No description available.

>Magister Scientiae - MSc / Antimicrobials are chemical compounds that destroy or inhibit the growth of microorganisms. The majority of these antimicrobials are actually natural products or natural product derived with key examples being the pioneer antibiotics penicillin and cephalosporin. Antimicrobials are an extremely important class of therapeutic agents; however, the development of drug resistance and slow pace of new antibiotic discovery is one of the major health issues facing the world today. There is therefore a crucial need to discover and develop new antibacterial agents. In this study, the potential of marine algae as a source of new antibiotics was explored. Crude organic extracts and chromatographic fractions obtained from small-scale extraction of 17 different marine algae were used to prepare a pre-fractionated library that would be tested against several disease causing microorganisms. The activity of the pre-fractionated library and purified compounds was determined against a panel of drug resistant microorganisms namely Acinetobacter baumannii ATCCBAA®-1605™, Enterococcus faecalis ATCC® 51299™, Escherichia coli ATCC® 25922™, Staphylococcus aureus subsp. aureus ATCC® 33591™ and Candida albicans ATCC® 24433™. Finally, cytotoxicity tests of 50 selected library extracts and isolated compounds were done against two cell lines namely MCF-7 (breast cancer) and HEK-293 (kidney embryonic). Based on their antimicrobial activity and interesting chemical profiles, the seaweeds Plocamium sp. and Stypopodium multipartitum were selected for further study. Three new and unusual halogenated monoterpenes (4.16, 4.17 and 4.18) were isolated from Plocamium sp., and an unusual meroditerpenoid (5.8) was isolated from Stypopodium multipartitum. The metabolites were purified using preparative (silica gel) chromatography as well as semipreparative normal phase HPLC. The structures of purified compounds were determined from spectroscopic data, including nuclear magnetic resonance (NMR) spectroscopy. A small library of 153 fractions was generated from collections of South African marine algae. Pre-fractionated crude extracts showed excellent antimicrobial activity against all microbes but particularly against Staphylococcus aureus. The compounds were generally active against the Gram positive bacteria and the yeast. In conclusion, three antimicrobial halogenated monoterpenes and an unusual monoterpene were isolated from a Plocamium sp. and Stypopodium multipartitum respectively. Antimicrobial activity of crude fractions was excellent but that of isolated compounds was not as great as anticipated.

Orientador: Christiane Pienna Soares / Banca: Cleslei Fernando Zanelli / Banca: Andreimar Martins Soares / Resumo: Diversos estudos buscam identificar compostos com atividade seletiva para celulas tumorais e que possuam mecanismo de acao para desencadear a apoptose. Dentre as substancias isoladas de Cryptocarya sp, algumas estirilpironas, como a goniotalamina, apresentam atividade antiproliferativa e apoptogenica em diferentes linhagens celulares. No presente estudo, foram avaliadas as atividades citotoxica e pro-apoptotica da estirilpirona (criptomoscatona D2) isolada de Cryptocarya mandioccana, em linhagens celulares de carcinoma cervical humano infectada por HPV (HeLa e SiHa), nao infectada (C33A) e fibroblasto pulmonar humano transformado pelo SV-40 (MRC-5). A atividade citotoxica foi avaliada pelo ensaio do MTT e a apoptose foi avaliada, respectivamente, pelos ensaios de anexina V e a expressao de bak/bcl-2, por citometria de fluxo. Para o ensaio do MTT, as celulas foram tratadas com estirilpirona (criptomoscatona D2) nas concentracoes de 15, 30, 60 e 90-ÊM por 6, 24 e 48 horas e por 6 horas com periodo de recuperacao de 24, 48 e 72 horas pos tratamento. Para os ensaios de apoptose, as celulas foram tratadas