazione di alfa-sinucleina è indotto dai seeds, evidenziando un altro possibile meccanismo di tossicità dovuto all’aggregazione. Tutti i risultati ottenuti in cellule sono in buon accordo con i risultati ottenuti in vitro e precedentemente riportati; questo rafforza ulteriormente l’idea che le proteine 14-3-3 e in particolare l’isoforma eta siano particolarmente interessanti nel contesto dello studio dell’aggregazione di alfa-sinucleina e che potrebbero essere utilizzare per interferire con il processo di aggregazione e ridurne gli effetti tossici.

Bone deformities are a common problem in veterinary medicine. These problems are frequently related to the main hind limb pathologies that commonly affect our animals, such as hip dypslasia, cranial cruciate ligament rupture and patellar luxation. It has been demonstrated that a precise and accurate preoperative planning is crucial to the success of the corrective surgeries. The assessment of hind limb deformities has been studied in the past years and up till now there is still a lot of confusion in understanding what could be the best method to apply for a correct evaluation of the deformity. Several methods have been suggested to measure femoral and tibial angles, some studies suggested even an assessment using computed tomography and magnetic resonance imaging. During the last years new methods combining traditional images with reverse engineering technique have been suggested too but although the use of different techniques have been described, radiographic measurement still represents the most common method used for the interpretation of hind limb deformities. This study aimed to compare a 3-dimensional model with standard radiographic evaluation of femoral and tibial angles. Cadavers of eight adult dogs, deceased for reason unrelated to this study, were obtained. Radiographs were obtained using four standard projections: an elevated-torso/hip-extended radiograph, a mediolateral radiograph of the femur, a caudocranial view of the stifle joint and a mediolateral radiograph of the stifle. All radiographs included in the study were made by two single individuals and reviewed and approved in terms of quality and positiong by three different examiners. Evaluation of the neck-shaft angle, the aLPFA, mLPFA, aMDFA and mMDFA, the angle of version and the varus angle for the femur and of the mMPTA, mMDTA and MAD for the tibia, was performed applying the main methods available in literature. After femoral and stifle radiographs were made of each cadaver, the femurs and tibia were harvested and freed of all of the soft tissues sparing the articular cartilage. Every bone was then scanned to create a 3-dimensional computed model. Using RAPIDFORM 2006 (Inus Technology INC.) we could manipulate the shell to evaluate all of the angles previously determined in the 2-dimensional model. The average error in assessing mLPFA , mMDFA , mMPTA and mMDTA, was less than 5% comparing the 2-dimensional method with our 3-dimensional model. Based on these findings, we feel that the reported radiographic methodologies and values may be used to diagnose and quantify hindlimb deformities with a good accuracy. aLPFA and aMDFA values were acceptable for three of the four methods and neck-shaft angle was better represented from the combination of Symax method for the neck axis and Kowalesky's method for the anatomic one.
Le deformita' ossee rappresentano un problema relativamente comune in mediacina veterinaria. Queste alterazioni sono frequentemente associate ad alcune delle principali patologie ortopediche dell'arto posteriore che comunemente affliggono i nostri animali, come per esempio la displasia dell'anca, la rottura del legamento crociato craniale e la lussazione di rotula. E' stato dimostrata la necessita' di eseguire planning preoperatori accurati e precisi, cio' risulta fondamentale per il successo di eventuali chirurgie correttive. Le principali linee guida per la misurazione delle deviazioni ossee sono state studiate nel corso degli anni e ad oggi persiste un ampio dibattito su quale possa essere considerato il metodo migliore per una valutazione delle deformita'. Sono stati suggeriti diversi metodi per la misura degli angoli femorali e tibiali, alcuni lavori suggeriscono l'impiego di tac e risonanza magnetica. Durante gli ultimi anni sono stati proposti metodi innovativi che combinano le metodologie tradizionali con l'impiego di elaborati software ingegneristici per la rielaborazione delle immagini e creazione di modelli tridimensionali. Nonostante sia stato suggerito l'impego di diverse tecniche, nel panorama odierno la radiografia continua a rappresentare il metodo piu' comunemente utilizzato per l'interpretazione delle deformita' scheletriche dell'arto posteriore. Questo studio vuole confrontare un modello 3-dimensionale e il metodo radiografico standard nella valutazione degli angoli femorali e tibiali. Sono stati ottenuti otto cadaveri di cani adulti, deceduti per cause esterne allo studio. Tutti i soggetti sono stati sottoposti a studio radiografico di entrambi gli arti posteriori, eseguendo quattro proiezioni standard: una proiezione ventrodorsale “a cane seduto”, una proiezione mediolaterale del femore, una caudocraniale ed una mediolaterale del ginocchio e della gamba. Tutte le radiografie incluse nel presente studio sono state selezionate in termini di qualita' e posizionamento da tre diversi operatori. E' stata quidi eseguita la valutazione dell'angolo cervico-diafisario, dell'aLPFA, mLPFA e mMDFA, l'angolo di versione e l'angolo di varismo femorale per il femore, e dell'angolo mMPTA, mMDTA e MAD, per la tibia, utilizzando alcuni dei principali metodi descritti in letteratura. Dopo l'esecuzione delle proiezioni radiografiche, sono stati scheletrizzati i femori e le tibie risparmiando la cartilagine articolare. L'immagine di ogni osso e' stata quindi acquisita tramite l'impiego di uno scanner per creare successivamente un modello tridimensionale. Usando il programma RAPIDFORM 2006 (Inus Technology INC.) abbiamo potuto lavorare con il nostro modello per valutare tutti I valori precedentemente calcolati nell'immagine radiografica. L'errore medio nella valutazione del mLPFA , mMDFA , mMPTA and mMDTA, e' stato inferiore al 5% confrontando il modello bidimensionale con quello tridimensionale. Basandoci su questi risultati ci sentiamo di suggerire che le metodologie radiografiche descritte possono essere utilizzate per diagnosticare e quantificare le deformita' degli arti posteriori con una buona accuratezza. I valori ottenuti per gli angli aLPFA e aMDFA sono stati accettabili in tre dei quettro metodi applicati mentre il metodo che meglio ha rappresentato l'angolo crevico-diafisario e' risultato dalla combinazione dell'utilizzo dell'asse cervicale Symax con l'anatomico di Kowalesky . I metodi utilizzati per il calcolo degli angoli di varismo femorale, verione e MAD sono risultati non accettabili con un valore del parametro p significativamente > 0.1 in tutti I casi.

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A sentença em muitos dos processos judiciais trabalhistas envolvendo lides provocadas por patologias pertencentes ao grupo LER/DORT são tomadas mediante a produção de prova pericial que além de se tratar de uma matéria de grande complexidade exigindo olhares multidisciplinares, ainda se defronta com questões como a formação profissional do perito nomeado e diretrizes a serem utilizadas como metodologia investigativa. Devido a esses fatores e a necessidade de evitar laudos periciais imprecisos procurou-se analisar possibilidades de auxílio na fase de reconhecimento de nexo causal a partir da adoção da norma ISO 11228-3 e do Decreto 6.957/09 utilizando-se de processos judiciais trabalhistas oriundos da intranet da 2ª Vara do Trabalho em Sergipe e de pesquisas praticadas na biblioteca digital da CAPES e do portal Google Acadêmico. As análises técnicas mostraram-se favoráveis a implantação da norma e do decreto como recurso auxiliar quando no enquadramento de nexo causalpor LER/DORT. A simulação do uso do decreto nos casos analisados revelou aumento no reconhecimento destas patologias ocupacionais em três vezes quando comparada as conclusões na íntegra produzidas pelo corpo pericial. Apenas um perito judicial possui formação de Pós-graduação na área de ergonomia. Disto confere ao Decreto 6.957/09 e a ISO 11228-3 valor como recurso auxiliar técnico em perícias judiciais de LER/DORT.
The sentence in many of the labor lawsuits involving labors caused by diseases belonging to the group LER/DORT are made by expert evidence production that besides it is a matter of great complexity requiring multidisciplinary looks, still faces issues such as training professional appointed expert and guidelines to be used as research methodology. Due to these factors and the need to avoid inaccurate expert reports sought to examine aid opportunities in the causal nexus of recognition phase with the adoption of ISO 11228-3 and Decree 6.957/09 using labor lawsuits arising intranet 2nd Labor Court in Sergipe and research practiced in the digital library and CAPES Scholar portal. Technical analysis showed their support for implementation of the standard and the decree as auxiliary resource when the nexus of causal framework for LER/DORT. The simulation of the use of the decree in the cases analyzed showed an increase in the recognition of these occupational diseases by three times compared to the conclusions in full produced by the expert body. Only one legal expert has Postgraduate training in ergonomics area. This gives the Decree 6,957 / 09 and ISO 11228-3 value as a resource technical assistant in legal expertise of RSI / MSDs.

