L’adénocarcinome pancréatique canalaire s’accompagne d’une néoexpression de la mucine membranaire MUC4 et d’une surexpression des mucines membranaires MUC1 et MUC16. Ces O-glycoprotéines de haut poids moléculaire sont codées par des ARNm possédant des particularités inhabituelles par rapport aux autres transcrits humains, comme une longue demi-vie et une très grande taille. La galectine-3, une lectine endogène également surexprimée au cours du cancer pancréatique, exerce de très nombreuses fonctions biologiques, en particulier dans le domaine du trafic intracellulaire des glycoprotéines et de l’épissage des pré-ARNm. Cependant, l’implication de cette galectine à un autre niveau du cycle de vie des ARNm n’avait pas été explorée jusque-là dans la littérature. De précédents travaux du laboratoire ont démontré que la suppression de l’expression de la galectine-3 dans la lignée cellulaire cancéreuse pancréatique humaine CAPAN-1 s’accompagne d’une diminution de l’expression des transcrits de certaines mucines membranaires. L’objectif de ce travail a donc été d’étudier les mécanismes de régulation de l’expression des mucines membranaires, et plus particulièrement MUC4, par la galectine-3.Nous avons démontré que la galectine-3, in vitro, régule l’expression de MUC4 au niveau post-transcriptionnel en stabilisant les transcrits de cette mucine. Ceci passe par la potentialisation de la fixation de la RNA Binding Protein hnRNP-L sur l’élément cis-régulateur CA repeat présent dans le 3’UTR de MUC4. Nos résultats indiquent que cette régulation est présente in vivo au niveau physiologique dans des tissus épithéliaux digestifs murins. Par ailleurs, nous avons mis en évidence que la galectine-3 interagit avec hnRNP-L dans le cytoplasme mais qu’elle interagit faiblement avec des marqueurs de P-Bodies ou de granules de stress. Concernant le rôle de la galectine-3 dans le cycle de vie des ARNm, nos données révèlent que celle-ci se lie à aux transcrits matures de MUC4 au niveau périnucléaire, probablement dans des granules de stockage qui ne sont ni des granules de stress ni des P-bodies et dont le type reste à déterminer. Nous avons également élargi nos résultats en étudiant l’implication de cette lectine dans le métabolisme d’autres ARNm et nos analyses indiquent que la galectine-3 serait impliquée dans la régulation post-transcriptionnelle positive ou négative d’un ensemble de transcrits dont les fonctions convergent vers les voies UPR (Unfolded protein response) et ERAD (Endoplasmic-reticulum-associated protein degradation) mais également plus généralement vers le processing des protéines en réponse au stress du réticulum endoplasmique.En conclusion, nos travaux mettent en évidence un nouveau rôle de la galectine-3 en tant que RNA binding protein dans la stabilisation des ARNm de MUC4 mais aussi un nouveau rôle dans la coordination de l’expression de répertoires de transcrits matures ayant des rôles biologiques communs (RNA regulon) permettant à la cellule de s’adapter au plan morphologique, métabolique et biologique à des changements physiopathologiques. Ceci renforce les interconnexions largement décrites dans la littérature entre mucines, galectine-3 et les grandes fonctions cellulaires qui sont perturbées en situation cancéreuse. / Pancreatic ductal adenocarcinoma is characterized by a neo expression of the membrane-bound mucin MUC4 and an overexpression of membrane-bound mucins MUC1 and MUC16. These high molecular weight O-glycoproteins are encoded by mRNA sharing unusual features among human transcripts, such as a long half-life and a very large size. Galectin-3, an endogenous lectin frequently over-expressed in pancreatic cancer, has many biological functions, especially in intracellular glycoprotein trafficking and pre-mRNA splicing. However, the involvement of this lectin in another step of mRNA life cycle has not been explored in literature yet. Previous works performed in the laboratory have demonstrated that LGALS3 gene knock-down in a human cancerous pancreatic cancer cell line is followed by a decrease of the expression of several membrane-bound mucin mRNAs. The aim of this present work was to study the mechanism of the regulation of mucins expression, especially for MUC4, by galectin-3.We have demonstrated that galectin-3, in vitro, regulates MUC4 expression at the post-transcriptionnal level through the stabilization of the transcripts of this mucin. Galectin-3 potentiates the binding of hnRNP-L, a RNA-Binding protein, on the CA repeat region present in MUC4 3’UTR. Our results show that this regulation occurs physiologically in vivo in mice digestive epithelial tissues. Moreover, we have demonstrated that galectin-3 interacts with hnRNP-L in cell cytoplasm but scarcely with protein markers of P-Bodies or stress granules markers. Regarding the influence of galectin-3 in mRNA life cycle, our results suggest that it binds to mature MUC4 transcripts in the perinuclear area, probably in storage granules whose type should to be determined. We have also broadened our results by studying this lectin’s involvement in the metabolism of other mRNA. Our analyzes suggest that galectin-3 could be involved in the positive or negative post-transcriptionnal regulation of a mRNA subset whose functions are linked to unfolded protein response (UPR) and Endoplasmic-reticulum-associated protein degradation (ERAD) pathways, but also more generally towards protein processing in response to endoplasmic reticulum stress.In conclusion, our work highlights a new function for galectin-3 as a RNA binding protein in the stabilization of MUC4 mRNA, but also a new function in the coordination of the expression of repertories of mature transcripts with shared functions or (RNA regulon) allowing morphological, biological and metabolic cell adaptation to physiopathological changes. These results strengthen the interplay between mucins, galectin-3 and cellular functions which are disturbed in cancer.