por 6 horas e periodo de recuperacao de 24, 48 e 72 horas. O tratamento com a estirilpirona (criptomoscatona D2) ocasionou elevada citotoxicidade dose-resposta e tempo-resposta em HeLa, SiHa, C33A e MRC-5. Embora nao haja diferenca estatisticamente significativa de citotoxicidade entre as linhagens, aparentemente a citotoxicidade foi maior em HeLa e C33A (tratamento de 24 e 48 horas) que em MRC-5 e SiHa. Ainda, no periodo de recuperacao, HeLa e SiHa aparentemente restabelecem sua capacidade proliferativa, que e diretamente proporcional ao tempo de recuperacao, enquanto o mesmo comportamento nao e observado em C33A. Ao avaliar a expressao de duas proteinas da via intrinseca de apoptose (bcl-2 e bak), nao foi observada modulacao dessa expressao entre as linhagens celulares, nas diferentes tempos de recuperação pos-tratamento. / Abstract: Several attempts have been made to identify chemical compounds with selective cytotoxicity against cancer cells and apoptosis trigger activity. Among the substances isolated from Cryptocarya sp, some styrylpyrones, such as goniothalamine, demonstrate antiproliferative and apoptotic activity in abroad human cell lines. In the present study, we evaluated the antiproliferative and apoptotic activities of the styrylpyrone (cryptomoschatone D2) isolated from Cryptocarya mandioccana in HPV-infected (HeLa and SiHa) and non-infected (C33A) human cervical carcinoma cell lines, and in human lung's fibroblast immortalized with SV-40 (MRC-5). The antiproliferative activity was evaluated by the MTT assay and the apoptotic activity was investigated by measuring the expression levels of annexin V and bak/bcl-2 by flow cytometry. In the MTT assay, cells were treated with styrylpyrone (cryptomoschatone D2) at a 15, 30, 60 or 90ìM concentration for 6, 24 or 48 hours as well as for 6 hours followed by a recovery posttreatment period of 24, 48 or 72 hours. In the apoptotic assays, cells were treated for 6 hours followed by a recovery posttreatment period of 24, 48 or 72 hours. High cytotoxicity (dose-response and time-response) was observed in HeLa, SiHa, C33A and MRC-5 cell lines. Although the styrylpyrone cytotoxicity was not significantly different among the cell lines tested, the citotoxicity was apparently higher in HeLa and C33A than MRC-5 and SiHa in the case of treatments for 24 or 48 hours. Moreover, HeLa and SiHa were able to recover their prolifetative status, which were directly proportional to the posttreatment recovery time. On the other hand, C33A did not demonstrate a similar posttreatment recovery. Despite the posttreatment recovery time, the expression of the apoptotic proteins bcl-2 and bak seems not to be modulated by the treatment. / Mestre

Orientador: Brenda Paula Figueiredo de Almeida Gomes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-04T04:19:03Z (GMT). No. of bitstreams: 1 Berber_VanessaBellocchio_M.pdf: 2399742 bytes, checksum: 5b61f74adaec8d2617407274d650019d (MD5) Previous issue date: 2005 / Resumo: Uma infecção pulpar pode resultar na colonização microbiana de todo sistema de canais radiculares incluindo os túbulos dentinários. Estes microrganismos e seus produtos metabólicos tóxicos são responsáveis pelo desenvolvimento e persistência de periodontite apical de origem endodôntica. Este estudo teve como objetivo testar in vitro o uso de substâncias químicas auxiliares do preparo mecânico (clorexidina (CLX) gel e líquida 2%, hipoclorito de sódio (NaOCl) 5,25%, 2,5% e 0,5% e soro fisiológico) e técnicas de instrumentação (Híbrida; Cérvico-Apical (FOP-UNICAMP); instrumentação rotatória com HERO 642) na redução de Enterococcus faecalis no canal radicular e nos túbulos dentinários. Para tanto, 270 raízes de pré-molares inferiores foram autoclavadas e contaminadas por 21 dias com Enterococcus faecalis. Em seguida, foram divididas em 18 grupos nos quais as técnicas de instrumentação