Aquesta dissertació doctoral es centra en les discussions acadèmiques que afecten a l'organització de processos en entorns de desastre. En particular, la recerca principal és ‘de quina manera la interacció dels atributs a nivell individual i col·lectiu de les accions improvisades de les organitzacions expliquen com aconsegueixen els seus objectius, p.e. ser eficaços, en contextos extrems?'. Fent servir una estratègia metodològica d’estudis de casos, emergeixen narratives de dos perfils d'organitzacions que eren crítiques per a la fase de resposta i de recuperació del Tifó Haiyan del 2013. La contribució principal d’aquest treball té dos vessants. Primer, es proposa que la improvisació, malgrat la seva naturalesa emergent, està guiada per un propòsit concret, doncs facilita a les organitzacions aconseguir els seus objectius preestablerts. Això succeeix a través de dos efectes: (a) l'efecte buffering, i (b) l'efecte connection-seeking. Segon, s’infereix que les improvisacions amb un propòsit concret estan promogudes per diverses narracions que interaccionen a nivell individual i col·lectiu. Tres narracions sorgeixen: (a) una interacció complementària entre els atributs a nivell individual i col·lectiu, on tots dos, l'individual i l'organitzatiu juguen una funció similar per promoure una improvisació amb un propòsit concret (b) una interacció gestionada a nivell individual, en que l’acció individual apareix més pronunciada en la decisió d'improvisar a nivell de cada propòsit concret, i (c) una interacció que s’adhereix la norma, on el paper de l'organització precedeix la posició individual en la decisió d'improvisar en el propòsit concret.
Esta disertación doctoral se sitúa la discusión académica que afecta a la organización de procesos en entornos de desastre. En particular, la investigación principal es ‘de qué manera la interacción de los atributos a nivel individual y colectivo de las acciones improvisadas de las organizaciones explican cómo pueden esas alcanzar sus objetivos, i.e. ser eficaz, en contextos extremos?' Utilizando una estrategia metodológica de estudio de casos, emergen narrativas de dos perfiles de organizaciones que fueron críticas en la fase de respuesta y de recuperación del Tifón Haiyan del 2013. La contribución principal del trabajo tiene dos vertientes. Primero, se propone que la improvisación, a pesar de su naturaleza emergente, está guiada por propósito concreto, ya que facilita a las organizaciones conseguir sus objetivos preestablecidos. Esto ocurre a través de dos efectos: (a) el efecto buffering, y (b) el efecto connection-seeking. Segundo, se infiere que una improvisación con propósito concreto está promovida por varias narrativas de interacción a nivel individual y colectivo. Surgen tres narrativas: (a) una interacción complementaria entre los atributos a nivel individual y colectivo, donde ambos, el individual y la organización juegan una función similar al promover la improvisación con propósito concreto (b) una interacción gestionada a nivel individual, en que la acción individual resulta más pronunciada en la decisión de improvisar con propósito concreto, y (c) una interacción que se adhiere a la norma, en que el rol de la organización precede la posición individual en la decisión de improvisar con un propósito concreto.
This dissertation positions itself within the scholarly conversations on organizing processes in disaster environments. In particular, the main research inquiry is ‘how does the interaction of individual and collective level attributes among improvised actions of the organizations explain how they can realize their goals, i.e. be effective, in extreme contexts?’. Using a case strategy approach, it surfaces narratives of two profiles of organizations that were critical to the response and recovery phase of the 2013 Typhoon Haiyan. The main contribution of the work is two-fold: First, it infers that improvisation, despite its emergent nature, is purpose-driven, as it allows organizations to consciously realize their pre-established goals. This occurs through two effects: (a) the buffering effect, and (b) the connection-seeking effect. Second, it infers that purpose-driven improvisation is enabled by various narratives of interaction between the individual and collective level attributes. Three narratives are emergent: (a) a complementary interaction between the individual- and collective-level attributes, where both the individual and the organization play an equal role in enabling purpose-driven improvisation (b) an individually-maneuvered interaction, where the individual positionality is more pronounced in the enactment of purpose-driven improvisation, and (c) rule-abiding interaction, where the role of the organization precedes the individual positionality in the enactment of purpose-driven improvisation.

Timp-3, in quanto inibitore fisiologico di metalloproteasi di matrice (MMP) e di TACE (TNF-alpha Converting Enzyme), ha un importante ruolo nella regolazione dell’attività immunitaria e conseguentemente nell’omeostasi metabolica. Nell’ambito del nostro laboratorio è stato recentemente scoperto che la diminuzione di tale proteina, in concomitanza con l’aploinsufficienza del recettore insulinico, potenzia l’insulino-resistenza, l’infiammazione e le disfunzioni epatiche (NAFLD, non-alcoholic fatty liver disease) indotte da obesità. In questo progetto di ricerca sono stati condotti studi metabolici al fine di valutare il ruolo della deficienza di Timp-3 nella regolazione della sensibilità insulinica in modelli animali nutriti con una dieta ricca di grassi (HFD). Allo scopo di individuare i potenziali meccanismi molecolari coinvolti nell’attività di Timp-3, il proteoma epatico di topi WT e TIMP-3-/- ad HFD è stato analizzato tramite nLC-ESI-MSE e i dati ottenuti sono stati interpretati mediante un programma di bioinformatica (IPA). Tra i risultati ottenuti i dati più significativi riguardano l’incremento di stress ossidativo e la forte alterazione del metabolismo della metionina, i quali potrebbero avere un ruolo predominante nel potenziamento d’insulino-resistenza e NAFLD riscontrata nei topi knockout.
Timp-3, as physiological inhibitor of matrix metalloproteinases (MMP) and of TACE (TNF-alpha Converting Enzyme), has an important role in the regulation of immunity and metabolic homeostasis. In our laboratory it was recently discovered that the decrease of this protein, together with the aploinsufficiency of Insr, enhance insulin resistance, inflammation and hepatic dysfunction (NAFLD, non-alcoholic fatty liver disease) induced by obesity. In this research project we performed metabolic analyses to evaluate the role of Timp-3 in regulation of insulin sensitivity in obese animal models. Moreover, to discover the molecular mechanisms involved in the effects of Timp-3, we investigated the hepatic proteome of WT and TIMP-3-/- mice on high fat diet (HFD) by using nLC-ESI-MSE and we performed a functional analysis with a bioinformatics tool (IPA). Among the results obtained of particular interest is the oxidative stress increment and the strong alteration of methionine metabolism, which could have a prominent role in enhancing insulin resistance and NAFLD in knockout mice.

Many psychophysical studies suggest that target depth and direction during reaches are processed independently, but the neurophysiological support to this view is so far limited. Here, we investigated the representation of reach depth and direction by single neurons in an area of the medial posterior parietal cortex (V6A). Single-unit activity was recorded from V6A in two Macaca fascicularis monkeys performing a fixation-to-reach task to targets at different depths and directions. We found that in a substantial percentage of V6A neurons depth and direction signals jointly influenced fixation, planning and arm movement-related activity in 3D space. While target depth and direction were equally encoded during fixation, depth tuning became stronger during arm movement planning, execution and target holding. The spatial tuning of fixation activity was often maintained across epochs, and this occurred more frequently in depth. These findings support for the first time the existence of a common neural substrate for the encoding of target depth and direction during reaching movements in the posterior parietal cortex. Present results also highlight the presence in V6A of several types of cells that process independently or jointly eye position and arm movement planning and execution signals in order to control reaches in 3D space. It is possible that depth and direction influence also the metrics of the reach action and that this effect on the reach kinematic variables can account for the spatial tuning we found in V6A neural activity. For this reason, we recorded and analyzed behavioral data when one monkey performed reaching movements in 3-D space. We evaluated how the target spatial position, in particular target depth and target direction, affected the kinematic parameters and trajectories describing the motor action properties.