L'inflammation allergique de la peau est un état dans lequel l'hôte réagit de manière excessive à des allergènes en induisant une inflammation de type Th2. Un certain nombre d’acteurs cellulaires et moléculaires ont été impliqués dans cette pathologie, mais la façon dont ils agissent dans le réseau inflammatoire demeure largement inconnue. Les basophiles ont été reconnus pour leurs fonctions effectrices en allergie, cependant, comment ils sont recrutés et activés, ainsi que comment ils interagissent avec d'autres cellules dans l’inflammation allergique cutanée demeurent mal caractérisés. L'objectif de ce travail de thèse est d'étudier le recrutement, l'activation et la fonction des basophiles dans le réseau inflammatoire de la peau allergique. En employant un modèle expérimental murin de dermatite de contact allergique, combiné à des outils génétique de souris, d’immunologie et d’approches biologiques cellulaires/moléculaires, l’étude de ma thèse a démontré que l’IL-3 joue un rôle crucial dans l’extravasation des basophiles dans la peau allergique des souris (Partie I), et que l’invalidation des basophiles chez les souris Mcpt8DTR entraîne une réduction systémique des éosinophiles et des neutrophiles (Partie II). Ces études fournissent ainsi de nouvelles connaissances sur le recrutement, l'activation et la fonction des basophiles dans l'inflammation allergique de la peau. / Allergic skin inflammation is a state in which the host overreacts to otherwise innocuous allergens by inducing T helper type 2 inflammation. A number of cellular and molecular players have been implicated in the generation of allergic skin inflammation, but how they act and crosstalk in the inflammatory network remains largely unknown. Basophils have been recognized for their effector functions in allergy, however, how they are recruited to the inflamed tissue, get activated and crosstalk with other cells in inflammatory skin remain still incompletely understood. The objective of this PhD study is to investigate the recruitment, activation and function of basophils in the inflammatory network of allergic skin. Using experimental mouse model for allergic contact dermatitis, combined with mouse genetic tools, immunology and cellular/molecular biology approaches, the study of my thesis discovered that IL-3 plays a crucial role in basophil extravasation to mouse allergic skin (Part I), and that the depletion of basophils in Mcpt8DTR mice leads to a systemic reduction of eosinophils and neutrophils (Part II). These studies provide novel insights into the recruitment, activation and function of basophils in allergic skin inflammation.

L’exploration des interactions fondamentales entre les particules subatomiquesa abouti à la construction du Modèle Standard de la physique des particules qui n’a été misen défaut par aucune expérience en laboratoire. Cependant, de sérieuses indications théoriqueset cosmologiques révèlent des insuffisances au Modèle Standard et des déviationssont attendues. Cette nouvelle physique est recherchée auprès des grands collisionneursmettant en jeu des énergies de l’ordre de l’échelle électrofaible et au-delà. A contre-courant,la nouvelle physique pourrait aussi se manifester à très basse énergie, nécessitant des techniquesexpérimentales atypiques. Cette thèse traite des nouvelles forces de courte portéedépendantes du spin, sujet au coeur de la physique de précision à basse énergie. Un gazd’hélium 3 hyperpolarisé a été utilisé comme sonde de cette nouvelle interaction de portéesubmillimétrique. Ce manuscrit présente la meilleure exclusion sur l’intensité du couplagescalaire-pseudoscalaire gsgp pour des portées entre 1 μm et 100 μm correspondant à desmasses inférieures à 1 eV. / The exploration of fundamental interactions between subatomic particles ledto the particle physics Standard Model which remains unchallenged by any lab experiment.However, some serious theoretical and cosmological clues reveal shortcomings inthe Standard Model and deviations are expected. This new physics is searched for at largecolliders with energies of the order of the electroweak scale and beyond. With a differentphilosophy, one can expect the new physics to show up at very low energy using atypicalexperimental techniques. This thesis deals with short-range spin-dependent new forceswhich are at the heart of the low energy precision physics. A hyperpolarized helium 3 gaswas used as a probe of this new sub-mm interaction. This manuscript present the bestexclusion limit on the strength of a scalar-pseudoscalar coupling gsgp for ranges between1 μm and 100 μm, which correspond to sub-eV masses.