foram testadas variando o uso das substâncias químicas auxiliares. Amostras bacteriológicas do canal radicular foram coletadas antes e imediatamente após o preparo químico-mecânico, as quais foram cultivadas a fim de se determinar as unidades formadoras de colônia (UFC). Após a instrumentação, as raízes foram seccionadas em 3 blocos: apical, médio e cervical. Amostras de dentina foram removidas seqüencialmente com brocas tronco-cônicas. As raspas obtidas foram coletadas em tubos com BHI e plaqueadas em BHI ágar-sangue. As placas foram incubadas por 48 horas para posterior contagem das UFC. Na luz do canal radicular, todas as substâncias, inclusive o soro fisiológico quando associados à instrumentação mecânica, promoveram uma redução de quase 100% nas coletas microbiológicas imediatamente após o preparo químico-mecânico. Nas coletas resultantes das raspas de dentina, em todos os terços, técnicas e profundidades o NaOCl 5,25% e a CLX gel 2% obtiveram os melhores resultados na redução bacteriana dos túbulos dentinários seguidos do NaOCl 2,5%, CLX líquida 2% e NaOCl 0,5%. Concluiu-se que, o NaOCl 5,25% seguido pela CLX gel 2% juntamente com as técnicas Cérvico-Apical da FOP-UNICAMP e HERO, foram mais efetivos na eliminação do E.faecalis dos túbulos dentinários e do canal radicular / Abstract: An infection of the pulp can result in microbial colonization of the entire root canal system, together with the dentinal tubules adjacent to the canal. These microorganisms and their toxic metabolic products are responsible for the development and persistence of apical periodontitis of endodontal origin. The aim of this study was to evaluate in vitro the efficacy of 6 chemical agents used during chemo-mechanical preparation (0.5%, 2.5%, 5.25% sodium hypochlorite; 2% chlorhexidine liquid and gel; sterile saline ¿negative control) and 3 instrumentation techniques (Hybrid, Crown-Down by FOP-UNICAMP; HERO 642 system) against Enterococcus faecalis within root canals and dentinal tubules.Two hundred and seventy freshly extracted human mandibular premolars were infected in vitro for 21 days with E.faecalis. The specimens were divided into 18 groups, according to the auxiliary chemical substances and instrumentation techniques used. Canals were sampled before and immediately after preparation. After serial dilution, samples were plated onto BHI agar, and the colony forming units (CFU) that were grown were counted. Then, the teeth were sectioned in thirds, and dentine chips were removed from the canals with sequential sterile conic burs at low speed. The samples obtained with each bur were immediately collected in separate test tubes containing BHI broth. The tubes were incubated at 37°C and plated onto BHI agar. The CFU were counted and analyzed. In the canal samples, all substances including sterile saline, promoted almost 100% of CFU reduction immediately after biomechanical procedures. In samples of dentinal chips, in all instrumentation techniques, in all thirds, in all dentinal depths tested, 5.25% NaOCl and 2% chlorhexidine gel obtained the best results, followed by 2.5% NaOCl, 2% chlorhexidine liquid and 0.5% NaOCl. It can be concluded that 5.25% NaOCl followed by 2% chlorhexidine gel together with Crown-Down by FOP-UNICAMP or HERO 642 system seem to be more effective in eliminating E.faecalis in the canal and dentinal tubules than the other ones tested in this study / Mestrado / Endodontia / Mestre em Clínica Odontológica

