Le mouvement brownien fractionnaire (mbf) constitue un important outil de modélisation utilisé dans plusieurs domaines (biologie, économie, finance, géologie, hydrologie, télécommunications, etc.) ; toutefois, ce modèle ne parvient pas toujours à donner une description suffisamment fidèle de la réalité, à cause, entre autres, des deux limitations suivantes : d’une part le mbf est un processus gaussien, et d’autre part, sa rugosité locale (mesurée par un exposant de Hölder) reste la même tout le long de sa trajectoire, puisque cette rugosité est partout égale au paramètre de Hurst H qui est une constante. En vue d'y remédier, S. Stoev et M.S. Taqqu (2004 et 2005) ont introduit le mouvement multifractionnaire stable linéaire (mmsl) ; ce processus stochastique strictement α-stable (StαS), désigné par {Y(t)}t, est obtenu en remplaçant la mesure brownienne par une mesure StαS et le paramètre de Hurst H par une fonction H(.) dépendant de t. On se place systématiquement dans le cas où cette fonction est continue et à valeurs dans l’intervalle ouvert ]1/α,1[. Il convient aussi de noter que l’on a pour tout t, Y(t)=X(t,H(t)), où {X(u,v)}(u,v) est le champ stochastique StαS, tel que pour tout v fixé, le processus {X(u,v)}u est un mouvement fractionnaire stable linéaire. L'objectif de la thèse est de mener une étude approfondie du mmsl, au moyen de méthodes d’ondelettes ; elle consiste principalement en trois parties. (1) On détermine de fins modules de continuité, globaux et locaux de {Y(t)}t ; cela repose essentiellement sur une nouvelle représentation de {X(u,v)}(u,v), sous la forme d’une série aléatoire, dont on montre la convergence presque sûre dans certains espaces de Hölder. (2) On introduit, via la base de Haar, une autre représentation de {X(u,v)}(u,v) en série aléatoire ; cette dernière permet la mise en place d'une méthode de simulation efficace du mmsl, ainsi que de ses parties hautes et basses fréquences. (3) On construit des estimateurs par ondelettes du paramètre fonctionnel H(.) du mmsl, ainsi que de son paramètre de stabilité α. / Fractional Brownian Motion (FBM) is an important tool in modeling used in several areas (biology, economics, finance, geology, hydrology, telecommunications, and so on); however, this model does not always give a sufficiently accurate description of reality, two important ones among its limitations, are the following: on one hand, FBM is a Gaussian process, and on the other hand, its local roughness (measured through a Hölder exponent) remains the same all along its path, since this roughness is everywhere equal to the Hurst parameter H which is a constant. In order to overcome the latter two limitations, S. Stoev and M.S. Taqqu (2004 and 2005) introduced Linear Multifractional Stable Motion (LMSM); this strictly α-stable (StαS) stochastic process, denoted by {Y(t)}t , is obtained by replacing the Brownian measure by a StαS one and the Hurst parameter H by a function H(.) depending on t. One assumes the latter function to be continuous and with values in the open interval (1/α,1). Also, it is worth noticing that one has for all t, Y(t)=X(t,H(t)), where {X(u,v)}(u,v) is the StαS stochastic field, such that for all fixed v, the process {X(u,v)}u is a Linear Fractional Stable Motion. The goal of the thesis is to conduct a thorough study on LMSM by making use of wavelet methods; it mainly consists into three parts. (1) One determines, sharp global and local moduli of continuity for {Y(t)}t; this mainly relies on a new representation of {X(u,v)}(u,v), as a random series which almost surely converges in some Hölder spaces. (2) One introduces, via the Haar basis, another random series representation of {X(u,v)}(u,v); the latter representation allows to derive an efficient simulation for LMSM as well as its high and low frequency parts. (3) One constructs wavelet estimators of the functional parameter H(.) of LMSM and of its stability parameter α.