Problemställning: År 2005 trädde IFRS 3 – Rörelseförvärv, vars syfte är att reglera rapporteringen av rörelseförvärv, ikraft. Denna standard innehöll även nya regler om identifiering av immateriella tillgångar som innebär att sådana i större utsträckning ska särskiljas från goodwill. Trots detta visar tidigare studier att beloppet av goodwill i genomsnitt motsvarar mer än hälften av köpeskillingen. Det var även så att av 92 förvärvande bolag år 2016 saknade 52 specifikationer av identifierade immateriella tillgångar. Dessa två förhållanden leder till informationsasymmetri som får konsekvenser för kapitalmarknaden. Syfte: Syftet är att undersöka branschmässiga skillnader mellan företag i olika branscher på den svenska börsen gällande fördelning av köpeskilling på tillgångsslagen finansiella och materiella, immateriella samt goodwill vid rörelseförvärv. Studien syftar även till att undersöka om det finns skillnader gällande vilka typer av immateriella tillgångar som presenteras. Forskningsfrågor: Skiljer sig branscherna åt gällande fördelningen av köpeskilling på finansiella och materiella tillgångar, immateriella tillgångar och goodwill? Vid rörelseförvärv, vilka olika typer av immateriella tillgångar identifierar företagen i de olika branscherna? Metod: Studien är av kvantitativ karaktär och bygger på en sammanställning av samtliga rörelseförvärv år 2016, genomförd av Björn Gauffin. Det empiriska materialet har statistiskt bearbetats med en variansanalys och sedermera analyserats med hjälp av den teoretiska referensramen. Resultat: I samtliga branscher, förutom i materialbranschen, hänfördes cirka 50 % av köpeskillingen till goodwill. Det finns signifikanta skillnader mellan branschen Material och branscherna Hälsovård, Informationsteknologi och Telekomoperatörer gällande fördelning av köpeskillingen på finansiella och materiella tillgångar, immateriella tillgångar och goodwill. Kundrelaterade immateriella tillgångar var den typ som förekom mest frekvent i samtliga branscher förutom materialbranschen. Kunskapsbidrag: Resultatet överensstämmer med tidigare studier: det går ej att bekräftabranschpraxis gällande fördelning av köpeskillingen vid rörelseförvärv. / Presentation of the problem: In 2005, IFRS 3 – Business Combinations, which aims to regulate the reporting of business combinations, became effective. This standard also included rules on the identification of intangible assets, to be more comprehensively distinguished from goodwill. Nevertheless, previous studies show that the amount of goodwill on average equals more than half of the purchase price. It was also the case that 52, of the 92 acquiring companies in 2016, lacked specifications of identified intangible assets. These two conditions lead to information asymmetry that has consequences for the capital market. Purpose: The purpose is to examine whether there are differences between Swedish listed companies in different industries regarding Purchase Price Allocations. The purpose is also to examine whether there are differences regarding identifications of intangible assets. Research questions: Do the companies in different industries differ regarding allocation of purchase price on financial and tangible assets, intangible assets and goodwill? What different types of intangible assets do companies in different industries identify? Methodology: The study has a quantitative research strategy and is based on a compilation of acquisitions made by listed Swedish companies during 2016. The empirical data have been statistically processed using a variance analysis and subsequently analyzed using the theoretical reference frame. Results: In all industries except one, approximately 50 % of the purchase price was attributed to goodwill. There are only significant differences between a limited number of industries regarding the allocation of the purchase price of financial and tangible assets, intangible assets and goodwill. Customer-related intangible assets were the most frequently occurring in all industries except in the materials industry. Contributions to knowledge: The results of the study are consistent with previous studies: it is not possible to confirm that there are industry practices regarding the allocation of the purchase price for business combinations.

Existem diversos trabalhos publicados sobre a utilização de diferentes métodos imunológicos para diagnosticar infecções do trato respiratório causadas por vírus parainfluenza bovino tipo 3 (bPIV-3). Entretanto, é escassa a literatura sobre a utilização da técnica de isolamento viral. Até o presente momento não havia sido relatada a utilização da Transcrição Reversa - Reação em Cadeia da Polimerase (RT-PCR), na detecção de bPIV-3. O objetivo deste estudo foi contribuir para uma melhor caracterização dos bPIV-3 através do desenvolvimento de técnicas de RTPCR para a sua detecção. Utilizando-se uma amostra referência de bPIV-3 (SF-4) e uma amostra de bPIV-3 isolada no Rio Grande do Sul (amostra DIO) foram desenvolvidas técnicas de RT-PCR para a amplificação de diferentes regiões do gene da hemaglutinina-neuraminidase (HN). Após seqüenciamento parcial do gene HN e alinhamento das seqüências da amostra DIO, os resultados revelaram homologia de 99% em relação à amostra referência, 98% a 91% quando comparada com outras amostras de bPIV-3 previamente publicadas na rede e 79% a 80% quando comparada com as amostras de hPIV-3. Foi desenvolvida, também, uma técnica de RT-PCR para amplificação de parte do gene da HN do bPIV-3 e do hPIV-3. Nos experimentos de otimização, a técnica de RT-PCR, comparada com o isolamento viral, apresentou sensibilidade de 140 DICC50, boa especificidade e reprodutibilidade. Os resultados, após seqüenciamento da amostra de vírus bPIV-3 isolada no RS, apresentaram, grande homologia com os das amostras de bPIV-3 comparadas, especialmente a amostra referência. Os dados obtidos neste estudo mostraram que a RT-PCR desenvolvida para a detecção de PIV-3 foi capaz de amplificar um