Esta pesquisa foi realizada com o objetivo de caracterizar cafeeiros de sete espécies de Coffea e das respectivas variedades pertencentes a C. canephora e C. liberica presentes no Banco de Germoplasma de Café do Instituto Agronômico de Campinas, visando à possibilidade do seu agrupamento, bem como a sua utilização no melhoramento das espécies C. arabica e C. canephora. Para o referido estudo foram utilizadas cento e dez plantas pertencentes a sete espécies e treze variedades, tendo sido avaliadas em função das características químicas de sementes como: sólidos solúveis, lipídios, trigonelina, ácidos clorogênicos e cafeína. Com base nos resultados destas variáveis observou-se uma grande variação entre e dentro dos diferentes materiais analisados, com valores extremos de 24,12% a 31,00% para sólidos solúveis; 6,61% a 17,49% para lipídios; 0,32% a 2,15% para trigonelina; 2,58% a 6,38% para ácidos clorogênicos e 0,80% a 3,29% para cafeína, indicando que estes atributos podem ser adotados na seleção de plantas com potencial para o melhoramento das espécies C. arabica e C. canephora. Os resultados evidenciam que as variáveis: (i) sólidos solúveis, lipídios, ácidos clorogênicos e cafeína permitem o agrupamento das variedades de C. canephora; (ii) sólidos solúveis, lipídios e trigonelina possibilitam discriminar as variedades de C. liberica; e, (iii) lipídios, ácidos clorogênicos, trigonelina e cafeína foram eficientes no agrupamento das sete espécies de Coffea. As variedades de C. canephora não apresentaram diferenças para o teor de trigonelina, enquanto as de C. liberica não variaram em relação aos teores de ácidos clorogênicos e cafeína. O conjunto dos dados obtidos para as variáveis químicas analisadas indica que há possibilidade das variedades Uganda e Bangelan serem híbridos entre as espécies C. congensis e C. canephora. / The objective of this work was to characterize seven coffee species and varieties from C. canephora and C. liberica presents in Germplasm Bank of the Instituto Agronômico in order to determine the possibility of its grouping as well its use on breeding of C. arabica and C. canephora species. A total of a hundred ten plants belonging to seven species and thirteen varieties were analysed for some chemical components of seeds (soluble solids, lipids, trigonelline, chlorogenic acids and caffeine). The results evidenced the existence of great variation among and within the materials analyzed, with values ranging from 24,12% to 31,00% for soluble solids; 6,61% to 17,49% for lipids; 0,32% to 2,15% for trigonelline; 2,58% to 6,38% for chlorogenic acids and 0,80% to 3,29% for caffeine, indicating that these variables can be used in selection of plants for the improvement of C. arabica and C. canephora. The results also showed that (i) soluble solids, lipids, chlorogenic acids and caffeine allowed to group C. canephora varieties, (ii) soluble solids, lipids and trigonelline permited discriminate C. liberica varieties.and (iii) lipids, chlorogenic acids, trigonelline and caffeine allowed to group coffee species. The C. canephora varieties did not show differences in relation to trigonelline while C. liberica varieties did not varied in relation to caffeine and chlorogenic acids. The hole group of obtained data for chemical variables analysed show that there is the possibility that Uganda and Bangelan varieties been hybrids between C. congensis and C. canephora.

Esta pesquisa foi realizada com o objetivo de caracterizar cafeeiros de sete espécies de Coffea e das respectivas variedades pertencentes a C. canephora e C. liberica presentes no Banco de Germoplasma de Café do Instituto Agronômico de Campinas, visando à possibilidade do seu agrupamento, bem como a sua utilização no melhoramento das espécies C. arabica e C. canephora. Para o referido estudo foram utilizadas cento e dez plantas pertencentes a sete espécies e treze variedades, tendo sido avaliadas em função das características químicas de sementes como: sólidos solúveis, lipídios, trigonelina, ácidos clorogênicos e cafeína. Com base nos resultados destas variáveis observou-se uma grande variação entre e dentro dos diferentes materiais analisados, com valores extremos de 24,12% a 31,00% para sólidos solúveis; 6,61% a 17,49% para lipídios; 0,32% a 2,15% para trigonelina; 2,58% a 