A pele humana é o maior e um dos mais importantes órgãos, esta é a principal barreira entre o corpo e o meio externo, sempre que há um desequilíbrio no mecanismo de defesa inata, o risco de uma infecção é proeminente. A queimadura é um quadro clínico em que a barreira epitelial é danificada, resultado em uma defesa imunológica deficitária e uma grande abundância de nutrientes, o que favorece a proliferação de micro-organismos. No presente estudo foi desenvolvido e caracterizado uma suspensão inovadora de nanocápsulas poliméricas de poli(ε-caprolactona) com triclosan e α-bisabol, revestida com quitosana (NCCQ) para aplicação tópica em feridas de queimadura, com intuito de reduzir a contaminação microbiana. Foram realizados ensaios de caracterização físico-química, microbiológica e cutânea. Os resultados obtidos para os ensaios de caracterização mostraram que a suspensão de nanocápsulas apresentou diâmetro de partícula adequado, ausência de cristais ou contaminação micrométrica, potencial zeta catiônico, pH levemente ácido, com uma alta eficiência de encapsulação e estável no período de trinta dias. Os ensaios microbiológicos foram realizados frente Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa e Candida albicans, através de microdiluição seriada e teste do desafio. O primeiro ensaio mostrou uma redução no valor da concentração inibitória mínima e melhores resultados para quase todas as cepas analisadas frente aos controles e dados da literatura, inclusive P. aeruginosa, a qual possui uma alta resistência ao triclosan. O teste do desafio serviu para comprovar que o efeito antimicrobiano da suspensão, se manteve ao longo de 28 dias, mesmo quando incorporada ao curativo de hemi-celulose. Os ensaios cutâneos de permeação/penetração e lavabilidade mostraram que a suspensão de nanocápsulas NCCQ teve uma retenção no estrato córneo, o que é desejável para uma formulação tópica. / The human skin is the largest and one of the most important organs, this is the main barrier between the body and the external environment, whenever there is an imbalance in innate defense mechanism, the risk of infection is prominent. Burn is a clinical condition in which the epithelial barrier is damaged, resulting in a deficient immune defense, also the abundance of nutrients favors to proliferation of microorganisms. In the present study it was developed and characterized a novel suspension of polymeric nanocapsules of poly (ε-caprolactone) with triclosan and α-bisabol coated with chitosan (NCCQ) for topical application in burn wounds, in order to reduce microbial contamination. Physical-chemical, microbiological and skin tests were performed in order to prove its applicability. The results obtained for the characterization tests showed that the suspension of nanocapsules had suitable particle diameter, no crystals or micrometric contamination cationic zeta potential, slightly acid pH with a high encapsulation efficiency and stable within thirty days. Microbiological tests were carried forward Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, via serial microdilution and challenge test. The first experiment showed a reduction in the value of the minimum inhibitory concentration and best results for nearly all strains analyzed when compared with controls and data from the literature, including P. aeruginosa, which has a high resistance to triclosan. The challenge test was used to demonstrate that the antimicrobial effect of the suspension was maintained over 28 days even when incorporated into the dressing hemi-cellulose. The skin permeation/penetration and washability tests of NCCQ nanocapsule suspension showed that it had retention in the stratum corneum, which is desirable for a topical formulation.

No description available.

First published online: 2012-03-10 / 名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成24年4月27日 森谷隆氏の博士論文として提出された

No description available.

α-Synuclein is an abundant nerve terminal protein and a primary component of the Lewy body pathology seen in Parkinson’s disease. While the precise biological and pathological role of α-synuclein remains unclear, its ability to bind to and dissociate from synaptic membranes may be linked to its function in these states. In this thesis, we characterized the role of the GTPase protein rab3a as a potential regulator of α-synuclein membrane binding and dissociation. We found evidence that GTP-bound rab3a sequesters α-synuclein on membranes during exocytosis, and that inhibition of rab3a dissociation from the membrane causes inhibition of α-synuclein dissociation as well. Furthermore, we found that the loss of rab3a in human neuroblastoma cells increases α-synuclein expression. This study identifies rab3a and proteins associated with its membrane dissociation as mediators of α-synuclein membrane binding and dissociation.

α-Synuclein is an abundant nerve terminal protein and a primary component of the Lewy body pathology seen in Parkinson’s disease. While the precise biological and pathological role of α-synuclein remains unclear, its ability to bind to and dissociate from synaptic membranes may be linked to its function in these states. In this thesis, we characterized the role of the GTPase protein rab3a as a potential regulator of α-synuclein membrane binding and dissociation. We found evidence that GTP-bound rab3a sequesters α-synuclein on membranes during exocytosis, and that inhibition of rab3a dissociation from the membrane causes inhibition of α-synuclein dissociation as well. Furthermore, we found that the loss of rab3a in human neuroblastoma cells increases α-synuclein expression. This study identifies rab3a and proteins associated with its membrane dissociation as mediators of α-synuclein membrane binding and dissociation.