fragmento de 1009 bp do gene da HN de amostras de PIV-3 isoladas de bovinos e humanos, possibilitando a sua utilização em diagnóstico e em estudos epidemiológicos. / There are several published studies on the application of different immunological methods for diagnosis of respiratory infections caused by bovine type 3 Parainfluenza virus (bPIV-3). However, the literature on viral isolation procedure is very scarce. At present there is no report about the utilization of reverse transcription-polymerase chain reaction (RT-PCR) for bPIV-3 detection. The aim of this study was to contribute to a better caracterization of bovine type 3 Parainfluenza virus by developing RT-PCR techniques to its detection. A reference sample (SF-4) and a sample of bPIV-3 isolated in Rio Grande do Sul (DIO sample) were used to develop RT-PCR techniques by amplification of different regions of hemagglutinin-neuraminidase gene (HN). After HN gene partial sequencing of DIO sample and sequence alignment, the results revealed 99% homology when compared to reference sample, 98% the 91% homology in relation to bPIV-3 samples previously published and 79% the 80% if compared to hPIV-3 samples. It was also developed a RT-PCR for amplification of a part of bPIV-3 and hPIV-3 NH gene. In optimization experiments, compared to viral isolation procedure, the RTPCR displayed a sensitivity of 140 DICC50, good specificity and reproductibility. Results after sequencing of bPIV-3 sample isolated in RS displayed strong homology with those of bPIV-3 tested samples, specially the reference sample. Data obtained in this study showed that the RT-PCR technique developed for PIV- 3 detection was able to amplify an 1009 bp HN gene fragment of bovine and human PIV-3 samples which enables its utilization in diagnostic and epidemiological studies.

Nanocápsulas poliméricas têm sido propostas como carreadores para filtros solares com o objetivo de prolongar o tempo de resistência desses no estrato córneo. O presente trabalho teve por objetivo avaliar comparativamente a permeação cutânea in vitro da benzofenona-3 (BZ3) a partir de nanocápsulas poliméricas incorporadas em diferentes formulações semi-sólidas. Primeiramente, suspensões de nanocápsulas contendo concentrações crescentes de BZ3 foram preparadas, caracterizadas físico-quimicamente e sua estabilidade foi determinada. A suspensão contendo 5 mg/mL de BZ3 apresentou diâmetro médio de 247 ± 4 nm, potencial zeta de -9,50 ± 1,02 mV, pH de 6,6 ± 0,1, teor e taxa de associação próximos a 100 % e foi selecionada para a continuidade do estudo. Na segunda etapa do trabalho a suspensão de nanocápsulas contendo 5 mg/mL de BZ3 foi incorporada em diferentes formulações semi-sólidas, hidrogel (HGNCBZ3), creme-gel (CGNCBZ3) e emulsão água em silicone (SNCBZ3). Estas formulações semi-sólidas foram caracterizadas segundo teor, pH, diâmetro médio das nanocápsulas incorporadas e comportamento reológico. As formulações semi-sólidas apresentaram pH adequado para aplicação cutânea, teores experimentais próximos aos valores teóricos e comportamento reológico não-newtoniano pseudoplástico. A permeação cutânea do filtro solar foi avaliada através de células de difusão de Franz e as formulações contendo BZ3 nanoencapsulada apresentaram maior quantidade e tempo de resistência do filtro no estrato córneo, comparando com as formulações contendo o ativo livre. A SNCBZ3 apresentou maior quantidade da BZ3 no estrato córneo quando comparada ao HGNCBZ3 e ao CGNCBZ3. Em uma última etapa do trabalho avaliou-se a fotoestabilidade da BZ3 nanoencapsulada e livre frente à irradiação por UVA e concluiu-se que a nanoencapsulação aumenta a fotoestabilidade da BZ3. Também foi avaliada a alergenicidade do filtro solar químico e observou-se que, nem o filtro nanoencapsulado ou livre, nem as nanocápsulas sem o ativo, apresentaram sensibilização cutânea. Em conclusão, o conjunto dos resultados obtidos demonstra que estas formulações são sistemas promissores para a aplicação cutânea de filtros solares. / Polymeric nanocapsules have been proposed as carriers for sunscreens in order to prolong the residence time of these substances in the stratum corneum. The aim of this work was to evaluate in vitro the skin permeation of benzophenone-3 (BZ3) from polymeric nanocapsules incorporated in different semi-solid formulations. Nanocapsule suspensions containing different concentrations of BZ3 were prepared, physico-chemically characterized and their stability was determinated. The suspension containing 5 mg/mL of the BZ3 presented average size of 247 ± 4 nm, zeta potential of - 9,50 ± 1,02 mV and pH values of 6,6 ± 0,1. This sample showed an amount of BZ3 and drug entrapment of 100 %. The nanocapsule suspension containing 5 mg/mL was incorporated in different semi-solid formulations, hydrogel (HGNCBZ3), cream-gel (CGNCBZ3) and emulsion water in silicon (SNCBZ3). The amount of BZ3, pH, average size and rheological properties of these formulations were evaluated. The formulations presented pH adequate for skin application and content of BZ3 close to 100 %. All semi-solid formulations showed pseudoplastic reological behavior. The skin permeation of BZ3 was evaluated by Franz diffusion cells and the formulation containing nanoencapsulated BZ3 showed an increase in the residence time of sunscreen in the stratum corneum and high amount of BZ3 compared to the formulations containing the free sunscreen. SNCBZ3 presented greater BZ3 quantity in the stratum corneum compared to HGNCBZ3 and CGNCBZ3. Finally, the photostability of free and nanoencapsulated BZ3 under UVA irradiation was evaluated and the results indicated that the nanoencapsulation increased the BZ3 photostability. The allergenicity of free and nanoencapsulated BZ3 was also determinated. Nanoencapsulated or free BZ3, and empty nanocapsules, did not present skin sensibilization. In conclusion, the results indicated that these formulations are promising delivery systems for the cutaneous applications of sunscreens.

