No description available.

O objetivo foi avaliar a ação do ácido indol-3-acético (AIA) na ausência e presença da peroxidase de raiz forte (HRP) sobre a viabilidade de cepas certificadas de Staphylococcus aureus ATCC 6538 (produtora de biofilme), ATCC 14458 (portadora do gene seb), ATCC 43300 (portadora do gene mecA) e ATCC 29213 (controle). Foi determinada a concentração inibitória mínima (CIM) e, após, escolheu-se uma concentração subinitória para avaliar a capacidade de formação de biofilme, atividade de enzimas antioxidantes, integridade de DNA, integridade de membrana para todas as cepas e avaliação da expressão do gene seb da cepa ATCC 14458. A CIM observada foi de 40 mM de AIA para cepa ATCC 6538, 60 mM para ATCC 43300,50 mM para ATCC 14458 e ATCC 29213. No entanto, houve diferença significativa (P<0,05), na presença de HRP, somente para as cepas ATCC 14458 e 43300. A concentração subinibitória de AIA utilizada foi de 30 mM para ATCC 6538 e ATCC 14458, e de 40 mM para ATCC 43300 e ATCC 29213.mM. A capacidade de formação de biofilme pelo S. aureus(biofilme positivo) não foi alterada pela presença de AIA ou AIA/HRP. A atividade específica de catalase e superóxido dismutase foi aumentada pela ação de AIA/HRP (P<0,05) para todas as cepas, porém, não houve diferença (P>0,05) entre AIA e AIA/HRP. O DNA manteve-se íntegro, porém, houve diminuição na integridade de membrana (P<0,05) para todas as cepas cultivadas na concentração subinibitória de AIA, sendo este efeito potencializado na presença de HRP somente para ATCC 43300. Houve redução na expressão do gene seb da cepa ATCC 14458 (P<0,05). Conclui-se que o AIA, associado à HRP apresenta efeito bacteriostático em cepas de Staphylococcus aureus portadoras de diferentes fatores de virulência. / The objective was to evaluate the action of indole-3-acetic acid (IAA) in the absence and presence of horseradish peroxidase (HRP) on the viability of certified strains of Staphylococcus aureus ATCC 6538 (producer of biofilm), ATCC 14458 (carrying the seb gene), ATCC 43300 (carrying the mecA gene) and ATCC29213 (control). Concentration was determined minimum inhibitory (CIM) and, after, we chose a concentration subinitória to assess the ability of biofilm formation, activity of antioxidant enzymes, integrity DNA, membrane integrity for all strains and evaluation of gene expression seb of strain ATCC 14458. The MIC for the strains ATCC 6538 was 40 mM IAA , for the strains ATCC 14458 and 29213 was 50 mM and for the strain ATCC 43300 was 60 mM IAA, however, significant differences (P<0.05) compared to the addition of HRP to the culture medium, was found only for the doses of MIC of the strains ATCC14458 and 43300. The concentration subinibitória IAA used was 30 mM for ATCC 6538 and ATCC 14458, and 40 mM to ATCC 43300 and ATCC 29213.mM for ATCC 6538 and ATCC 14458, and 50 mM to ATCC 43300 and ATCC 29213.The ability of biofilm formation by S. aureus(biofilm-positive) was not altered by the presence of IAA or IAA/HRP in the culture medium. The activity of catalase and superoxide dismutase was influenced by the action of IAA and IAA/HRP (P<0.05), but there was no difference (P≥0.05) between IAA and IAA/HRP. The DNA remained intact, however, there were changes (P<0.05) in the membrane integrity of microorganisms to sub-inhibitory concentrations of IAA and IAA/HRP, as well as the expression of the sebgene was lower (P<0.05) in microorganisms treated with IAA and IAA/HRP. It is concluded that the IAA has bacteriostatic effect on S. aureus carrying different virulence factors.