Many psychophysical studies suggest that target depth and direction during reaches are processed independently, but the neurophysiological support to this view is so far limited. Here, we investigated the representation of reach depth and direction by single neurons in an area of the medial posterior parietal cortex (V6A). Single-unit activity was recorded from V6A in two Macaca fascicularis monkeys performing a fixation-to-reach task to targets at different depths and directions. We found that in a substantial percentage of V6A neurons depth and direction signals jointly influenced fixation, planning and arm movement-related activity in 3D space. While target depth and direction were equally encoded during fixation, depth tuning became stronger during arm movement planning, execution and target holding. The spatial tuning of fixation activity was often maintained across epochs, and this occurred more frequently in depth. These findings support for the first time the existence of a common neural substrate for the encoding of target depth and direction during reaching movements in the posterior parietal cortex. Present results also highlight the presence in V6A of several types of cells that process independently or jointly eye position and arm movement planning and execution signals in order to control reaches in 3D space. It is possible that depth and direction influence also the metrics of the reach action and that this effect on the reach kinematic variables can account for the spatial tuning we found in V6A neural activity. For this reason, we recorded and analyzed behavioral data when one monkey performed reaching movements in 3-D space. We evaluated how the target spatial position, in particular target depth and target direction, affected the kinematic parameters and trajectories describing the motor action properties.

Reaching and grasping an object is an action that can be performed in light, under visual guidance, as well as in darkness, under proprioceptive control only. Area V6A is a visuomotor area involved in the control of reaching movements. V6A, besides neurons activated by the execution of reaching movements, shows passive somatosensory and visual responses. This suggests fro V6A a multimodal capability of integrating sensory and motor-related information, We wanted to know whether this integration occurrs in reaching movements and in the present study we tested whether the visual feedback influenced the reaching activity of V6A neurons. In order to better address this question, we wanted to interpret the neural data in the light of the kinematic of reaching performance. We used an experimental paradigm that could examine V6A responses in two different visual backgrounds, light and dark. In these conditions, the monkey performed an istructed-delay reaching task moving the hand towards different target positions located in the peripersonal space. During the execution of reaching task, the visual feedback is processed in a variety of patterns of modulation, sometimes not expected. In fact, having already demonstrated in V6A reach-related discharges in absence of visual feedback, we expected two types of neural modulation: 1) the addition of light in the environment enhanced reach-related discharges recorded in the dark; 2) the light left the neural response unmodified. Unexpectedly, the results show a complex pattern of modulation that argues against a simple additive interaction between visual and motor-related signals.

Reaching and grasping an object is an action that can be performed in light, under visual guidance, as well as in darkness, under proprioceptive control only. Area V6A is a visuomotor area involved in the control of reaching movements. V6A, besides neurons activated by the execution of reaching movements, shows passive somatosensory and visual responses. This suggests fro V6A a multimodal capability of integrating sensory and motor-related information, We wanted to know whether this integration occurrs in reaching movements and in the present study we tested whether the visual feedback influenced the reaching activity of V6A neurons. In order to better address this question, we wanted to interpret the neural data in the light of the kinematic of reaching performance. We used an experimental paradigm that could examine V6A responses in two different visual backgrounds, light and dark. In these conditions, the monkey performed an istructed-delay reaching task moving the hand towards different target positions located in the peripersonal space. During the execution of reaching task, the visual feedback is processed in a variety of patterns of modulation, sometimes not expected. In fact, having already demonstrated in V6A reach-related discharges in absence of visual feedback, we expected two types of neural modulation: 1) the addition of light in the environment enhanced reach-related discharges recorded in the dark; 2) the light left the neural response unmodified. Unexpectedly, the results show a complex pattern of modulation that argues against a simple additive interaction between visual and motor-related signals.

This thesis deals with the efficient resolution of Vehicle Routing Problems (VRPs). The first chapter faces the archetype of all VRPs: the Capacitated Vehicle Routing Problem (CVRP). Despite having being introduced more than 60 years ago, it still remains an extremely challenging problem. In this chapter I design a Fast Iterated-Local-Search Localized Optimization algorithm for the CVRP, shortened to FILO. The simplicity of the CVRP definition allowed me to experiment with advanced local search acceleration and pruning techniques that have eventually became the core optimization engine of FILO. FILO experimentally shown to be extremely scalable and able to solve very large scale instances of the CVRP in a fraction of the computing time compared to existing state-of-the-art methods, still obtaining competitive solutions in terms of their quality. The second chapter deals with an extension of the CVRP called the Extended Single Truck and Trailer Vehicle Routing Problem, or simply XSTTRP. The XSTTRP models a broad class of VRPs in which a single vehicle, composed of a truck and a detachable trailer, has to serve a set of customers with accessibility constraints making some of them not reachable by using the entire vehicle. This problem moves towards VRPs including more realistic constraints and it models scenarios such as parcel deliveries in crowded city centers or rural areas, where maneuvering a large vehicle is forbidden or dangerous. The XSTTRP generalizes several well known VRPs such as the Multiple Depot VRP and the Location Routing Problem. For its solution I developed an hybrid metaheuristic which combines a fast heuristic optimization with a polishing phase based on the resolution of a limited set partitioning problem. Finally, the thesis includes a final chapter aimed at guiding the computational evaluation of new approaches to VRPs proposed by the machine learning community.

La valutazione del merito di credito da parte degli istituti bancari è da sempre oggetto di dibattito tra gli esperti. Ad oggi, i temi che suscitano maggiori perplessità gravitano attorno alla prossima entrata in vigore della nuova regolamentazione Basilea 3 ed al mantenimento dei sistemi di valutazione della precedente Basilea 2. Le conseguenze dell’adeguamento ai nuovi requisiti non sono ancora del tutto concrete ma dalle analisi delle autorità emergono aspetti positivi e numerose critiche, per alcune delle quali la ricerca ha già provveduto diffondendo le soluzioni realizzabili nell’immediato nonché individuando gli strumenti idonei ad implementarle. Il passaggio di tali conoscenze agli operatori è necessario per migliorare l’allocazione del credito e i relativi criteri di valutazione in un mercato altamente diversificato e complesso come è il mondo delle PMI in Italia, per limitare ulteriori danni derivanti dall’utilizzo di metodi inefficienti nella rilevazione del rischio di credito.