A acidúria 3-hidróxiisobutírica é uma doença metabólica hereditária causada pela deficiência da atividade da 3-hidroxiisobutiril-Coenzima A desidrogenase. Acúmulo tecidual e alta excreção urinária do ácido 3- hidroxiisobutírico são as características bioquímicas desta desordem. As manifestações clínicas são heterogêneas, geralmente incluindo achados dismórficos, atraso no desenvolvimento motor, retardo mental severo e encefalopatia aguda. A acidemia láctica também ocorre nos pacientes afetados, indicando que uma disfunção mitochondrial pode estar envolvida na fisiopatogenia desta doença. Portanto, o objetivo do presente trabalho foi investigar o efeito in vitro do ácido 3-hidroxiisobutírico (0,1; 0,5 e 1,0 mM) sobre a atividade de enzimas essenciais para o metabolismo energético, tais como os complexos I-V da cadeia respiratória, creatina quinase total, citosólica e mitocondrial e Na+, K+-ATPase em homogeneizados de córtex cerebral de ratos de 30 dias de idade. Também medimos o consumo de oxigênio em preparações mitocondriais de cérebro total na presença de ácido 3-hidroxiisobutírico. O ácido 3-hidroxiisobutírico inibiu significativamente o complexo I-III (20%), sem afetar as outras atividades da cadeia respiratória Além disso, o ácido 3-hidroxiisobutírico não alterou o estado III, estado IV e o índice de controle respiratório na presença de glutamato/malato ou sucinato, sugerindo que seu efeito sobre a respiração celular foi de pequena intensidade. Por outro lado as atividades da creatina quinase total e mitocondrial, mas não citosólica, foram inibidas (30%) pelo ácido 3- hidroxiisobutírico. Também verificamos que a inibição provocada por esse ácido sobre a atividade mitocondrial da creatina quinase foi completamente prevenida por pré-incubação dos homogeneizados com glutationa reduzida, α-tocoferol ou a combinação de superóxido desmutase com catalase, indicando que esta inibição foi mediada por oxidações de grupos tióis essenciais para a enzima e provavelmente pelos radicais superóxido, peróxido de hidrogênio e/ou peroxilas. Também demonstramos que a atividade da Na+, K+-ATPase da membrana plasmática sináptica foi marcadamente reduzida (37%) pelo ácido 3-hidroxiisobutírico e que esse efeito foi prevenido pela co-incubação com α-tocoferol, sugerindo que radicais peroxilas estavam provavelmente envolvidos nesta ação. Considerando a importância das atividades enzimáticas afetadas pelo ácido 3-hidroxiisobutírico para o metabolismo cerebral e para a neurotransmissão, sugerimos que os aumentos teciduais das concentrações desse ácido possam contribuir para a neurodegeneração dos pacientes afetados pela acidúria 3-hidroxiisobutírica, e possivelmente expliquem achados prévios de elevada produção e excreção de lactato. / 3-Hydroxyisobutyric aciduria is an inherited metabolic disease caused by 3- hydroxyisobutyryl-CoA dehydrogenase deficiency. Tissue accumulation and high urinary excretion of 3-hydroxyisobutyric acid is the biochemical hallmark of this disorder. Clinical phenotype is heterogeneous and generally includes dysmorphic features, delayed motor development, profound mental impairment, and acute encephalopathy. Lactic acidemia is also found in the affected patients, indicating that mitochondrial dysfunction may be involved in the pathophysiology of this disorder. Therefore, the aim of the present work was to investigate the in vitro effect of 3-hydroxyisobutyric acid (0.1, 0.5 and 1mM) on essential enzymes of energy metabolism, namely the activities of the respiratory chain complexes I-V, total, cytosolic and mitochondrial creatine kinase and Na+, K+-ATPase in cerebral cortex homogenates of 30-day-old rats. We also measured the rate of oxygen consumption in brain mitochondrial preparations in the presence of 3- hydroxyisobutyric acid. 3-Hydroxyisobutyric acid significantly reduced complex I-III (20%), without affecting the other activities of the electron transport chain. Furthermore, 3-hydroxyisobutyric acid did not change state III, state IV and the respiratory control ratio in the presence of glutamate/malate or succinate, suggesting that its effect on cellular respiration was weak. On the other hand, the activities of total and mitochondrial creatine kinase, but not cytosolic creatine kinase, were inhibited (30%) by 3-hydroxyisobutyric acid. We also observed that 3- hydroxyisobutyric acid-induced inhibition of mitochondrial creatine kinase activity was fully prevented by pre-incubation of the homogenates with reduced glutathione, α-tocopherol or the combination of superoxide dismutase plus catalase, suggesting that this inhibition was mediated by oxidation of essential thiol groups of the enzyme probably by superoxide, hydrogen peroxide and/or peroxyl radicals. It was also demonstrated that Na+, K+-ATPase activity from synaptic plasma membranes was markedly suppressed (37 %) by 3-hydroxyisobutyric acid and that this effect was prevented by α-tocopherol co-incubation implying that peroxyl radicals were probably involved in this action. Considering the importance of the affected enzyme activities for brain metabolism homeostasis and neurotransmision, it is suggested that increased tissue levels of 3- hydroxyisobutyric acid may contribute to the neurodegeneration of patients affected by 3-hydroxyisobutyric aciduria and possibly explain previous reports describing elevated production and excretion of lactate.