Μελέτες αλληλεπίδρασης μικρών μονοστοιβαδιακών αρσονολιποσωμάτων τα οποία αποτελούνται από μίγματα αρσονολιπιδίων και φωσφολιπιδίων, έδειξαν ότι αυτά είναι ιδιαίτερα τοξικά απέναντι σε καρκινικά κύτταρα. Έχει προταθεί ότι το γεγονός αυτό μπορεί να συνδέεται με την ιδιότητα των αρσονολιπιδίων As (V) να ανάγονται σε As(III) από μεμβρανικές ή κυτταροπλασματικές θειόλες. Το γεγονός ότι τα HL-60 κύτταρα τα οποία είναι πολύ ευαίσθητα στα αρσονολιποσώματα βρέθηκαν να περιέχουν υψηλά επίπεδα γλουταθειόνης, έρχεται σε πλήρη συμφωνία με τη θεωρία αυτή. Στην παρούσα εργασία μελετήθηκε in vitrο, η επίδραση μιας θειόλης, της γλουταθειόνης, η οποία αποτελεί την κύρια θειόλη των κυττάρων, στη σταθερότητα των αρσονολιποσωμάτων, με σκοπό να διαλευκανθεί η αλληλεπίδραση μεταξύ θειολών και αρσονολιποσωμάτων. Αν η γλουταθειόνη αλληλεπιδρά με το As (V) των αρσονολιπιδίων, τότε είναι πιθανόν να μεταβάλλεται η μεμβρανική τους σταθερότητα. Επιπλέον, η κυτταροτοξικότητα αυτών των αρσονολιποσωμάτων απέναντι σε σε μια καρκινική σειρά (PC3) μελετήθηκε με σκοπό να εξακριβωθεί εάν ένα in vitro τεστ με γλουταθειόνη μπορεί να χρησιμοποιηθεί για την πρόβλεψη της τοξικότητας αρσονολιποσωμάτων απέναντι σε καρκινικά κύτταρα. Τα αποτελέσματα της μελέτης αυτής δείχνουν ότι η επίδραση της γλουταθειόνης στη σταθερότητα των αρσονολιποσωμάτων είναι μεγαλύτερη όταν το περιεχόμενο των λιποσωμάτων σε αρσονολιπίδιο αυξάνει, σαν συνέπεια αλληλεπίδρασης του αρσονολιπιδίου με τη γλουταθειόνη. Μάλιστα σε κάποιες περιπτώσεις που εξαρτώνται από τη σκληρότητα της λιπιδικής μεμβράνης, η αλληλεπίδραση αυτή οδηγεί σε αποσταθεροποίηση του αρσονολιποσώματος. Η αρνητική επίδραση της γλουταθειόνης στη σταθερότητα των λιποσωμάτων είναι μεγαλύτερη στα PC-αρσονολιποσώματα απ’ ότι στα DSPC. Πιθανόν η αυξημένη σταθερότητα των DSPC-αρσονολιποσωμάτων παρουσία γλουταθειόνης σε σχέση με τα PC να σχετίζεται με το γεγονός ότι τα πρώτα είναι πιο σταθερά ακόμα και στην περίπτωση που αυτά επωάζονται σε διάλυμα ορού. Για τα σταθεροποιημένα με PEG αρσονολιποσώματα η επίδραση της γλουταθειόνης στη σταθερότητα της μεμβράνης είναι πολύ μικρότερη σε σχέση με τα μη σταθεροποιημένα γεγονός που μπορεί να σχετίζεται είτε με την υψηλή σταθερότητα που έχουν εμφανίσει σε προηγούμενες μελέτες, είτε με την ιδιότητα της πολυαιθυλενογλυκόλης να περεμποδίζει στερεοχημικά την προσέγγιση και άρα την αλληλεπίδραση των αρσονολιποσωμάτων με τη γλουταθειόνη. Με σκοπό να εξακριβώσουμε αν τα PEG-αρσονολιποσώματα παρουσιάζουν κυταροτοξικότητα, μετρήσαμε τη % βιοσημότητα των καρκινικών κυττάρων ύστερα από επώαση αυτών παρουσία και απουσία (control) αρσονολιποσωμάτων. Τα αποτελέσματα έδειξαν ότι τα PC-αρσονολιποσώματα εμφανίζουν αυξημένη κυτταροτοξικότητα σε σχέση με τα DSPC, γεγονός που συμφωνεί απόλυτα με τη μειωμένη σταθερότητά τους παρουσία γλουταθειόνης. Παρόλαυτά τα PEG- αρσονολιποσώματα τα οποία είναι σταθερά παρουσία γλουταθειόνης, εμφανίζουν παρόμοια κυτταροτοξικότητα με τα μη σταθεροποιημένα PC-αρσονολιποσώματα. (Πειράματα που έγιναν με συμβατικά PEG-λιποσώματα δείχνουν ότι η παρουσία πολυαιθυλενογλυκόλης δεν προκαλεί κυτταροτοξικότητα). Συμπερασματικά τα αποτελέσματα αυτής της εργασίας δείχνουν ότι όντος πραγματοποιείται αλληλεπίδραση μεταξύ του As της πολικής κεφαλής του αρσονολιπιδίου και της θειολομάδας της γλουταθειόνης. Για τα μη σταθεροποιημένα αρσονολιποσώμτα τα αποτελέσματα από τη μελέτη της γλουταθειόνης συμφωνούν με την κυτταροτοξικότητα που εμφανίζουν στα PC3 κύτταρα, γεγονός που δεν ισχύει για τα σταθεροποιημένα. Αυτό μπορεί να οφείλετα σε διαφορετικό μηχανισμό υπεύθυνο για την κυταροτοηικότητα των PEG-αρσονολιποσωμάτων. / In cell culture studies, sonicated liposomes composed of phospholipid-arsonolipid mixtures (arsonoliposomes) demonstrate a specific toxicity against cancer cells. It has been previously proposed that this may be linked with the ability of arsonolipid As(V) to be reduced to As(III) by membrane-bound or cytoplasmic thiols. The fact that HL-60 cells which are very sensitive towards arsonoliposomes were found to have high basal glutathione concentrations, is in correlation with this theory. Here we studied in vitro, the effect of a thiol-containing compound, glutathione, on the integrity of arsonoliposomes, in order to gain some information about the interaction between thiols and arsonoliposomes. If GSH interacts with the As(V) of arsonoliposomes, this may alter their membrane stability. Furthermore, the cytotoxicity of these arsonoliposome types towards a cancer cell line (PC3) was measured in order to see if the results from the in vitro test with GSH can predict arsonoliposome toxicity towards cancer cells. The results of this study show that the effect of glutathione on arsonoliposome integrity is higher when their arsonolipid content increases, indicating that arsonolipid molecules interact with glutathione. In some cases, depending on the rigidity of their membranes, this interaction leads to a destabilization of arsonoliposomes. The destabilizing effect of GSH was higher for PC-based arsonoliposomes compared to DSPC-based ones. ). Perhaps the enhanced stability of the DSPC arsonoliposomes in presence of glutathione compared to the PC-based-ones is related with the fact that they are also significantly more stable during incubation in serum, as previously proven. For pegylated-arsonoliposomes membrane destabilization was minimal and this may be related to the high stability demonstrated previously for these specific arsonoliposomes, or, it may indicate that pegylation results in prevention (total or partial) of arsonolipid interaction