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Η αποαδενυλίωση είναι συνήθως στάδιο που καθορίζει το ρυθμό της αποικοδόμησης και καταστολής της μετάφρασης του mRNA. Το γεγονός αυτό καθιστά την αποαδενυλίωση ως το κυριότερο σημείο ελέγχου για τις δύο αυτές διαδικασίες. Οι αποαδενυλάσες είναι εξωριβονουκλεάσες εξαρτώμενες από Mg2+ που υδρολύουν το mRNA με κατεύθυνση 3’-5’, παράγοντας 5’-AMP. Ο αριθμός των γνωστών αποαδενυλασών έχει επεκταθεί πρόσφατα, κυρίως μέσω βιοχημικών και γενετικών μελετών. Όλες οι γνωστές αποαδενυλάσες ανήκουν σε μία από της δύο ομάδες, την DEDD ή την exonuclease– endonuclease–phosphatase (EEP) υπέρ-οικογένεια. ∆εν είναι ακόμη πλήρες κατανοητό το πλεονέκτημα της ύπαρξης τόσων πολλών αποαδενυλασών. Υποψήφιες αποαδενυλάσες έχουν ταυτοποιηθεί με χρήση προγραμμάτων βιοπληροφορικής, ωστόσο η δράση τους δεν έχει αποδειχθεί, όπως η PNLDC1. Αρχική βιοπληροφορική ανάλυση του γονιδίου pnldc1 έδειξε ότι κωδικοποιεί μια πρωτεΐνη η οποία παρουσιάζει ομολογία με την PARN με πετιδικό σήμα που σχετίζεται με μια διαμεμβρανική περιοχή της πρωτεΐνης. Επιπρόσθετα φαίνεται ότι ο υποκινητής του γονιδίου περιέχει νησίδες CpG. Με μοντελοποίηση με βάση την ομολογία με την ανθρώπινη PARN της περιοχής με δράση νουκλεάσης προβλέφθηκε μία δομή που περιέχει ενεργό κέντρο με τα χαρακτηριστικά κατάλοιπα DEDD να κατευθύνονται σωστά για να αλληλεπιδρούν με τα ιόντα Mg2+ και να υποστηρίζουν ενζυμική δράση. Το γονίδιο pnldc1 κλωνοποίηθηκε και η ανασυνδυασμένη πρωτεΐνη καθαρίσθηκε με χρωματογραφία συγγένειας. Πειράματα αποαδενυλίωσης έδειξαν ότι η PNLDC1_1 είναι μία αποαδενυλάση με υψηλή συγγένεια γα το υπόστρωμα poly(A) αλλά χαμηλή Vmax σε σχέση με την PARN. Η έκφραση του pnldc1 μπορεί να ρυθμιστεί μέσω μεθυλίωσης του υποκινητή. Ύστερα από χορήγηση του απομεθυλιωτικού 5-Aza- Deoxycytidine, η έκφραση του pnldc1 αυξήθηκε 4 φορές στις κυτταρικές σειρές HaCaT και HEK 293 T ενώ τα επίπεδα των PARN και CNOT7 mRNA παρέμειναν αμετάβλητα, υποδεικνύοντας πιθανόν έναν μοναδικό ρόλο της νέας αποαδενυλάσης στην γονιδιακή έκφραση κατά την διάρκεια της εμβρυογένεσης και διαφοροποίησης. Η αποαδενυλάση PAN2 είναι μέλος της οικογένειας των DEDD νουκλεασών και ξεκινάει την βράχυνση του poly(A) απομακρύνοντας σχεδόν την μισή ουρά μέχρι άλλες αποαδενυλάσες να αποικοδομούν την υπόλοιπη. Παρόλο που ο ρόλος της PAN2 στην αποικοδόμηση του mRNA είναι διευκρινισμένος ο βιολογικός ρόλος του ενζύμου δεν έχει αποσαφηνισθεί. Μετά από αποσιώπηση της PΑΝ2 παρατηρήθηκε σημαντική μείωση στα επίπεδα έκφρασης των mRNA PARN, Papola, CBP20 και MYC ενώ δεν παρατηρήθηκε σημαντική αλλαγή στα επίπεδα EIF4E και CNOT7. Τα αποτελέσματα μας υποδεικνύουν ότι η PAN2 εμπλέκεται στην γονιδιακή έκφραση πιθανόν με το να διεγείρει την αποικοδόμηση των ήδη υπαρχόντων μορίων mRNA επιτρέποντας την παραγωγή νέων. / Deadenylation is often a rate-limiting step for mRNA decay and translational silencing, making it an ideal control point for both processes. Deadenylases are Mg2+ -dependent exoribonucleases that hydrolyse RNA in the 3′→5′ direction, which results in release of 5′- AMP. The number of known deadenylases has recently expanded, mainly through biochemical and genetics studies. All known deadenylases belong to one of two groups, the DEDD or the exonuclease–endonuclease–phosphatase (EEP) superfamilies. The advantage of having such a range of deadenylases is not yet completely understood. Putative deadenylases have been identified through bioinformatics, but their functions has not yet been established such as the mammalian PNLDC1 which is a Poly(A)-specific Ribonuclease homolog (termed PARN-like). Preliminary bioinformatic analysis of the pnldc1 gene revealed that it encodes a PARN-similar protein with a signal peptide which corresponds to a trans-membrane region. In addition several CpG islands within the gene’s promoter were identified. Homology modeling of the nuclease domain with its human PARN homolog predicted a structure that forms an active site which contains the characteristic DEDD residues orientated correctly to interact with Mg2+ ions to support enzymatic activity. Pnldc1 was cloned and the recombinant protein was purified via affinity chromatography. Deadenylation