As nanocápsulas poliméricas têm sido alvo de grande interesse por permitirem alterações no tempo de permanência das substâncias no estrato córneo da pele. O objetivo deste trabalho foi avaliar a influência da carga de superfície positiva das nanocápsulas revestidas com quitosana sobre as características físico-químicas e avaliar a penetração cutânea in vitro da benzofenona-3 a partir destas nanocápsulas incorporadas em hidrogéis. As nanocápsulas preparadas com Span 60® (NC-S: 234 nm; NC-S-B3: 260 nm; NC-Q-S-B3: 295 nm) apresentaram diâmetros médios maiores do que as preparadas com Epikuron 170® (NC-E: 174 nm; NC-E-B3: 175; NC-Q-E-B3: 202 nm), e todas apresentaram baixos valores de polidispersão (< 0,2), o que foi confirmado por de microscopia eletrônica de transmissão. O revestimento de quitosana conduziu a um potencial de superfície positivo (NC-Q-S-B3: 21 mV, NC-Q-E-B3: 21 mV), enquanto nas nanocápsulas não revestidas esses foram negativos (NC-S-B3: -12 mV, NC-E-B3: -8 mV). O teor de benzofenona-3 e as taxas de associação foram de aproximadamente 100 % para todas as amostras após preparação e após 30 dias de armazenamento. Análises em Turbiscan LAb® mostraram que as nanocápsulas sem revestimento de quitosana preparadas com Span 60® (NC-S-B3) apresentaram tendência à coalescência. As nanocápsulas incorporadas em hidrogéis de Natrosol® em total substituição à água não alteraram o comportamento pseudoplástico originalmente conferido ao Natrosol®. Análises em Turbiscan LAb® dos hidrogéis mostraram que não há tendência à desestabilização para todas as formulações. Estudos de penetração cutânea usando células de difusão de Franz demonstraram que as nanocápsulas contendo benzofenona-3 revestidas com quitosana foram eficazes em prolongar o tempo de residência do filtro solar sobre o estrato córneo da pele e, também, em reduzir a sua chegada ao meio receptor das células de Franz. Em conclusão, o conjunto de resultados indica que as nanocápsulas contendo benzofenona-3 e revestidas com quitosana são sistemas promissores para aplicação cutânea da benzofenona-3. / Polymeric nanocapsules have been considered to be of great interest because of their ability to promote variation in time-residence of substances on the skin stratum corneum. The aim of this work was to evaluate either the positive charged surface, due to chitosan coating, plays any influence in the nanocapsules physico-chemical properties and to evaluate the in vitro skin penetration of benzophenone-3 from these nanocapsules after incorporation in hydrogels. The nanocapsules prepared with Span 60® (NC-S: 234 nm; NC-S-B3: 260 nm; NC-Q-S-B3: 295 nm) presented higher average diameter than the ones prepared with Epikuron 170® (NC-E: 174 nm; NC-EB3: 175; NC-Q-E-B3: 202 nm), and all of them presented low polidispersity values (under 0,2), results that were confirmed by transmission electronic microscopy. The chitosan coating led to a positive zeta potential (NC-Q-S-B3: 21 mV, NC-Q-E-B3: 21 mV) while the uncoated nanocapsules presented negative zeta potential (NC-S-B3: - 12 mV, NC-E-B3: -8 mV). All the samples showed an amount of benzophenone-3 of 100 % after preparation and 30 days of storage. The drug entrapment was 100 % for all formulations. Turbiscan LAb® analyses demonstrated that the uncoated nanocapsules, prepared with Span 60®, have the tendency to coalescence. The incorporation of the nanocapsules in Natrosol® hydrogels, by substitution of the water for the nanocapsules suspension, did not cause any alteration in the originally observed pseudoplastic reological behavior of Natrosol®. Turbiscan LAb® analyses have also shown no tendency to destabilization for all hydrogel formulations. Skin penetration studies using Franz diffusion cells, have shown that the chitosan coated nanocapsules containing benzophenone-3 were considered to be efficient in prolonging the time-residence of the sunscreen in the stratum corneum and also in reducing its presence in the receptor medium of the Franz cells. In conclusion, the results indicate that the chitosan coated nanocapsules containing benzophenone-3 are promising systems for cutaneous application of the referred sunscreen.