with thiols (perhaps because of steric repulsion). In order to see if PEG-arsonoliposomes are cytotoxic towards cancer cells, we measured by MTT assay, the proliferation of PC3 cells after incubation in presence and absence (control) of different types and amounts of arsonoliposomes. Results show that DSPC-based arsonoliposomes are slightly, but significantly less cytotoxic compared to the equivalent PC-based ones, in agreement with the higher effect of GSH on PC-based arsonoliposomes. However although the pegylated arsonoliposomes studied were basically not affected by GSH, their PC3 cytotoxicity is equal with that measured for the PC-based arsonoliposomes, (PEG-related cytotoxicity was excluded by control experiments). To conclude the results of this study show that interaction between thiol groups and As-containing headgroups of arsonoliposomes take place. For the non-pegylated-arsonoliposomes the results of the GSH- study agree with the relative cytotoxicity of the corresponding arsonoliposomes towards PC3 cells. However, this is not the case for pegylated arsonoliposomes. Perhaps this implies that another mechanism is responsible for the pegylated liposome cytotoxicity.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-01-30T11:04:36Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Arthur Francisco Júnior.pdf: 1488392 bytes, checksum: 48308d252d06dcc28ec7f91907c90eec (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-30T13:36:00Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Arthur Francisco Júnior.pdf: 1488392 bytes, checksum: 48308d252d06dcc28ec7f91907c90eec (MD5) / Made available in DSpace on 2015-01-30T13:36:00Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Arthur Francisco Júnior.pdf: 1488392 bytes, checksum: 48308d252d06dcc28ec7f91907c90eec (MD5) Previous issue date: 2014-08-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / This study was conducted to evaluate the effect of adding L-acetyl-cysteine (CIS) as anti oxidant agent to extender used for sperm cryopreservation of equine Mangalarga Marchador. Three semen samples from seven healthy stallions were obtained by artificial vagina, diluted in commercial extender and distributed into two treatments: control without addition and CIS, containing the antioxidant concentration of 2.5 mM. After the stabilization period, the samples were filled into 0.5 mL straws and subjected to cryopreservation protocol manually. Sixty days after the samples were thawed and evaluated in vitro by means of computerized analysis of sperm kinetics (CASA) and the evaluation of the integrity of plasma and acrosomal membranes by fluorescent probes. Regarding the kinetic variables related to sperm was found that in samples supplemented with CIS percentage of total and progressive motility (TM) (MP) was higher (P <0.01) compared to control samples (23.95±3 , 33 X 11 .38±2.35 and 4.66 ± 0.84 X 2.04 ± 0.36, respectively), but variable for straightness (STR) and linearity (LIN) the percentages were highe r (p ≤ 0.01) for the control samples (86.95 ± 1.02 X 82.28 ± 1.02 and 49.85 ± 1.32 X 45.19 ± 1.17, respectively). As those related to the integrity of the membranes of the sperm variables was superior (P <0.05) in the number of sperm with intact acrosome reaction (ICRA) in the samples compared to those CIS Control (4.7 ± 1.6 x 0.8 ± 0.2, respectively). The results suggest that the addition of acetyl-L-cysteine concentration of 2.5 mM in the diluent medium before cryopreservation can improve both the motility as the integrity of the sperm plasma membrane equine Mangalarga Marcher. More studies should be done to confirm this effect in vivo. / Esse estudo foi conduzido com o objetivo de avaliar o efeito da adição de L-acetilcisteína (CIS) como agente anti oxidante ao meio diluente utilizado para criopreservação de espermatozóides de equinos da raça Mangalarga Marchador. Três amostras seminais de sete garanhões hígidos foram obtidas por meio de vagina artificial, diluídas em meio comercial e distribuídas em dois tratamentos: controle, sem adição e CIS, contendo o antioxidante na concentração de 2,5 mM. Após o período de estabilização as amostras foram envasadas em palhetas de 0,5 mL e submetidas ao protocolo de criopreservação manual. Sessenta dias após as amostras foram descongeladas e avaliadas in vitro por meio da análise computadorizada da cinética espermática (CASA) e da avaliação da inte gridade das membranas plasmática e acrosomal por meio de sondas fluorescentes. Em relação às variáveis relacionadas à cinética espermática verificou-se que nas amostras suplementadas com CIS o percentual de motilidade total (MT) e progressiva (MP) foi superior (P<0,01) em relação às amostras controle (23,95±3,33 X 11,38±2,35 e 4,66±0,84 X 2,04±0,36, respectivamente), mas para as variáveis retilinearidade (STR) e linearidade (LIN) os percentuais foram maiores (P≤0,01) para as amostras Controle (86,95±1,02 X 82,28±1,02 e 49,85±1,32 X 45,19±1,17, respectivamente). Quanto às variáveis relacionadas à integridade das membranas do espermatozóide houve superioridade (P<0,05) no número de espermatozóides íntegros com reação acrossomal (ICRA) nas amostras CIS em relação àquelas controle (4,7±1,6 X 0,8±0,2, respectivamente). Os resultados sugerem que a adição de L-acetil-cisteína na concentração de 2,5 mM ao meio diluente antes da criopreservação pode melhorar tanto a motilidade quanto a integridade da membrana plasmática de espermatozóides de equinos da raça Mangalarga Marchador. Mais estudos devem ser feitos para comprovar esse efeito in vivo.