assays showed that PNLDC1_1 is a deadenylase with high affinity for poly(A) substrates but relatively low Vmax compared to PARN. Interestingly, pnldc1 expression can be regulated through promoter methylation. Following treatment with the demethylating agent, 5-Aza-Deoxycytidine, pnldc1 expression was increased 4-fold in both HaCaT and HEK 293 T cell lines, whereas PARN and CNOT7 mRNA expression was unchanged, thus implying a possible unique role of this new deadenylase in gene expression during embryogenesis and differentiation. PAN2 deadenylase, a member of the DEDD nucleases initiates poly(A) degradation removing almost half of the tail when other nucleases degrade the rest of the tail. Although the role of PAN2 in mRNA degradation has been revealed, its biologic function has not been studied. On knockdown of PAN2 a significant reduction was observed in the levels of PARN, Papola, CBP20 and MYC, but no significant change changes were observed in the levels of EIF4E and CNOT7 mRNA expression. Our results indicate that PAN2 is involved in gene expression, this probably by triggering the degradation of the current messengers allowing production of new messengers.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-10-03T12:12:48Z No. of bitstreams: 1 THIAGO JOSÉ FIGUEIRA RAMOS.pdf: 1733228 bytes, checksum: c1606fa7a4e1569d3cc660ae62c32331 (MD5) / Made available in DSpace on 2013-10-03T12:12:48Z (GMT). No. of bitstreams: 1 THIAGO JOSÉ FIGUEIRA RAMOS.pdf: 1733228 bytes, checksum: c1606fa7a4e1569d3cc660ae62c32331 (MD5) Previous issue date: 2012-10-10 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A silicose é uma doença ocupacional causada pela inalação de sílica em sua forma livre e cristalina. A inflamação e fibrose pulmonar constituem as principais características da patologia, que envolve uma variedade de mediadores inflamatórios, como o TNF-α, um mediador de ações amplas e que exerce seus efeitos em um grande número de tipos celulares. LASSBio-468 é um análogo da talidomida já descrito como capaz de modular a produção de TNF-α e inibir o choque endotóxico e artrite reumatóide em modelos animais. Neste estudo, nós investigamos o potencial efeito do LASSBio-468 em modelo de silicose experimental em camundongos. Animais Swiss-webster machos (18 – 20g) foram instilados intranasalmente com uma suspensão de sílica (10 mg/50 μL) e veículo (salina). O tratamento consistiu na administração por via oral de LASSBio-468 (12,5 – 100 mg/kg) e talidomida (50 mg/Kg) durante 7 dias consecutivos, do vigésimo primeiro ao vigésimo oitavo dia após a instilação de sílica. Foram analisadas a função pulmonar (resistência e elastância) e hiperreatividade das vias aéreas à aerolização de metacolina (3 – 27 mg/mL), através de pletismógrafo invasivo de corpo inteiro (Finepoint, Buxco System), além de análises morfológicas e morfométricas do tecido pulmonar. A produção de colágeno tecidual foi acessada pelo método de sircol, enquanto que a quantificação de citocinas/quimiocinas foi realizada pelo ensaio de ELISA. A técnica de imunohistoquímica permitiu a identificação da população de miofibroblastos no pulmão. Através de uma digestão mecânica e enzimática do tecido pulmonar, obtivemos uma cultura primária de miofibroblastos que, assim como macrófagos alveolares AMJ2C11 e células epiteliais A549, foram pré-tratados com diferentes concentrações de LASSBio-468 e talidomida, estimulados com IL-13 ou sílica e avaliados quanto a proliferação celular, viabilidade e produção de mediadores inflamatórios in vitro. Demonstramos que animais silicóticos apresentaram níveis basais elevados de resistência pulmonar e elastância, bem como hiperreatividade frente à aerolização de metacolina. Resposta inflamatória tecidual, extensiva deposição de colágeno, formação de granuloma e produção de quimiocinas (KC and MCP-1) e citocinas (TNF- αand TGF-β) também foram detectadas em pulmões de animais silicóticos, assim como o aumento do número de miofibroblastos no tecido. O tratamento com LASSBio-468 reduziu o comprometimento da função pulmonar e hiperreatividade, formação de granulomas, expressão de miofibroblastos e deposição de colágeno no tecido