As acidúrias 3-hidroxi-3-metilgutárica (HMGA) e 3-metilglutacônica (MGTA) são doenças que afetam o catabolismo da leucina e a cetogênese. Os pacientes afetados pela HMGA apresentam acúmulo e elevada excreção urinária dos ácidos 3-hidroxi-3-metilglutárico (HMG), 3-metilglutárico (MGA), 3- metilglutacônico (MGT) e 3-hidroxiisovalérico (OHIVA), ao passo que a MGTA é bioquimicamente caracterizada pelo acúmulo e aumento na excreção urinária de MGT, MGA e OHIVA. Os indivíduos afetados por MGTA apresentam predominantemente sintomas neurológicos. Já os pacientes acometidos pela HMGA também apresentam hepatomegalia, principalmente em situações de descompensação metabólica, quando ocorrem elevações drásticas nas concentrações dos metabólitos acumulados. Considerando que os mecanismos patogênicos responsáveis pelo dano neurológico encontrado nos pacientes portadores da HMGA e da MGTA são pouco conhecidos, inicialmente investigamos os efeitos in vitro dos ácidos HMG, MGA, MGT e OHIVA sobre importantes parâmetros de estresse oxidativo em córtex cerebral de ratos jovens. Verificamos que os metabólitos testados induziram peroxidação lipídica (aumento de substâncias reativas ao ácido tiobarbitúrico (TBA-RS) e da quimioluminescência) e dano oxidativo protéico (formação de carbonilas e oxidação de grupamentos sulfidrila). Além disso, a indução de peroxidação lipídica causada pelo HMG e pelo MGT foi prevenida pela adição de antioxidantes. Também foi demonstrado que o HMG, MGA, MGT e OHIVA diminuíram as defesas antioxidantes não-enzimáticas (diminuição das concentrações de glutationa reduzida (GSH), do potencial antioxidante total e da reatividade antioxidante total) e que a diminuição das concentrações de GSH causada pelo HMG e pelo MGA foi prevenida por antioxidantes. Por outro lado, nenhum dos ácidos orgânicos foi capaz de oxidar grupamentos sulfidrila de uma solução comercial de glutationa e de membranas mitocondriais isoladas de córtex cerebral. Na segunda parte do trabalho, comparamos a vulnerabilidade do estriado cerebral e do fígado ao dano oxidativo causado pelo HMG, MGA, MGT e OHIVA. Verificamos que todos os ácidos orgânicos aumentaram a medida de TBA-RS no estriado, sendo que apenas o HMG, além de apresentar o efeito mais pronunciado, induziu peroxidação lipídica no fígado. Esses metabólitos também diminuíram as concentrações de GSH em estriado. Novamente apenas o HMG reduziu as defesas antioxidantes não-enzimáticas no fígado. Finalmente, nossos resultados demonstraram que o HMG, MGA e o MGT induziram a oxidação de grupamentos sulfidrila em estriado, ao passo que nenhum dos metabólitos alterou esse parâmetro em fígado. Os efeitos detectados no presente estudo demonstram que os ácidos orgânicos acumulados na HMGA e na MGTA induzem estresse oxidativo em córtex cerebral, estriado e fígado de ratos jovens. Também evidencia que os tecidos cerebrais apresentam maior vulnerabilidade que o fígado ao dano oxidativo provocado por esses metabólitos, o que está de acordo com a sintomatologia apresentada pelos pacientes que é predominantemente neurológica. Portanto, presumimos que o estresse oxidativo pode contribuir, ao menos em parte, para a fisiopatologia dos danos teciduais encontrados nos pacientes acometidos pela HMGA e MGTA. / 3-Hydroxy-3-methylglutaric (HMGA) and 3-methylglutaconic (MGTA) acidurias are disorders that affect leucine catabolism and ketogenesis. HMGAaffected patients present tissue accumulation and high urinary excretion of 3- hydroxy-3-methylglutaric (HMG), 3-methylglutaric (MGA), 3-methylglutaconic (MGT) and 3-hydroxyisovaleric (OHIVA) acids, whereas MGTA is biochemically characterized by accumulation of MGT, MGA and OHIVA. MGTA- and HMGAaffected patients predominantly present neurologic symptoms. HMGA patients also present hepatomegaly, especially during metabolic crisis, which is characterized by a dramatic increase of the concentrations of the accumulating organic acids. Considering that the mechanisms of brain damage in HMGA and MGTA are poorly known, we first investigated the in vitro effects of HMG, MGA, MGT and OHIVA on important parameters of oxidative stress in cerebral cortex from young rats. We verified that the organic acids induced lipid peroxidation (thiobarbituric-acid reactive species, TBA-RS, and chemiluminescence increase) and protein oxidative damage (carbonyl formation and sulfhydryl oxidation) in cortical supernatants. The lipid peroxidation induced by HMG and MGT was