O objetivo foi avaliar a ação do ácido indol-3-acético (AIA) na ausência e presença da peroxidase de raiz forte (HRP) sobre a viabilidade de cepas certificadas de Staphylococcus aureus ATCC 6538 (produtora de biofilme), ATCC 14458 (portadora do gene seb), ATCC 43300 (portadora do gene mecA) e ATCC 29213 (controle). Foi determinada a concentração inibitória mínima (CIM) e, após, escolheu-se uma concentração subinitória para avaliar a capacidade de formação de biofilme, atividade de enzimas antioxidantes, integridade de DNA, integridade de membrana para todas as cepas e avaliação da expressão do gene seb da cepa ATCC 14458. A CIM observada foi de 40 mM de AIA para cepa ATCC 6538, 60 mM para ATCC 43300,50 mM para ATCC 14458 e ATCC 29213. No entanto, houve diferença significativa (P&lt;0,05), na presença de HRP, somente para as cepas ATCC 14458 e 43300. A concentração subinibitória de AIA utilizada foi de 30 mM para ATCC 6538 e ATCC 14458, e de 40 mM para ATCC 43300 e ATCC 29213.mM. A capacidade de formação de biofilme pelo S. aureus(biofilme positivo) não foi alterada pela presença de AIA ou AIA/HRP. A atividade específica de catalase e superóxido dismutase foi aumentada pela ação de AIA/HRP (P&lt;0,05) para todas as cepas, porém, não houve diferença (P&gt;0,05) entre AIA e AIA/HRP. O DNA manteve-se íntegro, porém, houve diminuição na integridade de membrana (P&lt;0,05) para todas as cepas cultivadas na concentração subinibitória de AIA, sendo este efeito potencializado na presença de HRP somente para ATCC 43300. Houve redução na expressão do gene seb da cepa ATCC 14458 (P&lt;0,05). Conclui-se que o AIA, associado à HRP apresenta efeito bacteriostático em cepas de Staphylococcus aureus portadoras de diferentes fatores de virulência. / The objective was to evaluate the action of indole-3-acetic acid (IAA) in the absence and presence of horseradish peroxidase (HRP) on the viability of certified strains of Staphylococcus aureus ATCC 6538 (producer of biofilm), ATCC 14458 (carrying the seb gene), ATCC 43300 (carrying the mecA gene) and ATCC29213 (control). Concentration was determined minimum inhibitory (CIM) and, after, we chose a concentration subinitória to assess the ability of biofilm formation, activity of antioxidant enzymes, integrity DNA, membrane integrity for all strains and evaluation of gene expression seb of strain ATCC 14458. The MIC for the strains ATCC 6538 was 40 mM IAA , for the strains ATCC 14458 and 29213 was 50 mM and for the strain ATCC 43300 was 60 mM IAA, however, significant differences (P&lt;0.05) compared to the addition of HRP to the culture medium, was found only for the doses of MIC of the strains ATCC14458 and 43300. The concentration subinibitória IAA used was 30 mM for ATCC 6538 and ATCC 14458, and 40 mM to ATCC 43300 and ATCC 29213.mM for ATCC 6538 and ATCC 14458, and 50 mM to ATCC 43300 and ATCC 29213.The ability of biofilm formation by S. aureus(biofilm-positive) was not altered by the presence of IAA or IAA/HRP in the culture medium. The activity of catalase and superoxide dismutase was influenced by the action of IAA and IAA/HRP (P&lt;0.05), but there was no difference (P&ge;0.05) between IAA and IAA/HRP. The DNA remained intact, however, there were changes (P&lt;0.05) in the membrane integrity of microorganisms to sub-inhibitory concentrations of IAA and IAA/HRP, as well as the expression of the sebgene was lower (P&lt;0.05) in microorganisms treated with IAA and IAA/HRP. It is concluded that the IAA has bacteriostatic effect on S. aureus carrying different virulence factors.

No description available.