Methods based on pairwise comparison matrices (PCMs) form a significant part of multi-criteria decision making (MCDM) methods. These methods are based on structuring pairwise comparisons (PCs) of objects from a finite set of objects into a PCM and deriving priorities of objects that represent the relative importance of each object with respect to all other objects in the set. However, the crisp PCMs are not able to capture uncertainty stemming from subjectivity of human thinking and from incompleteness of information about the problem that are often closely related to MCDM problems. That is why the fuzzy extension of methods based on PCMs has been of great interest. In order to derive fuzzy priorities of objects from a fuzzy PCM (FPCM), standard fuzzy arithmetic is usually applied to the fuzzy extension of the methods originally developed for crisp PCMs. %Fuzzy extension of the methods based on PCMs usually consists in simply replacing the crisp PCs in the given model by fuzzy PCs and applying standard fuzzy arithmetic to obtain the desired fuzzy priorities. However, such approach fails in properly handling uncertainty of preference information contained in the FPCM. Namely, reciprocity of the related PCs of objects in a FPCM and invariance of the given method under permutation of objects are violated when standard fuzzy arithmetic is applied to the fuzzy extension. This leads to distortion of the preference information contained in the FPCM and consequently to false results. Thus, the first research question of the thesis is: ``Based on a FPCM of objects, how should fuzzy priorities of these objects be determined so that they reflect properly all preference information available in the FPCM?'' This research question is answered by introducing an appropriate fuzzy extension of methods originally developed for crisp PCMs. That is, such fuzzy extension that does not violate reciprocity of the related PCs and invariance under permutation of objects, and that does not lead to a redundant increase of uncertainty of the resulting fuzzy priorities of objects. Fuzzy extension of three different types of PCMs is examined in this thesis - multiplicative PCMs, additive PCMs with additive representation, and additive PCMs with multiplicative representation. In particular, construction of PCMs, verifying consistency, and deriving priorities of objects from PCMs are studied in detail for each type of these PCMs. First, well-known and in practice most often applied methods based on crisp PCMs are reviewed. Afterwards, fuzzy extensions of these methods proposed in the literature are reviewed in detail and their drawbacks regarding the violation of reciprocity of the related PCs and of invariance under permutation of objects are pointed out. It is shown that these drawbacks can be overcome by properly applying constrained fuzzy arithmetic instead of standard fuzzy arithmetic to the computations. In particular, we always have to look at a FPCM as a set of PCMs with different degrees of membership to the FPCM, i.e. we always have to consider only PCs that are mutually reciprocal. Constrained fuzzy arithmetic allows us to impose the reciprocity of the related PCs as a constraint on arithmetic operations with fuzzy numbers, and its appropriate application also guarantees invariance of the methods under permutation of objects. Finally, new fuzzy extensions of the methods are proposed based on constrained fuzzy arithmetic and it is proved that these methods do not violate the reciprocity of the related PCs and are invariant under permutation of objects. Because of these desirable properties, fuzzy priorities of objects obtained by the methods proposed in this thesis reflect the preference information contained in fuzzy PCMs better in comparison to the fuzzy priorities obtained by the methods based on standard fuzzy arithmetic. Beside the inability to capture uncertainty, methods based on PCMs are also not able to cope with situations where it is not possible or reasonable to obtain complete preference information from DMs. This problem occurs especially in the situations involving large-dimensional PCMs. When dealing with incomplete large-dimensional PCMs, compromise between reducing the number of PCs required from the DM and obtaining reasonable priorities of objects is of paramount importance. This leads to the second research question: ``How can the amount of preference information required from the DM in a large-dimensional PCM be reduced while still obtaining comparable priorities of objects?'' This research question is answered by introducing an efficient two-phase method. Specifically, in the first phase, an interactive algorithm based on weak-consistency condition is introduced for partially filling an incomplete PCM. This algorithm is designed in such a way that minimizes the number of PCs required from the DM and provides sufficient amount of preference information at the same time. The weak-consistency condition allows for providing ranges of possible intensities of preference for every missing PC in the incomplete PCM. Thus, at the end of the first phase, a PCM containing intervals for all PCs that were not provided by the DM is obtained. Afterward, in the second phase, the methods for obtaining fuzzy priorities of objects from fuzzy PCMs proposed in this thesis within the answer to the first research question are applied to derive interval priorities of objects from this incomplete PCM. The obtained interval priorities cover all weakly consistent completions of the incomplete PCM and are very narrow. The performance of the method is illustrated by a real-life case study and by simulations that demonstrate the ability of the algorithm to reduce the number of PCs required from the DM in PCMs of dimension 15 and greater by more than 60\% on average while obtaining interval priorities comparable with the priorities obtainable from the hypothetical complete PCMs.

Nel presente lavoro è stata finemente studiata la distribuzione e la localizzazione dei trasportatori del GABA, GAT-1 e GAT-3 nella corteccia cerebrale di ratto, al fine di estendere le attuali conoscenze riguardo la loro espressione. A tale scopo sono stati studiati sistematicamente la distribuzione sub-cellulare, l’organizzazione sinaptica e i livelli sinaptici di GAT-1 e GAT-3 nella corteccia cerebrale mediante microscopia elettronica con tecniche di pre- e post-embedding e microscopia confocale tridimensionale ad alta risoluzione. In generale gli studi di pre-embedding hanno permesso di evidenziare una complessa localizzazione di entrambi i trasportatori GAT-1 e GAT-3. Sebbene GAT-1 sia principalmente neuronale, è largamente espresso anche nei processi gliali, mentre GAT-3 è prevalentemente gliale e solo una piccola frazione è presente anche nei neuroni. L’espressione di GAT-1, e non di GAT-3, è polarizzata nelle sinapsi corticali. A livello sinaptico, GAT-1 è prevalentemente espresso nei terminali assonici di sinapsi simmetriche compresi diversi processi gliali, seppure una frazione non trascurabile di GAT-1 sia presente anche a livello di sinapsi asimmetriche (nei processi gliali perisinaptici e dendriti postsinaptici). Al contrario GAT-3, a livello sinaptico, è maggiormente espresso nei processi perisinaptici ed è equamente distribuito tra sinapsi simmetriche e asimmetriche. Gli studi di post-embedding dimostrano che l’espressione di GAT-1 nelle sinapsi GABAergiche è all’incirca nel 60% dei casi nei terminali, nel 29% nei soli astrociti perisinaptici e nell’11% in entrambi i comparti. Mentre nel caso di GAT-3 è nel 78% negli astrociti perisinaptici, 13% nei terminali e 8% in entrambi. Inoltre nei terminali assonici, GAT-1 è distribuito nell’area peri-sinaptica ed extrasinaptica, mentre nei processi astrocitari è presente maggiormente nella zona extrasinaptica. Per quanto 2 riguarda GAT-3, nei processi astrocitari è maggiormente concentrato nell’area extrasinaptica, mentre nei terminali è sparso, senza formare un vero e proprio agglomerato. Infine, i livelli di GAT-1 e GAT-3 nelle sinapsi GABAergicche sono stati stimati attraverso microscopia confocale-3D ad alta risoluzione applicata ai terminali VGAT: a livello delle sinapsi GABAergiche, GAT-1 è significativamente più espresso (~1,5 volte) rispetto a GAT-3. In conclusione, i risultati sull’espressione e sulla fine localizzazione di GAT-1 e GAT-3 nelle sinapsi GABAergiche, consentono da un lato di elucidare il ruolo dei due trasportatori nella regolazione dei fenomeni sia fasici che tonici del GABA e dall'altro di suggerire la possibilità di un dualismo funzionale di GAT-1 (determinato dalla regolazione dei livelli di GABA nei siti sinaptici ed extrasinaptici), mentre GAT-3 mostrerebbe una singola attività funzionale (regolazione dei livelli di GABA nei siti extrasinaptici).
In the present work has been carefully studied the distribution and localization of GABA transporters, GAT-1 and GAT-3 in the rat cerebral cortex, in order to extend the current knowledge about their expression, by studying systematically the distribution of sub-cellular, synaptic organization and synaptic levels of GAT-1 and GAT-3 in the brain cortex by pre and post-embedding electron microscopy techniques and threedimensional high resolution confocal microscopy. In general, pre-embedding studies have revealed a complex localization of both transporters. Although GAT-1 is mainly neuronal, is also widely expressed in glial processes, while GAT-3 is predominantly glial and only a small fraction is also present in neurons. The expression of GAT-1, and not of GAT-3, is polarized in the cortical synapses. At the synaptic level, GAT-1 is predominantly expressed in axon terminals of symmetrical synapses including several glial processes, although a non-negligible part of GAT-1 is present at the level of asymmetric synapses (glial processes in perisynaptic and postsynaptic dendrites). In contrast GAT-3, at the synaptic level, is more expressed in the perisynaptic processes and is equally distributed between symmetric and asymmetric synapses. Post-embedding studies show that the expression of GAT-1 in GABAergic synapses is approximately in 60% of cases in terminals, in 29% only in peri-synaptic astrocytes and 11% in both segments. While in the case of GAT-3, is in 78% of perisynaptic astrocytes, 13% in terminals and 8% in both. Also, in axon terminals GAT-1 is distributed in the peri-synaptic and extra-synaptic area, while in the astrocytic processes is present more in the extra-synaptic area. As regards GAT-3, in astrocytic processes is more concentrated in extra-synaptic area, while in the terminals is scattered, without forming a real agglomeration. 2 Finally, GAT-1 and GAT-3 levels at GABA-ergic synapses were estimated by 3D quantitative confocal microscopy applied to VGAT positive terminals: estimated levels indicated that at GABA-ergic synapses GAT-1 expression was significantly higher (~1.5 times) than that of GAT-3. In conclusion, results on the expression and localization of GAT-1 and GAT-3 in GABA-ergic synapses, allow on the one hand to elucidate the role of the two transporters in the regulation of both phasic and tonic transmission, on the other hand to suggest the possibility of a functional duality of GAT-1 (determined by the adjustment of the levels of GABA in synaptic and extra-synaptic sites), while GAT-3 would show a single functional activity (regulation of GABA levels in the extrasynaptic sites).