prevented by the addition of free radical scavengers, suggesting that free radicals are involved in these effects. It was also demonstrated that HMG, MGA, MGT and OHIVA reduced non-enzymatic antioxidant defenses (reduced glutathione, GSH, concentrations, total radical-trapping antioxidant potential and total antioxidant reactivity) and that GSH decrease caused by HMG and MGA was prevented by free radical scavengers. On the other hand, the metabolites did not oxidize sulfhydryl groups from a commercial solution of GSH and from purified membrane protein-bound thiol groups, indicating that the decrease of GSH levels caused by the organic acids was not due to a direct oxidative effect. Next, we evaluated the vulnerability of a central (striatum) and a peripheral (liver) tissue to the oxidative damage caused by HMG, MGA, MGT and OHIVA. It can be observed that HMG, MGA, MGT and OHIVA induced lipid peroxidation (TBA-RS increase) in striatum. Among the various acids, only HMG increased TBA-RS in the liver. Furthermore, HMG induced the highest degree of lipid peroxidation in the striatum. It was also verified that the organic acids diminished GSH concentrations. Again, HMG presented the strongest effect compared to the other metabolites, reducing GSH levels in the striatum and in the liver. Finally, we observed that HMG, MGT and MGA reduced the sulfhydryl content (protein oxidation) in the striatum, whereas the metabolites did not alter this parameter in the liver. The present data indicate the HMG, MGA, MGT and OHIVA induce oxidative stress in cerebral cortex, striatum and liver from young rats. It can be also observed that the brain is more vulnerable than liver to lipid and protein oxidative damage induced by these organic acids, which is in agreement with the fact that the patients present predominantly neurologic symptoms. Therefore, it may be presumed that oxidative stress elicited in vitro by HMG, MGA, MGT and OHIVA contributes, at least in part, to the pathophysiology of the brain and hepatic damage found in HMGA and MGTA.

Made available in DSpace on 2015-07-22T20:50:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-28 / Alguns estudos apontam para o efeito benéfico do uso isolado de Ginkgo biloba ou ômega 3 na cognição de idosos. Desta forma, o objetivo desta pesquisa foi verificar se o uso associado destas duas substâncias potencializa a melhora cognitiva em mulheres idosas (n=43), de 60 a 70 anos, saudáveis e com escolaridade igual ou superior a oito anos. Realizou-se um estudo duplo-cego, placebo controlado e randomizado. As substâncias foram administradas por 3 meses, de acordo com o grupo de tratamento: placebo+placebo (n=12), ginkgo+placebo (n=10), ômega3+placebo (n=11) e, ômega3+ginkgo (n=10). Os comprimidos continham 80 mg de extrato seco de Ginkgo biloba e as cápsulas, 1 g de óleo de peixe (rico em ômega 3). Exames laboratoriais, testes cognitivos e escalas para ansiedade, depressão e qualidade de vida foram aplicadas no início e ao final do tratamento. Não encontramos melhoras na funções cognitivas avaliadas em nenhum dos quatro grupos. A ação conjunta de uma série de fatores podem ser destacados para auxiliar a compreensão dos resultados finais, como o tempo de tratamento, doses insuficientes das substâncias, alta escolaridade das idosas e pequeno número de sujeitos em cada grupo. A comparação da literatura com nossos achados demonstrou que não há um consenso sobre os testes cognitivos a serem utilizados, as doses e tempo de uso das duas substâncias. / Several studies point to the beneficial effect of Ginkgo biloba and omega 3 on cognition in the elderly. The objective of this research was to verify whether the combined use of these substances enhances the cognitive functioning. The study was conceived as a double-blind, placebo-controlled, randomized trial. Altogether, 43 subjects between 60 and 70 years old, female, healthy, with more than 8 years of formal education, have ingested substances on a daily basis, for three months, according to the assigned treatment group: placebo+placebo (n=12), ginkgo+placebo (n=10), ômega3+placebo (n=11), ômega3+ginkgo (n=10), in the form of capsules that could contain 80 mg of ginkgo, 1 gram of fish oil (rich in omega 3) or placebo. Unlike other studies, we found no improvement in cognition of/in any of the four groups. The hypotheses suggested are: time of treatment or insufficient doses of substances. / TEDE / BV UNIFESP: Teses e dissertações