Οι κυκλοδεξτρίνες είναι υδρόφιλα μόρια ολιγοσακχαριτών που έχουν την ικανότητα να σχηματίζουν σύμπλοκες ενώσεις με δυσδιάλυτα στο νερό μόρια φαρμάκων, εγκλωβίζοντας τα μέσω ασθενών διαμοριακών δυνάμεων στην υδρόφιλη κοιλότητα του μορίου της κυκλοδεξτρίνης (Frank 1975). Η ιδιότητα τους αυτή να σχηματίζουν σύμπλοκες ενώσεις με δυσδιάλυτα στο νερό φάρμακα έχει πολλές εφαρμογές στη φαρμακευτική βιομηχανία, καθώς ο σχηματισμός συμπλόκων των φαρμάκων αυτών με τις κυκλοδεξτρίνες έχει σημαντική επίδραση στις φυσικοχημικές τους ιδιότητες (διαλυτότητα, χημική σταθερότητα, βιοδιαθεσιμότητα). Τα λιποσώματα από την πλευρά τους είναι μορφές χορήγησης φαρμάκων με πολλά πλεονεκτήματα. Παρουσιάζουν όμως το πρόβλημα της διαφυγής των λιπόφιλων φαρμάκων (που διέρχονται από τη μεμβράνη), κυρίως μετά από in vivo χορήγηση (Kirby and Gregoriadis, 1983; Takino et al, 1994). Έτσι για την παρασκευή σταθερών μορφών χορήγησης φαρμάκων προτάθηκε ο εγκλωβισμός συμπλόκων του φαρμάκου με κυκλοδεξτρίνη στην υδατική φάση του λιποσώματος σχηματίζοντας σύστημα φάρμακο-σε-κυκλοδεξτρίνη-σε-λιπόσωμα (McCormack and Gregoriadis, 1994). Ωστόσο και το σύστημα αυτό εμφανίζει προβλήματα λόγω ικανότητας των κυκλοδεξτρινών να απομακρύνουν λιπίδια από τη μεμβράνη και να σχηματίζουν με αυτά σύμπλοκες ενώσεις. Καθώς όμως τα λιπίδια απομακρύνονται από τη μεμβράνη και εισέρχονται στην κοιλότητα της κυκλοδεξτρίνης αντικαθιστούν το φάρμακο που βρίσκεται εντός της κυκλοδεξτρίνης, το οποίο με τη σειρά του απελευθερώνεται από το λιπόσωμα λόγω αποδιοργάνωσης της λιπιδικής μεμβράνης. Στη παρούσα εργασία μελετήθηκε η σταθερότητα της μεμβράνης (προσδιορισμός συγκράτησης καλσεΐνης) και η σχετική θολερότητα διαφορετικών λιποσωμικών διασπορών κατά την επώασή τους παρουσία διαφορετικών κυκλοδεξτρινών. Συγκεκριμένα παρασκευάστηκαν DRV, MLV και SUV λιποσώματα αποτελούμενα από διαφορετικά φωσφολιπίδια και τα οποία περιέχουν ή όχι χοληστερόλη. Όλες οι παραπάνω λιποσωμικές μορφές παρασκευάστηκαν έτσι ώστε να περιέχουν καλσεΐνη (100mM) στην υδατική φάσης τους και έπειτα προσδιορίστηκε η συγκράτηση της καλσεΐνης από τα λιποσώματα κατά τη επώασή τους για 24 ώρες παρουσία των κυκλοδεξτρινών ΗΡ-β-CD, HP-γ-CD και Methyl-β- CD. Τα αποτελέσματα της μελέτης της σταθερότητας της μεμβράνης έδειξαν ότι η σταθερότητα των λιποσωμάτων στα διαλύματα των κυκλοδεξτρινών εξαρτάται από τον τύπο του λιποσώματος και τη λιπιδική σύσταση. Έτσι στα λιποσώματα με την ίδια λιπιδική σύσταση η διαφυγή καλσεΐνης από τα λιποσώματα κατά την επώασή τους παρουσία κυκλοδεξτρινών, ακολούθησε την εξής σειρά MLV>DRV >SUV. Η σταθερότητα των MLV και SUV λιποσωμάτων επηρεάστηκε περισσότερο από τη Methyl-β-CD σε σχέση με τις άλλες κυκλοδεξτρίνες. Μάλιστα είναι χαρακτηριστικό ότι η σταθερότητα των SUV λιποσωμάτων επηρεάστηκε μόνο από τη Me-β-CD. Όσον αφορά στη επίδραση της λιπιδικής σύστασης, τα DSPC λιποσώματα βρέθηκε να είναι σταθερά παρουσία και των τριών κυκλοδεξτρινών, ακόμα και όταν η λιπιδική τους μεμβράνη περιείχε χοληστερόλη. Επιπλέον τα HPC λιποσώματα αποδείχτηκαν σταθερότερα από τα PC, γεγονός που καταδεικνύει την επίδραση του κορεσμού των λιπιδίων, αλλά και της ακαμψίας της μεμβράνης στη σταθερότητα των λιποσωμάτων. Τέλος η χοληστερόλη βρέθηκε να αυξάνει τη σταθερότητα των PC και να αποσταθεροποιεί τα λιποσώματα που αποτελούνται από HPC. Επιπλέον οι μετρήσεις θολερότητας των λιποσωμικών διασπορών παρουσία αυξανόμενης συγκέντρωσης κυκλοδεξτρινών έδειξαν ότι η Me-β-CD προκάλεσε τη διαλυτοποίηση των λιποσωμάτων. Μάλιστα τα DRV λιποσώματα βρέθηκε να επηρεάζονται περισσότερο σε σχέση με τα SUV λιποσώματα. Χαρακτηριστικό είναι ότι τα αποτελέσματα από της μελέτης της απελευθέρωσης καλσεΐνης δε συμφωνούσαν πάντα με τα αποτελέσματα μέτρησης της σχετικής θολερότητας. Έτσι εξάγεται το συμπέρασμα ότι η απελευθέρωση καλσεΐνης είναι ανεξάρτητη της λιποσωμικής διαλυτοποίησης. / Cyclodextrins (CDs) are hydrophilic water-soluble oligosaccharides that can accommodate water-insoluble drugs in the hydrophobic cavities they form (Frank, 1975). They received considerable attention in the pharmaceutical field because of the improved characteristics (as aqueous solubility, chemical stability and bioavailability) observed for several drug molecules through inclusion complex formation. Previous indications that highly lipophilic drugs are rapidly released from liposomes after in-vivo administration (Kirby and Gregoriadis, 1983; Takino et al, 1994) prompted the consideration of an alternative approach for stable encapsulation of lipophilic drugs in the aqueous interior of liposomes, utilizing CDs (McCormack and Gregoriadis, 1994). This approach established a novel system in drug delivery, combining liposomes and cyclodextrin complexes of lipophilic drugs by forming drug-in-cyclodextrin-in-liposome preparations. A recently observed problem of such systems is the release of drug from the CD-drug complex encapsulating liposomes. This has been connected with the known ability of CDs to remove lipid components from cell membranes by forming inclusion complexes with the lipids (Fauvelle et al, 1997, Nishijo and Mizuno, 1998). In other words, membrane lipids may be entering in the CD cavity replacing the drug, which is in turn released from the vesicles as a consequence of the lipid membrane reorganization. The membrane integrity (calcein retention) and relative turbidity of different liposomal dispersions during incubation in presence of various cyclodextrin (CD) molecules was studied. DRV, MLV and SUV liposomes, composed of different phospholipids and containing or not cholesterol were prepared. All liposomes were formulated to encapsulate calcein (100 