6,38% para ácidos clorogênicos e 0,80% a 3,29% para cafeína, indicando que estes atributos podem ser adotados na seleção de plantas com potencial para o melhoramento das espécies C. arabica e C. canephora. Os resultados evidenciam que as variáveis: (i) sólidos solúveis, lipídios, ácidos clorogênicos e cafeína permitem o agrupamento das variedades de C. canephora; (ii) sólidos solúveis, lipídios e trigonelina possibilitam discriminar as variedades de C. liberica; e, (iii) lipídios, ácidos clorogênicos, trigonelina e cafeína foram eficientes no agrupamento das sete espécies de Coffea. As variedades de C. canephora não apresentaram diferenças para o teor de trigonelina, enquanto as de C. liberica não variaram em relação aos teores de ácidos clorogênicos e cafeína. O conjunto dos dados obtidos para as variáveis químicas analisadas indica que há possibilidade das variedades Uganda e Bangelan serem híbridos entre as espécies C. congensis e C. canephora. / The objective of this work was to characterize seven coffee species and varieties from C. canephora and C. liberica presents in Germplasm Bank of the Instituto Agronômico in order to determine the possibility of its grouping as well its use on breeding of C. arabica and C. canephora species. A total of a hundred ten plants belonging to seven species and thirteen varieties were analysed for some chemical components of seeds (soluble solids, lipids, trigonelline, chlorogenic acids and caffeine). The results evidenced the existence of great variation among and within the materials analyzed, with values ranging from 24,12% to 31,00% for soluble solids; 6,61% to 17,49% for lipids; 0,32% to 2,15% for trigonelline; 2,58% to 6,38% for chlorogenic acids and 0,80% to 3,29% for caffeine, indicating that these variables can be used in selection of plants for the improvement of C. arabica and C. canephora. The results also showed that (i) soluble solids, lipids, chlorogenic acids and caffeine allowed to group C. canephora varieties, (ii) soluble solids, lipids and trigonelline permited discriminate C. liberica varieties.and (iii) lipids, chlorogenic acids, trigonelline and caffeine allowed to group coffee species. The C. canephora varieties did not show differences in relation to trigonelline while C. liberica varieties did not varied in relation to caffeine and chlorogenic acids. The hole group of obtained data for chemical variables analysed show that there is the possibility that Uganda and Bangelan varieties been hybrids between C. congensis and C. canephora.

Η διαδικασία της αδειοδότησης της αντιγραφής του DNA περιλαμβάνει αρχικά το σχηματισμό του προ-αντιγραφικού συμπλόκου στις αφετηρίες της αντιγραφής, με τελικό αποτέλεσμα την προσέλκυση των ελικασών MCM2-7 (Mini Chromosome Maintenance Complex) και την έναρξη της αντιγραφής. Ο παράγοντας Cdt1 διαδραματίζει σημαντικό ρόλο στη διαδικασία αδειοδότησης και για τον λόγο αυτό ρυθμίζεται αυστηρά κατά την διάρκεια του κυτταρικού κύκλου. Στους ανώτερους ευκαρυωτικούς οργανισμούς, εκτός από την αποικοδόμησή του, υπάρχει ένας επιπλέον τρόπος ρύθμισής του, μέσω της πρωτεΐνης της Geminin. Η Geminin προσδένεται στο Cdt1 και με αυτόν τον τρόπο αναστέλλει τη στρατολόγηση των MCM2-7 στη χρωματίνη και επομένως, την αδειοδότηση της αντιγραφής. Εκτός από την δράση της στην ρύθμιση του κυτταρικού κύκλου, η Geminin παίζει σημαντικό ρόλο και στην κυτταρική διαφοροποίηση, αλληλεπιδρώντας με μεταγραφικούς παράγοντες και πρωτεϊνικά σύμπλοκα αναδιοργάνωσης της δομής της χρωματίνης. Η γονιδιωματική αστάθεια είναι χαρακτηριστικό των καρκινικών κυττάρων, στην οποία συντελεί απορύθμισης παραγόντων του συστήματος αδειοδότησης της αντιγραφής και συγκεκριμένα των κύριων ρυθμιστών του, Geminin και Cdt1. Η υπερέκφραση του Cdt1 οδηγεί σε επαναντιγραφή και, συνεπώς, υπερδιπλασιασμό του DNA, ενώ την ίδια επίπτωση εμφανίζει και η αποσιώπηση της Geminin. Η σημαντικότητα της