The aim of this thesis is to explore different aspects of the female retirement process. Adopting a life-course perspective, this study investigates the development of women's life course to understand the influence of previous life experience on different dimensions of the retirement process itself. More specifically, the thesis consists of three contributions; the first one aims to analyse the influence that the career trajectories have on female retirement timing, in three different European countries (Italy, Germany and Denmark); the second contribution aims to understand the effects of the retirement transition on female life satisfaction, depending on the working pathways. Finally, the third contribution focuses on the influence of work-family life trajectories on life satisfaction in retirement. Empirically, I analyse longitudinal data from the Survey of Health, Ageing and Retirement in Europe (SHARE). In the first contribution, I observed a negative and significant probability of retirement for the discontinuous and part-time working trajectories compared to the full-time one, in Italy. In Germany and particularly in Denmark, no substantial differences were found between the working pathways in terms of retirement timing. In the second contribution, I found that some of the working trajectories constituted by discontinuity or part-time periods, showed a continuous increase in life satisfaction, passing from employment (or unemployment) to retirement. For other working trajectories, such as the full-time one, retirement seems to not have implications for subjective wellbeing. In the third contribution, the results show that all the life course trajectories characterized by part-time work show a higher general level of life satisfaction compared to the other pathways (discontinuous or full-time ones). Moreover, a part-time trajectory was associated with higher life satisfaction in retirement when combined with a stable marriage and two children, than when it was coupled with one child or three or more children.

This thesis gives an overview of the history of gold per se, of gold as an investment good and offers some institutional details about gold and other precious metal markets. The goal of this study is to investigate the role of gold as a store of value and hedge against negative market movements in turbulent times. I investigate gold’s ability to act as a safe haven during periods of financial stress by employing instrumental variable techniques that allow for time varying conditional covariance. I find broad evidence supporting the view that gold acts as an anchor of stability during market downturns. During periods of high uncertainty and low stock market returns, gold tends to have higher than average excess returns. The effectiveness of gold as a safe haven is enhanced during periods of extreme crises: the largest peaks are observed during the global financial crises of 2007-2009 and, in particular, during the Lehman default (October 2008). A further goal of this thesis is to investigate whether gold provides protection from tail risk. I address the issue of asymmetric precious metal behavior conditioned to stock market performance and provide empirical evidence about the contribution of gold to a portfolio’s systematic skewness and kurtosis. I find that gold has positive coskewness with the market portfolio when the market is skewed to the left. Moreover, gold shows low cokurtosis with the market returns during volatile periods. I therefore show that gold is a desirable investment good to risk averse investors, since it tends to decrease the probability of experiencing extreme bad outcomes, and the magnitude of losses in case such events occur. Gold thus bears very important and under-researched characteristics as an asset class per se, which this thesis contributed to address and unveil.

This thesis gives an overview of the history of gold per se, of gold as an investment good and offers some institutional details about gold and other precious metal markets. The goal of this study is to investigate the role of gold as a store of value and hedge against negative market movements in turbulent times. I investigate gold’s ability to act as a safe haven during periods of financial stress by employing instrumental variable techniques that allow for time varying conditional covariance. I find broad evidence supporting the view that gold acts as an anchor of stability during market downturns. During periods of high uncertainty and low stock market returns, gold tends to have higher than average excess returns. The effectiveness of gold as a safe haven is enhanced during periods of extreme crises: the largest peaks are observed during the global financial crises of 2007-2009 and, in particular, during the Lehman default (October 2008). A further goal of this thesis is to investigate whether gold provides protection from tail risk. I address the issue of asymmetric precious metal behavior conditioned to stock market performance and provide empirical evidence about the contribution of gold to a portfolio’s systematic skewness and kurtosis. I find that gold has positive coskewness with the market portfolio when the market is skewed to the left. Moreover, gold shows low cokurtosis with the market returns during volatile periods. I therefore show that gold is a desirable investment good to risk averse investors, since it tends to decrease the probability of experiencing extreme bad outcomes, and the magnitude of losses in case such events occur. Gold thus bears very important and under-researched characteristics as an asset class per se, which this thesis contributed to address and unveil.

Дисертація на здобуття наукового ступеня кандидата економічних наук за спеціальністю 08.00.09 – бухгалтерський облік, аналіз та аудит (за видами економічної діяльності). – Київський національний торговельно-економічний університет, Київ, 2014.