mM), and the retention of the liposome entrapped dye was followed during a 24 hour incubation period in presence of HP-β- CD, HP-γ-CD or Me-β-CD. The experimental results demonstrate that the integrity of liposome membranes in cyclodextrin solutions is affected by the liposome lipid composition and type. In general, for the same lipid composition calcein release from liposomes during incubation in CD’s was in the order MLV>DRV>SUV. The CD molecule that influences MLV and SUV liposome stability the most is Me-β-CD. In fact SUV liposomes, are affected only by Me-β-CD. Considering lipid composition, DSPC liposomes are always very stable, while cholesterol addition in their membrane either has no effect or decreases stability. HPC liposomes are more stable compared to PC liposomes, suggesting that phospholipid saturation and/or membrane rigidity influences their interaction with CD molecules, while cholesterol addition improved PC-liposome stability but destabilized HPC liposomes. Turbidity of some liposome dispersions was measured in presence of increasing concentrations of cyclodextrins and results show that Me-β-CD induces liposome solubilization. Aditionaly, they confirm that DRV liposomes are affected more than SUV. Decrease of relative turbidity does not always agree with calcein release, indicating that calcein release occurs irrespective of liposomal solubilization.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-03-27T14:27:58Z No. of bitstreams: 2 Dissertação - Laís Cristina da Silva - 2014.pdf: 4002977 bytes, checksum: c54a0aa96c3be46a95b8df8e9a407c1d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-27T15:49:49Z (GMT) No. of bitstreams: 2 Dissertação - Laís Cristina da Silva - 2014.pdf: 4002977 bytes, checksum: c54a0aa96c3be46a95b8df8e9a407c1d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-27T15:49:49Z (GMT). No. of bitstreams: 2 Dissertação - Laís Cristina da Silva - 2014.pdf: 4002977 bytes, checksum: c54a0aa96c3be46a95b8df8e9a407c1d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The main predictive models of absorption of potential new drugs in preclinical stage are focused on the gastrointestinal mucosa, given the predominance of this pathway in drug administration. Often, the fraction absorbed (Fa) can be predicted in ex vivo models (p.e. Ussing chambers), in vitro (p.e. Caco-2 cells monolayers), intestinal perfusion studies in situ and in vivo absorption. In the present study, from an adaptation of Snapwell ™ inserts, a new ex vivo model to evaluate the permeability of substances passively absorbed is proposed. High permeable drugs (metoprolol, caffeine and theophylline) and low permeable drugs (atenolol, ranitidine and cimetidine) were maintained in an incubator at 37 ° C under constant stirring (60 rpm) and carbogenic atmosphere (5% CO2). The viability of the jejunal membrane (52 Ω.cm2 ± 8.0) was observed remaining above 20 Ω.cm2 for 120 min incubation, under all conditions evaluated, including the addition of co-solvents (1% DMSO and 1% EtOH). Values of apparent permeability coefficients obtained (Papp) were characteristic of ex vivo permeation studies (3.8 to 12.6 x10-6 cm / s). Strong correlation was observed between the data obtained here versus data intestinal perfusion in vivo (r = 0.89), as well as the fraction absorbed in humans (r = 0.85), reported in the literature. Additionally, the model features high sensitivity and accuracy compared to other commonly used models in classification permeability of substances. In line, we can infer that the MTSSNAPWELL model demonstrates, yet, potential application in studies of screening for selection of low molecular weight, such as potential phytochemicals, as well as their synthetic analogues evaluated with low amount of sample (ca 10 mg). / Os principais modelos preditivos da absorção de potenciais novos fármacos na etapa pré-clinica são focados na mucosa gastrointestinal, haja vista a predominância desta via na administração medicamentosa. Frequentemente, a fração absorvida (Fa) pode ser predita em modelos ex vivo em câmaras de Ussing, in vitro em monocamadas de células Caco-2, perfusão intestinal in situ e estudos de absorção in vivo. No presente estudo, a partir de uma adaptação do aparato Snapwell™, um novo modelo ex vivo de avaliação da permeabilidade para substâncias absorvidas por difusão passiva é proposto. Substâncias de alta (metoprolol, cafeína e teofilina) e baixa (atenolol, ranitidina e cimetidina) permeabilidade, foram mantidos em incubadora à 37OC, sob agitação constante (60 rpm) e atmosfera carbogênica (5% CO2). A viabilidade da membrana jejunal (52 ± 8,0 Ω.cm2) foi observada mantendose acima de 20 Ω.cm2 por até 120 min de incubação, sob todas condições avaliadas incluindo a adição de co-solventes (DMSO 1% e EtOH 1%). Os valores de coeficientes de permeabilidade aparente obtidos (Papp) mostraram-se característicos de estudos ex vivo de permeação (3,8 – 12,6 x10-6 cm/s). Forte correlação foi observada entre os dados aqui obtidos versus dados de perfusão intestinal in vivo (r = 0,89), assim como da fração absorvida em humanos (r = 0,85), relatados na literatura. Adicionalmente, o modelo apresenta elevada sensibilidade e precisão frente aos demais modelos comumente utilizados na classificação da permeabilidade de substâncias. Em consonância, pode-se inferir que o modelo MTSSNAPWELL demonstra, até o momento, potencial aplicação em estudos de screening para seleção de moléculas de baixo peso molecular, tais como potenciais fitofármacos, assim como seus análogos sintéticos avaliados com baixa quantidade de amostra (c.a. 10 mg).