αλληλεπίδρασης των δυο αυτών μορίων οδήγησε στην αναζήτηση συνθετικών χημικών ουσιών με την ικανότητα διάσπασης του συμπλόκου Geminin-Cdt1. Οι συνθετικές αυτές χημικές ενώσεις μπορούν να χρησιμοποιηθούν ως μοριακά εργαλεία για την περαιτέρω μελέτη της in vivo αλληλεπίδρασης των Geminin και Cdt1 στις διαδικασίες του κυτταρικού πολλαπλασιασμού και διαφοροποίησης, καθώς επίσης και ως φαρμακολογικές ουσίες με πιθανή αντικαρκινική δράση. Η παρούσα εργασία εστιάστηκε στη μελέτη της συνθετικής χημικής ένωσης XIV, η οποία είχε ταυτοποιηθεί σε έναν προηγούμενο μαζικό έλεγχο υψηλής απόδοσης (High throughput Screening - HTS). Αρχικά καθορίστηκαν τα επίπεδα κυτταροτοξικότητάς της. Στη συνέχεια μελετήθηκε η ικανότητά της να μεταβάλλει την ενδοκυτταρική κατανομή της Geminin και τέλος, εξετάστηκε η επίδρασή της στον κυτταρικό κύκλο. Τα αποτελέσματα έδειξαν ότι η συνθετική χημική ένωση XIV παρουσιάζει περιορισμένη κυτταροτοξικότητα. Επιπλέον, αυξάνει τον πυρηνικό εντοπισμό της εξωγενώς εκφρασμένης Geminin, υποδεικνύοντας ότι μπορεί να ανταγωνίζεται το Cdt1 για την πρόσδεσή του στη Geminin. Πειράματα ανοσοφθορισμού και ανάλυσης περιεχομένου του DNA, μέσω FACS, έδειξαν ότι η συνθετική χημική ένωση XIV προκαλεί αναστολή του κυτταρικού κύκλου των καρκινικών κυττάρων κατά τα αρχικά στάδια της S φάσης. Συμπερασματικά, η συνθετική χημική ένωση XIV ενδέχεται να επηρεάζει την αλληλεπίδραση των Geminin και Cdt1. Χημικές ενώσεις ικανές να παρεμβαίνουν στην ειδικότητα αλληλεπίδρασης των δυο αυτών μορίων και συνεπώς στο σύστημα αδειοδότησης της αντιγραφής, μπορούν να αποτελέσουν τη βάση για την ανάπτυξη μια νέας γενιάς αντικαρκινικών φαρμάκων με βελτιστοποιημένες ιδιότητες. / The replication licensing begins with the formation of the pre-Replicative Complex (preRC) on the origins of replication (ori), leading to the recruitment of the MCM2-7 helicases onto chromatin and the initiation of DNA replication. Cdt1 plays a crucial role in the licensing and is therefore strictly regulated during the cell cycle. Cdt1 is primarily regulated via proteolytic degradation, while in higher eukaryotes, is also negatively regulated by Geminin. Geminin’s binding to Cdt1 inhibits the loading of the MCMs to chromatin and thus licensing of DNA replication. Geminin also has an additional role in differentiation processes by multiple interactions with transcriptional factors and chromatin remodeling complexes. Cancer cells are characterized by genomic instability. Deregulation of the components of the licensing system and especially of Geminin and Cdt1, leads to genomic instability and promotes malignant transformation. Geminin and Cdt1 complex could be used as could serve as target for the identification of chemical compounds that would be able to modulate proliferation. These chemical compounds can be used as molecular tools for further studying the in vivo interaction of these two molecules during the processes of cellular proliferation and differentiation, and ultimately serve as potential pharmacological agents with anti-cancer properties. In this study, we aimed to characterize the chemical compound XIV, which was identified in a previous High Throughput Screening (HTS). Specifically, we examined compound XIV in cellular assays in order to determine cytotoxity, its ability to alter the subcellular localization of Geminin and cell cycle profile of cancer cells. Our results, suggest that the chemical compound XIV exhibits limited cytotoxicity. It increases the nuclear localization of transiently expressed Geminin, suggesting that it might act by antagonizing Cdt1 for the binding of Geminin. Additionally, immunofluorescence and FACS experiments showed that chemical compound XIV causes cell cycle arrest during early S phase. Overall, in this study we propose that chemical compound XIV interferes with the Geminin and Cdt1 complex and affects proliferation of tumorigenic cells.