The development of Nano Drug Delivery Systems (NDDS) is a promising approach for developing intelligent therapeutic systems, which will bring significant advances in the diagnosis and treatment of disease, with the challenges to maximize therapeutic activity and to minimize undesirable side effects. The aim of this thesis was focused on the design of two kind of self-assembled nanocarrier for the delivery of hydrophobic drugs: Hyaluronan based nanohydrogel and pNIPAAm based micelles that both show hydrophobic internal domains and a surrounding hydrophilic shell. To achieve such self-assembled nanostructures, amphiphilic polymers were synthesized and extensively characterized. Hyaluronan (HA) and the thermosensitive di-block copolymer of methoxy poly(ethylene glycol)-b-(N-isopropylacrylamide)-co-(2-azidoethyl) methacrylate (mPEG-b-p(NIPAAm)-co-AzEMA) constituted the starting polymers on which riboflavin 2',3',4',5'- tetrabutyrate (Rfv), due to its interesting chemical-physical proprieties and due to its particularly biocompatibility, was conjugated as hydrophobic moiety via azide-alkyne click chemistry reaction. The HA-Prop derivatives were synthesized in order to provide the polymer of alkyne groups and make it versatile for “click” reaction. In this way, the azido-hexyl derivative of riboflavin tetrabutyrate was synthesized and covalently coupled to the propargyl derivative of hyaluronic acid by Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC), due to the high efficacy and selectivity of this kind of reaction. Sterile self-assembled NHs were obtained in one-step by thermal treatment in autoclave of the aqueous dispersion of the HA-c-Rfv polymers. Size and polydispersity of the obtained NHs resulted be influenced from the Mw and from the degrees of derivatization of the starting Hyaluronan. Micellar nano-assemblies composed of thermosensitive amphiphilic block polymers were formed by heating the aqueous solutions of the resulting poly(NIPAAm) block copolymer, in order to obtain temporal control of the release of the encapsulated drugs. For this purpose, mPEG-b-p(NIPAAm)-co-AzEMA block copolymer was synthesized by free radical polymerization and subsequently, a propargyl derivative of riboflavin (Rfv-Prop) was synthesized and covalently coupled to the azide modified diblock copolymers by Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC). The nanosystems thus formed were intended to be used for the delivery of hydrophobic drugs, thus taking advantages of the lipophilic core represented by riboflavin. Moreover, the potential π-π stacking interactions between the aromatic rings of the riboflavin in the core of such nanocarriers, may bring more stable nanostructures able to provide increased loading capacity. Highly hydrophilic and biocompatible nanocarriers based on polysaccharide hydrogels (nanohydrogels, NHs) were shown to be promising systems for drug delivery applications. Inspired by these emerging and promising drug carriers for therapeutic applications, in this work we aimed to develop self-assembled hydrogel nanoparticles based on amphiphilic derivative of hyaluronic acid (HA). For this purpose, new HA-Riboflavin (HA-c-Rfv) derivatives were synthetized by “click” Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction, requiring a previous HA derivatization with alkyne moieties and Riboflavin modification with azide groups. The resulting amphiphilic product was able to form nanohydrogels in aqueous environments by suitable treatments, in particular by an innovative method by using an autoclave cycle. Various HA molecular weights (Mw) and derivatization degrees of the starting polymers have been used in order to assess the effect of different parameters on the NHs formation. The derivative HA220-c-Rfv (Mw 220kDa, 40% of propargylic portions and 40% of Rfv moieties), was chosen as the most interesting NHs forming system; NHs were 150-200 nm in size and showed a ζ-potential in the range -40 / -50 mV, depending on the experimental conditions adopted. NHs resulted to be stable in water and at physiological pH. Moreover, by the addition of a suitable cryoprotectant, the NHs suspension can be freeze-dried and recovered by re-suspension in water. The developed system is intended to be used for the delivery of hydrophobic drugs, such us dexamethasone, piroxicam and paclitaxel, used as model drugs. Drug encapsulations were performed by hydrating drugs film with polymers aqueous suspensions, followed by an autoclave treatment, resulting in a high encapsulation efficiency (EE%). Moreover, the HA-propargyl backbone with 60% of propargylic portions and partially linked to Rfv, was capable to react with other molecules bearing an azide group, opening the route to a wide spectrum of functionalization opportunities: in this direction, PEG-N3 have been tested as model molecule for the NHs. Micellar nano-assemblies composed of thermosensitive amphiphilic block polymers are formed spontaneously above their CMC. For these types of polymers, the aqueous solubility properties depend on temperature, and micellar nanostructures are formed by self-assembly above their lower critical solution temperature (LCST) in aqueous media. For this purpose, poly N-isopropylacrylamide (pNIPAAm) block-copolymers were synthesized by free radical polymerization using a polyethylene glycol based macroinitiator. Upon dissolution in aqueous solvents and heating above the LCST, these polymers are able to form micelles. To stabilize these micelles, functional groups facilitating π-π stacking interactions were introduced into the polymers. For this purpose, riboflavin as aromatic moiety was coupled to the polymer backbone by azide-alkyne “click chemistry” reaction. Two block-copolymers, methoxy poly(ethylene glycol)-b-(N-isopropylacrylamide)-co-(2-azidoethyl) methacrylate (mPEG-b-p(NIPAAm)-co-AzEMA) and the corresponding derivative methoxy poly(ethylene glycol)-b-(N-isopropylacrylamide)-co-(2-azidoethyl) methacrylate-riboflavin (mPEG-b-p(NIPAAm)-co-AzEMA-Rfv), were synthesized and compared in terms of physico-chemical properties, micelle size, drug retention and release. Micelles were formed by heating the polymeric aqueous solution from 0 to 50°C, and paclitaxel (PTX) was encapsulated by mixing a concentrated drug solution in ethanol with the polymer solution in phosphate buffer followed by heating. Three different feed PTX loadings (feed drug loading concentration of 5, 10 and 20%) were tested in micelles of both block-copolymers. Upon introduction of riboflavin in the polymeric backbone, a lower critical micelle temperature was obtained by (26°C for mPEG-b-p(NIPAAm)-co-AzEMA and 24° C for the corresponding riboflavin containing polymer, respectively). An average size of ~50 nm for the mPEG-b-p(NIPAAm)-co-AzEMA and ~90 nm for the mPEG-b-p(NIPAAm)-co-AzEMA-Rfv micelles in water was observed respectively for empty micelles, which increased with ~20 nm in phosphate buffered saline. Drug loading resulted in an increase of the micelle size by approximately 10-20 nm (from 60 to 75 nm for mPEG-b-p(NIPAAm)-co-AzEMA based micelles, and from 150 to 170 nm for mPEG-b-p(NIPAAm)-co-AzEMA-Rfv based ones). Increased encapsulation efficiency (EE%) and drug loading (DL%) were obtained for micelles containing riboflavin; almost 100% encapsulation of PTX was found for a feed PTX loading of 5% . Nevertheless, drug release resulted be faster for the Rfv-derivative backbone based micelle, likely due to their lower polymer density. Overall, this novel Rfv containing system is very promising in bringing advantages with regard to drug loading, warranting further investigations in tunability of drug release profiles for further application in stimuli-responsive anticancer therapy.

It is difficult for policymakers to predict the behavior of people in response to a water rationing policy. The public may not necessarily behave as expected or in accordance with market rules or policy mandates. In this research, I will ask whether people were responsive to a summer 2011 City of Saskatoon legal restrictions to reduce their outdoor water consumption due to reduced capacity at the water treatment plant resulting from excessive solids in the river water. I will try to explore the policy response - which can be expressed as a reduction of outdoor water consumption in 2011 in response to the water mandate - while holding constant other factors, including environmental variables (temperature and rainfall), socio-economic factors (income and education level), lot size, and an annual downward trend in water consumption that appeared in many North American cities during the past two decades. Monthly water consumption data for the period from 2004 to 2012 for the City of Saskatoon were analyzed to detect if there is a policy response from the water mandate during June and July 2011. Regression analysis with water consumption as the dependent variable and lot size, temperature, rainfall, education index, income, consumption trend, and policy as independent variables was conducted to test whether there is a policy response in the Saskatoon water records, holding other factors relevant to water consumption constant. Results showed there was a statistically significant reduction in Saskatoon water consumption during June and July 2011 as a result of the water rationing mandate, with considerable variations through different neighborhoods. In addition, there is a positive relationship between water consumption and lot size and a reduction in water consumption over the research period from 2004 to 2012. The policy response varied widely across neighborhoods, and there was relationship between policy and annual income per capita, and household size; households with more income per capita are less responsive to the policy while bigger household sizes showed more policy responsiveness. Key words: City of Saskatoon, water rationing, water policy, water mandate, outdoor water use.