MSCAGR (Animal Science) / Department of Animal Science / The objectives of the study were to evaluate the effect of two extenders (Triladyl® and Bioxcell®) and the removal of seminal plasma on goat buck semen. Six ejaculates were collected from six indigenous bucks by means of electro-ejaculator method, and semen was pooled, and replicated 10 times. Raw semen were randomly allocated into six groups as follows: (i) Raw non-washed, (ii) Raw washed, (iii) Triladyl®-washed, (iv) Triladyl®-non-washed, (v) Bioxcell®-washed and (vi) Bioxcell®-non-washed. All six groups were analysed for spermatozoa motility rates using computer-aided sperm analysis (CASA). The spermatozoa viability for all groups were assessed using Eosin-Nigrosin, acrosome integrity using Spermac, chromatin structure using Acridine Orange, and mitochondria using JC-1 staining solutions. Both the Triladyl® and Bioxcell® washed semen groups were diluted (1:4) with Phosphate Buffered Saline (PBS) then centrifuged at 1500 x g for ten min and seminal plasma was aspirated using 1 mL sterile plastic pipette. Semen samples were diluted (1:4) as follows: Triladyl® (washed and non-washed) or Bioxcell® (washed and non-washed) and then equilibrated at 5 ºC for 2 hours. Following equilibration, semen parameters were analysed. Thereafter, the semen samples were loaded into straws and placed 5 cm above a liquid nitrogen vapour for 10 min, and then stored at -196 ºC until use. Following one month of storage, frozen semen straws per treatment group were thawed at 37 ºC for 30 seconds, then semen parameters were analysed again. Significant differences among the mean values of semen parameters were determined by Tukey’s test using ANOVA, GLM procedure of SAS version 12.1 of 2010. Total Spermatozoa motility rate of Bioxcell® (92.5±4.6), (68.2±13.5) and Triladyl® (94.9±5.5), (63.1±15.1) were significantly reduced (P < 0.05) following equilibration and freeze-thawing process, respectively on washed semen groups. Live and normal spermatozoa percentages were drastically reduced in Bioxcell® (5.2±4.9) and Triladyl® (6.9±8.6) washed semen groups, following freeze-thawing. There was a significantly lower number of spermatozoa with high mitochondrial membrane potential in non-washed semen extended with Triladyl® (68.7±26.8) compared to non-washed semen extended with Bioxcell® (49.8±20.1) following the freeze-thawing process. In conclusion, the freezing-thawing process did reduce the indigenous buck semen parameters irrespective of removal or non-removal of seminal plasma. However, Bioxcell® extender was found to be more suitable for preserving spermatozoa during equilibration and freezing/thawing process of buck semen.