pulmonar de animais doentes. Miofibroblastos oriundos de animais silicóticos apresentaram basal de proliferação superior, sendo responsivos também ao estímulo mitogênico da IL-13, que foi atenuado frente ao pré-tratamento com LASSBio-468. A estimulação de macrófagos alveolares e células epiteliais com sílica promoveu a liberação de TNF-α e IL-8, respectivamente, sendo o LASSBio-468 capaz de inibir de forma significativa esta produção. Em conjunto, nossos resultados mostraram que o tratamento com LASSBio-468 foi capaz de reduzir de forma curativa o comprometimento da função pulmonar e resposta granulomatosa, através de ações sobre células específicas, indicando que o composto em questão parece constituir uma promissora ferramenta para o tratamento de doenças pulmonares fibróticas crônicas como a silicose. / Silicosis, one of the oldest occupational diseases in the world, is a consequence of long-term exposure to inhaled dust containing silica in its free and crystalline form. Lung interstitial inflammation and fibrosis are the main features of the disease, involving a wide range of chemical mediators such as TNF-α. This is a pleiotropic molecule which exerts its effects on many cell types. LASSBio-468 is a thalidomide analogue which modulates TNF-α production and inhibits endotoxic shock and arthritis in animal models. In this study we investigated the effect of LASSBio-468 on experimental silicosis in mice. Anesthetized male Swiss-Webster mice (18-20g) received intranasal (i.n.) instillation of silica (10 mg/50 μL) and vehicle (saline). Treatment consisted of oral administration of the LASSBio-468 (12,5 - 100 mg/kg) and thalidomide (50 mg/Kg) during 7 consecutive days, from day 21 to 28 post-silica. The analyses included lung function (resistance and elastance) and airways hyperreactivity to aerosolized metacholine (3 -27 mg/mL) measured by whole body invasive plestimography (Finepoint, Buxco System). Morphological and morphometrical analyses included classical dyes such as Hematoxylin-Eosin and Picrus-Sirius. Collagen content and cytokine/chemokine generation were quantified by Sircol technique and ELISA, respectively. Immunohistochemistry was employed to identify the lung myofibroblastic population. Primary murine myofibroblasts cells were obtained after a mechanical and enzymatic digestion of the lung from saline and silicotic mice. Myofibroblasts, AMJ2C11 alveolar macrophages and A549 epithelial cells were pre-treated with LASSBio-468 and Thalidomide, stimulated with IL-13 or silica and evaluated thought proliferation, viability and production of inflammatory mediators in vitro. We showed that silicotic mice exhibited increased basal levels of lung resistance and elastance as well as airways hyperreactivity to methacholineaerosolization. Tissue inflammatory response, extensive collagen deposition, granuloma formation and chemokine (KC and MCP-1)/cytokine (TNF-α and TGF-β) generation were also detected in the silicotic lungs, as well as an increasing of myofibroblasts expression in the lung. Administration of LASSBio- 468 into silicotic mice reduced the decline lung function and airways hyperreactivity, myofibroblasts numbers, tissue collagen deposition and granulomatous area. The generation of cytokines and chemokines was also suppressed by the drug. The primary lung myofibroblasts obtained from silicotic mice showed a superior basal proliferation and IL-13, a pro-fibrotic cytokine, stimulated the (3H) incorporation in vitro, which was diminished by the treatment with LASSBio-468. Stimulation of alveolar macrophages and epithelial cells by silica in vitro increased the release of TNF-α and IL-8, respectively, and LASSBio-468 was able to reduce these inflammatory mediators production. The compound modulated key cells functions without decline in cell death. Altogether our findings show that the treatment of silicotic mice with LASSBio-468 reduced curatively the decline in lung function and granulomatous response, throught actions on myofibroblasts, macrophages and epithelial cells, indicating that this compound constitutes a promising tool for the treatment of chronic fibrotic pulmonary diseases such as silicosis.