Il presente documento intende studiare in maniera approfondita tecniche e proporre metodologie atte a ricucire la “dicotomia” tra forma architettonica e performance strutturale con l’ausilio e il riconoscimento dell’architettura di San Paolo (o Scuola Paulista – o ancora Scuola Paulista Brutalista), esaminando in maniera critica il periodo tra il 1945 e il 1970, attraverso due sezioni differenti e non immediatamente complementari. Tale difficoltà è rappresentata dalla differenza delle tematiche trattate nei successivi capitoli, all’interno dei quali ci si vuole focalizzare sui concetti pragmatici che conducono alla creazione di determinate soluzioni architettoniche, a loro volta guidate da rappresentazioni “ideali” fondamentali dell’architettura della Scuola di São Paulo. Il documento è suddiviso in due differenti macro-sezioni che consentono al lettore di essere guidato nella ricerca condotta, partendo dalle motivazioni per il quale determinate architetture si sono prestate più di altre nel fungere come “background” della ricerca, approfondendo allo stesso tempo eventi storici, punti salienti, caratteri distintivi e pratici nella definizione dell’architettura paulista. La prima sezione si propone di fornire alcune indagini bibliografiche, con approccio il più possibile critico. Al contempo, vengono raccolti riferimenti sparsi e sconnessi sull'Architettura Paulista, permettendo di indagare principalmente sull’incidenza della tematica forma-struttura in riferimento a specifiche opere esistenti e/o esistite nel panorama della Escola Paulista, le quali sono in grado di riflettere il valore intrinseco del linguaggio architettonico accostato con la stabilità e l’innovazione strutturale. In particolare, viene concentrata l’attenzione su specifiche opere dei maestri Vilanova Artigas e Paulo Mendes da Rocha – pionieri e fondatori della Scuola di San Paolo, il cui studio ha permesso di riconoscere le caratteristiche estetiche ed etiche dell'architettura Brutalista Paulista, sottolineando la fusione di caratteri morfologici e strutturali che caratterizzano tali opere. La seconda sezione esamina sistematicamente le caratteristiche dell’Architettura Paulista e, in special modo, di specifici casi di studio caratterizzati dall’ampia campata. Vengono così introdotte specifiche metodologie di Ottimizzazione Strutturale (SO) utili nella progettazione formale delle strutture - o parte di esse. Le metodologie studiate nel campo della SO offrono la possibilità al progettista di poter controllare (e dunque decidere) la forma finale di organismi architettonici e delle sue componenti, avendo allo stesso tempo la certezza di ottenere strutture performanti. I criteri di ottimizzazione proposti e sperimentati - riguardanti maggiormente casi di trave a sezione variabile - saranno applicati sui casi di studio scelti, dimostrando le potenzialità degli algoritmi sviluppati, i quali fungono da punto di connessione tra la progettazione della forma architettonica e quella della performance strutturale. La documentazione sviluppata – grazie anche agli approfondimenti condotti sui casi di studio – ha consentito di introdurre le prime osservazioni sull’argomento riguardante l’Ottimizzazione Strutturale e sull’importanza che riveste in ambito architettonico, costruttivo e sulla tematica del rapporto forma-struttura. Alla presentazione di metodologie tradizionali di SO segue l’introduzione di metodologie sperimentali applicate sull’elemento strutturale trave - oggetto di sperimentazioni - riportando le principali teorie che la caratterizzano. La valutazione della precisione dei metodi proposti è oggetto di studio nel quale, oltre a constatare l’errore effettivo commesso nell’utilizzo dei metodi riportati dalla letteratura classica sull’argomento, verrà effettuato un primo studio di SO nell’ambito di uno o più casi studio esistenti estrapolati dalle architetture della Scuola Paulista. L’attenzione è dunque focalizzata verso quei criteri di ottimizzazione strutturale che hanno le potenzialità di fungere da elemento di contatto tra una struttura correttamente realizzata e l’equivalente formale che ne deriva, procedendo tramite l’applicazione di criteri sperimentali di ottimizzazione volti alla determinazione di sezioni ottimali valutando differenti combinazioni di carico. Si tratterà dunque, nel corso della presente dissertazione, la determinazione di sviluppo ottimale di travi secondo la trattazione proposta da Timoshenko (il quale fa proprie le teorie di Eulero – Bernoulli, utilizzando la componente di sforzo data dal momento flettente agente per la determinazione della condizione di ottimo per lo sviluppo longitudinale della trave) e di studio e sviluppo di elementi strutturali dalla funzione autoportante (Modelli Comparativi - Appendice). In dettaglio, Nel Capitolo I saranno esaminati i caratteri fondanti della Escola Paulista nel tentativo introdurre le prime considerazioni sulla tematica forma-struttura. La finalità di tali analisi consiste nel voler comprendere la rilevanza del rapporto tra caratteri formali e strutturali dell’architettura in ambito linguistico e costruttivo, fissando le linee guida per la lettura dell’organismo architettonico attraverso la sua decostruzione nelle varie discipline/ragioni che ne concorrono alla formazione. Il Capitolo II descrive criticamente, attraverso il ridisegno e la rielaborazione della documentazione consultata, i casi di studio considerati nella ricerca, anche in questo caso rimarcando il ruolo che gli elementi strutturali hanno rappresentato nella creazione dell’organismo architettonico e di come nello stesso elemento strutturale sia racchiuso il linguaggio dell’architettura e la volontà di denunciare specifiche ideologie. La seconda parte del documento si sviluppa in due capitoli. Essi rappresentano il fulcro della ricerca in quanto vengono presentati i principali criteri sperimentali di ottimizzazione strutturale testati su travi a sezione variabile con geometrie vincolate attraverso l’applicazione di tecniche computazionali (geometria computazionale) accostate all’uso di algoritmi genetici evolutivi per la risoluzione del problema (Capitolo III). Le metodologie proposte verranno valutate attraverso la loro applicazione nel Capitolo IV, all’interno del quale viene discussa la validità dei processi e l’utilità in fase di concepimento di un’opera architettonica, sottolineando l’importanza e l’impatto che gli algoritmi presentati potrebbero avere nella definizione di un nuovo metodo progettuale che possa guidare simultaneamente la progettazione architettonica e quella strutturale. I risultati ottenuti nel capitolo finale e argomentati nella sezione dedicata alle conclusioni, non hanno la prepotenza di contestare la forma delle strutture esistenti, ma vogliono essere testimonianza di come sia possibile avvicinare due mondi, quelli dell’ingegneria e dell’architettura, che hanno subito una scissione nel tempo, ma che restano profondamente connessi. Inoltre, le indagini condotte nella dissertazione desiderano ardentemente sollevare alcuni argomenti iniziali, piuttosto che arrivare a conclusioni definitive, ancora ampiamente lontane data l’estrema complessità di opere che non sono ancora del tutto state raccontate, svelate e demistificate dal punto di vista pratico dell’architettura.
This document intends to study in-depth techniques and propose methodologies to mend the "dichotomy" between architectural shape and structural performance with the help and recognition of the architecture of São Paulo (or Paulist School - or Paulist Brutalist School), by critically examining the period between 1945 and 1970, through two different and not immediately complementary sections. This difficulty is represented by the difference in the themes dealt with in the following chapters, within which we want to focus on the pragmatic concepts that lead to the creation of specific architectural solutions, in turn, guided by fundamental "ideal" representations of the architecture of the School of São Paulo. The document is divided into two different macro-sections, allowing the reader to be guided in the research conducted. Starting from the reasons why specific architectures have lent themselves more than others in representing the "background" of the investigation, the dissertation also deepens historical events, salient points, distinctive and practical characters in the definition of Paulist architecture. The first section aims to provide bibliographic investigations with an approach that is as critical as possible. At the same time, scattered and disconnected references on Paulista Architecture are collected. This allows investigating mainly the incidence of the shape-structure theme concerning specific current and existing works in the panorama of the Escola Paulista, able to reflect the intrinsic value of the architectural language combined with stability and structural innovation. Attention is focused on specific works by the masters Vilanova Artigas and Paulo Mendes da Rocha - pioneers and founders of the School of São Paulo, whose study has allowed us to recognize the aesthetic and ethical characteristics of Paulist Brutalist architecture, underlining the fusion of morphological and structural characters that characterize these works. The second section systematically examines the characteristics of Paulista Architecture and of specific case studies characterized by wide spans. Specific Structural Optimization (OS) methodologies are thus introduced, useful in the shape-design of structures - or part of them. The methods developed in the OS field offer the possibility for the designer to control (and therefore decide) the final shape of architectural organisms and their components while having the certainty of obtaining performing structures. The proposed and tested optimization criteria - mainly concerning cases of variable section beams - will be applied to the selected case studies, demonstrating the developed algorithms' potential, representing the linking point between the design of the architectural shape and that of the structural performance. Thanks to the in-depth analysis conducted on the case studies, the developed documentation made possible the introduction to the first observations on the topic concerning Structural Optimization and its importance in architectural design, construction, and the theme of the shape-structure relationship. The presentation of traditional SO methodologies is followed by introducing experimental methods applied to the structural beam element - subject to experimentation - reporting the main theories that characterize it. The evaluation of the precision of the proposed methods is the subject of study. In fact, in addition to ascertaining the actual error committed to using the methods reported in the classical literature on the subject, one first study of OS will be carried out in the context of one or more case studies extrapolated from the architecture of the Paulista School. Attention is therefore focused on those structural optimization criteria that potentially represent a contact element between a correctly constructed structure and the resulting shape-equivalent, proceeding through the application of experimental optimization criteria to determine optimal sections by evaluating different load combinations. Therefore, in this dissertation, we will determine the optimal development of beams according to the treatment proposed by Timoshenko and the development of structural elements with a self-supporting function (Comparative Models - Appendix). In detail, in Chapter 1, the founding characteristics of the Escola Paulista, to introduce the first considerations on the form-structure theme, will be examined. The purpose of these analyses is to understand the relevance of the relationship between shape features and structural architecture characteristics in the linguistic and constructive field, setting the guidelines for reading the architectural organism through its deconstruction in the various disciplines/reasons that concur to its creation. Through the redraws and elaboration of the consulted documentation, Chapter 2 critically describes the case studies considered in the research, underlining the role of specific structural elements played in the creation of the architectural organism and how in the structural parts itself, the architectural language, and the will to denounce specific ideologies, are contained. The second part of the document is developed in two chapters. They represent the core of the research as the main experimental criteria of structural optimization tested on variable section beams with constrained geometries are presented through the application of computational techniques (computational geometry) combined with the use of evolutionary genetic algorithms for solving the problem. (Chapter III). The proposed methodologies will be evaluated through their application in Chapter IV. First, the validity of the processes and the usefulness in the conception phase of architectural work are discussed. Likewise, the impact of the algorithms presented in defining a new design method that can guide architectural and structural design simultaneously is debated. The results obtained in the final chapter and then discussed in the section dedicated to the conclusions do not have the pride to challenge the architectural shape of the case studies. Still, they want to testify how it is possible to bring two worlds, engineering, and architecture, which have undergone a split over time but remain deeply connected. Furthermore, the conducted investigations are eager to raise some initial arguments rather than reach definitive conclusions, still widely distant given the extreme complexity of works that have not yet been fully narrated, revealed and demystified from the practical architectural point of view.