Les procédés d'osmose inverse (OI) permettent la production d'eau recyclée de très haute qualité grâce à l'élimination de contaminants organiques et inorganiques et de micro-organismes. Le suivi du bon fonctionnement de ce procédé est nécessaire pour valider la rétention des virus pathogènes afin de protéger la santé des usagers. La présence de minéraux et matières organiques dans les effluents rend inévitable le colmatage des membranes lors de leur fonctionnement et diminue ainsi leur performance. Afin d'éviter et d'éliminer ces colmatages, les stations de traitements des eaux utilisent des produits chimiques. Ces derniers vont modifier les performances globales des membranes en polyamide comme par exemple la diminution de la perméabilité à l'eau, et plus particulièrement les performances de rétention des virus, or l'ensemble de ces perturbations n'est que très peu compris et donc peu maitrisé. L'abattement des virus par l'OI sur des membranes intègres ou modifiées (ex : colmatage) ont donc été déterminés en mesurant la rétention d'un virus modèle de type phage MS2 et de substituts comme les sels (mesurés par conductivité), la rhodamine-WT (R-WT) ou les sulfates. La conductivité est, en effet, la technique de contrôle standard dans les stations de traitement des eaux (échelle industrielle).Le premier objectif de ce travail est d'évaluer l'utilisation d'un autre paramètre, les matières organiques dissoutes (DOM) comme nouveau substitut de virus et de déterminer l'impact du dysfonctionnement des procédés d'OI sur l'abattement des DOM et des sels à l'échelle industrielle. Les DOM peuvent en effet également être utilisées comme indicateur de qualité des eaux en fonction de leurs compositions et de leurs concentrations. L'abattement des DOM est donc testé comme nouvelle technique de surveillance afin de distinguer les fuites des changements de performance des membranes. Il est conclu que les DOM peuvent être utilisées comme nouvelle technique de contrôle. De plus, une variation de l'abattement des DOM peut aider à identifier des fuites de manière plus robuste que par l'abattement des sels. Le deuxième objectif est de déterminer l'effet des défauts membranaires sur les abattements d'un virus modèle (phage MS2) et de quatre substituts (R-WT, DOM, sulfate et sels) à l'échelle de systèmes de laboratoire. Deux systèmes à flux longitudinal est utilisés : une membrane plane et un module à spirale. Dans un premier temps, l'effet du colmatage sur les abattements de ces différents virus et substituts est étudié. Le colmatage organique, créé en utilisant un mélange de matières organiques, a pour effet d'augmenter de plus de 0,1 log les abattements de la R-WT, des sels et des DOM. Cette augmentation générale peut être due au blocage des cavités de la membrane et/ou par la sorption des substituts sur les matières organiques.Le colmatage inorganique, créé en utilisant un mélange de sels, n'a pas d'effet sur le rejet des substituts sauf pour les sels qui montre un comportement différent entre les deux systèmes. Dans le système à membrane plane, la couche inorganique permet d'augmenter le passage des sels à travers la membrane. Par opposition, il n'y a pas d'effet sur leur abattement avec le module à spirale. Cette variation entre les deux systèmes peut être causée par la différence de configuration (module à spirale contre membrane plane). Dans un deuxième temps, l'effet du chlore (modes passif et actif) sur la rétention de ces cinq composés est mesuré. Après un contact de 9000 ppm.h de NaOCl à pH 7, la surface membranaire change chimiquement. La formation de liaison Cl dans la couche en polyamide et la rupture des liaisons NH provoquent l'augmentation de la perméabilité à l'eau et diminuent l'abattement de l'ensemble des substituts. Malgré une forte diminution de 1,2 log de l'abattement en sel, l'abattement minimum du phage MS2 reste de 3 log. / One of the major applications of reverse osmosis (RO) process is the production of high quality recycled water by providing a barrier to remove organic and inorganic contaminants as well as pathogens including viruses. In order to protect public health, validation and monitoring of the RO process integrity are necessary to ensure its correct operation. During operation a certain degree of fouling is inevitable and can reduce RO membrane performance. Thus, chemicals are often used in water treatment plants to prevent or remove the membrane fouling. However, these chemicals can modify the integrity of the polyamide layer on RO membrane overtime. Up-to-date, the impact of membrane's physical change on its virus removal efficiency cause by the chemical use during operation is still not well understood.A minimum virus removal efficiency of intact and impaired (e.g. by fouling) RO membranes can be ascertained by measuring the rejection of MS2 phage and virus surrogates such as salt as measured by conductivity, rhodamine-WT (R-WT) or sulphate. However, conductivity measurement is the only full-scale standard monitoring technique. The removal of dissolved organic matter (DOM), which has been used as an indicator of water quality, can possibly be used for this purpose.The first objective of this work was to assess the suitability of DOM as a virus surrogate and to determine the impact of process failure on salt and DOM rejection in full-scale plants. A change of the conductivity does not necessarily mean that the membrane integrity has been breached. Thus, DOM monitoring has been tested and combined with the conductivity monitoring in order to distinguish between leaks and changes in membrane performances. It was concluded that DOM could be used as new monitoring technique. Moreover, a variation of DOM rejection can help identifying leaks better than just conductivity profiling alone.The second objective was to determine the effect of membrane impairments on the rejection of one model virus (MS2 phage) and four virus surrogates (R-WT, DOM, sulphate and salt) using lab-scale RO set-ups. To this aim, two different cross-flow set-ups were used: a flat-sheet and a single 2.5” spiral-wound module.Firstly, the effects of organic fouling and scaling on the rejection of model virus and virus surrogates were studied separately. Organic fouling was created using a mix of organic foulants. The result of this study showed an increase of the rejection by more than 0.1 log for R-WT, salt and DOM. The general increase of the surrogates' rejection might be due to the blocking of cavities of the polyamide membrane and/or to the sorption of surrogates to the fouling layer, which was observed by different autopsy techniques.Scaling was created using a mix of inorganic salts in order to reconstitute the composition of a RO feed water and avoiding the presence of organic foulants. Scaling was found to have no impact on the rejection of all tested virus surrogates except for salt. Salt rejection showed a change of behaviour between different set-ups: with the 2.5” module set-up the inorganic layer led to a stabilisation of the salt rejection, whereas the salt rejection increased with the flat-sheet set-up. This could be explained by the variations of the systems configuration (i.e. spiral module versus flat-sheet, feed spacer height, etc.).Secondly, the long-term impact of membrane ageing by exposure to chlorine, either active under filtration or passive by soaking, on the rejection of the model virus and four surrogates was studied. After a contact time of 9000 ppm∙h NaOCl at pH 7, the membrane surface chemistry changed. The introduction of chlorine in the membrane chemistry and the breakage of amide bonds caused an increase of the water permeability and a decrease of the model virus and virus surrogates rejection.