Дисертація на здобуття наукового ступеня кандидата педагогічних наук за спеціальністю 13.00.10 – інформаційно-комунікаційні технології в освіті. – Інститут інформаційних технологій і засобів навчання НАПН України, Київ, 2017. У дисертації досліджено проблему проектування електронних освітніх ресурсів з математики для учнів початкової школи. Проаналізовано термінологічний апарат з теми дослідження та уточнено дефініції понять «електронний освітній ігровий ресурс» (ЕОІР), «проектування електронного освітнього ресурсу». Здійснено аналіз зарубіжного та вітчизняного досвіду використання електронних освітніх ресурсів (ЕОР) у початковій освіті; теоретично обґрунтовано та розроблено модель проектування ЕОІР для учнів початкової школи та функціональну модель ЕОІР з математики для учнів початкової школи; розроблено факторно-критеріальні моделі оцінювання якості ЕОІР з математики для учнів початкової школи та ефективності навчання з їх використанням; розроблено модель використання ЕОІР з математики для навчання учнів початкової школи. Визначено критерії та підібрано діагностичний інструментарій для оцінювання ефективності методики використання ЕОІР для навчання математики учнів початкової школи. Розроблено основні компоненти методики використання ЕОІР з математики для навчання учнів початкової школи та експериментально перевірено її ефективність. Загальні результати педагогічного експерименту показали, що використання ЕОІР для учнів початкової школи під час навчання математики на основі розробленої методики сприяє формуванню в них математичної та ключових компетентностей і заслуговує на впровадження у навчально-виховний процес початкової освіти.
Thesis for the candidate of Pedagogical Science Degree, specialty 13.00.10 – Information and Communication Technologies in Education. – Institute of Information Technologies and Learning Tools of NAPS of Ukraine, Kyiv, 2017. The thesis focuses on the problems of the designing and using of electronic educational resources on mathematics (EER) for primary school students. The author analyzes the terminology on the topic of research; foreign and Ukrainian experience in implementing of EER in the primary level of education; substantiates the theoretical and methodological bases of the designing of the electronic educational game resources (EEGR) in mathematics for primary school students; expands the stages of design of these EEGR; devises the technique of estimation of the effectiveness of the educational process with such EEGR; proposes the designed typical functional model of such EEGR that can help to build an individual educational path of every student according to their abilities; the model and methodology of using EEGR in mathematics for primary school students. The overall results of the pedagogical experiment confirm that the methodology of using EEGR in mathematics for primary school students developed by the author is effective and can be implemented in the educational process of primary schools.

L’ipertensione arteriosa rappresenta una delle condizioni più frequenti nella popolazione generale con una prevalenza stimata di 1 miliardo di persone, destinata ad aumentare con l’invecchiamento della popolazione globale. La denominazione di “killer silenzioso” sintetizza la sua frequente asintomaticità e le sue sequele in termini di complicanze cardiovascolari (infarto del miocardio), renali, cerebrali e del sistema arterioso periferico. La terapia antiaggregante e anticoagulante rappresenta uno dei cardini della prevenzione primaria e secondaria di queste condizioni, delle quali l’ipertensione è una delle cause principali. Nel 2010, il clopidrogel è stato il secondo farmaco più prescritto al livello mondiale, con un ricavato di 9 miliardi di dollari. Con il diffondersi dell’utilizzo di questi farmaci nella popolazione aumenta però l’incidenza di eventi avversi, specie emorragici, che rappresentano un temibile effetto collaterale, con un rischio di sanguinamento dal tratto gastrointestinale dall’1.5 al 4.5%. La malattia emorroidaria è la più frequente causa di sanguinamento anorettale e colpisce, con almeno un episodio, fino al 50% della popolazione sopra i 50 anni. In pazienti in terapia antiaggregante o anticoagulante gli episodi emorragici correlati all’emorroidi possono essere più severi e meno responsivi alla terapia medica. In questi casi, l’intervento ambulatoriale o chirurgico rappresenta l'unica opzione, ma comporta rischi aggiuntivi di eventi emorragici postoperatori, se la terapia viene continuata, o di nuovi eventi cardiovascolari e cerebrali per sospensione prudenziale della terapia nel periodo perioperatorio. La presente tesi è espressione di un filone di ricerca sulla malattia emorroidaria in termini di anatomia, biologia vascolare e fisiopatologia che il sottoscritto ha intrapreso durante il dottorato e che ha fatto da terreno di coltura a uno studio pilota su una nuova tecnica chirurgica, lo Sclerobanding, che in modo non invasivo, economico e senza anestesia (o se richiesto con anestesia locale) si propone di trattare la malattia emorroidaria e il sintomo principale, il sanguinamento, anche in pazienti ad alto rischio nei quali la sospensione della terapia anticoagulante/antiaggregante per un intervento tradizionale potrebbe comportare un rischio aggiuntivo per il presentarsi di eventi trombotici legati alla sospensione del trattamento e al fenomeno della rebound ipercoagulability. Tale studio è stato preceduto da una descrizione della tecnica sulla rivista ufficiale della Società Europea di Coloproctologia (ESCP), Colorectal Disease e da uno studio sulla fattibilità della metodica in pazienti in buone condizioni di salute pubblicato nel 2021 sul Journal of Clinical Medicine e che rappresenta il terzo capitolo di questa tesi. Il percorso del dottorato mi ha consentito una conoscenza più approfondita sul tema dell’ipertensione e delle sue conseguenze. La possibilità di unire queste nuove conoscenze, anche in termini di trattamenti anticoagulanti/antiaggreganti, per la realizzazione di uno studio su un tema, quello della malattia emorroidaria, a me caro in quanto chirugo colorettale, è stata per me fonte di soddisfazione e di arricchimento culturale. La tesi è cosi strutturata: Il Capitolo I, tratta dell’anatomia, biologia vascolare, fisiologia e fisiopatologia della malattia emorroidaria ed è stato pubblicato nel 2020 in Reviews on Recent Clinical Trials. Il Capitolo II descrive, in una chiave di evoluzione storica, gli interventi e le procedure ambulatoriali per il trattamento della malattia emorroidaria ed è stato pubblicato nel 2021 sulla rivista scientifica Frontiers in Surgery . È stato tra i 10 articoli più letti della rivista nel 2021. Il Capitolo III descrive uno studio di fattibilità e di sicurezza dello Sclerobanding su 97 soggetti non in terapia anticoagulante ed è stato pubblicato sul the Journal of Clinical Medicine. Il Capitolo IV è, infine, uno studio pilota sulla sicurezza, la fattibilità dello Sclerobanding in un campione di 51 pazienti ad alto rischio in terapia anticoagulante e antiaggregante, sottoposti alla procedura senza interrompere la terapia e che rappresenta la parte sperimentale della presente tesi.