Made available in DSpace on 2014-06-11T19:23:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-06-01Bitstream added on 2014-06-13T18:51:18Z : No. of bitstreams: 1 giocondo_mp_me_arafcf.pdf: 1362305 bytes, checksum: 5d252e88df73be4c96290a8a2c37f546 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / Diversos estudos buscam identificar compostos com atividade seletiva para celulas tumorais e que possuam mecanismo de acao para desencadear a apoptose. Dentre as substancias isoladas de Cryptocarya sp, algumas estirilpironas, como a goniotalamina, apresentam atividade antiproliferativa e apoptogenica em diferentes linhagens celulares. No presente estudo, foram avaliadas as atividades citotoxica e pro-apoptotica da estirilpirona (criptomoscatona D2) isolada de Cryptocarya mandioccana, em linhagens celulares de carcinoma cervical humano infectada por HPV (HeLa e SiHa), nao infectada (C33A) e fibroblasto pulmonar humano transformado pelo SV-40 (MRC-5). A atividade citotoxica foi avaliada pelo ensaio do MTT e a apoptose foi avaliada, respectivamente, pelos ensaios de anexina V e a expressao de bak/bcl-2, por citometria de fluxo. Para o ensaio do MTT, as celulas foram tratadas com estirilpirona (criptomoscatona D2) nas concentracoes de 15, 30, 60 e 90-ÊM por 6, 24 e 48 horas e por 6 horas com periodo de recuperacao de 24, 48 e 72 horas pos tratamento. Para os ensaios de apoptose, as celulas foram tratadas por 6 horas e periodo de recuperacao de 24, 48 e 72 horas. O tratamento com a estirilpirona (criptomoscatona D2) ocasionou elevada citotoxicidade dose-resposta e tempo-resposta em HeLa, SiHa, C33A e MRC-5. Embora nao haja diferenca estatisticamente significativa de citotoxicidade entre as linhagens, aparentemente a citotoxicidade foi maior em HeLa e C33A (tratamento de 24 e 48 horas) que em MRC-5 e SiHa. Ainda, no periodo de recuperacao, HeLa e SiHa aparentemente restabelecem sua capacidade proliferativa, que e diretamente proporcional ao tempo de recuperacao, enquanto o mesmo comportamento nao e observado em C33A. Ao avaliar a expressao de duas proteinas da via intrinseca de apoptose (bcl-2 e bak), nao foi observada modulacao dessa expressao entre as linhagens celulares, nas diferentes tempos de recuperação pos-tratamento. / Several attempts have been made to identify chemical compounds with selective cytotoxicity against cancer cells and apoptosis trigger activity. Among the substances isolated from Cryptocarya sp, some styrylpyrones, such as goniothalamine, demonstrate antiproliferative and apoptotic activity in abroad human cell lines. In the present study, we evaluated the antiproliferative and apoptotic activities of the styrylpyrone (cryptomoschatone D2) isolated from Cryptocarya mandioccana in HPV-infected (HeLa and SiHa) and non-infected (C33A) human cervical carcinoma cell lines, and in human lung's fibroblast immortalized with SV-40 (MRC-5). The antiproliferative activity was evaluated by the MTT assay and the apoptotic activity was investigated by measuring the expression levels of annexin V and bak/bcl-2 by flow cytometry. In the MTT assay, cells were treated with styrylpyrone (cryptomoschatone D2) at a 15, 30, 60 or 90ìM concentration for 6, 24 or 48 hours as well as for 6 hours followed by a recovery posttreatment period of 24, 48 or 72 hours. In the apoptotic assays, cells were treated for 6 hours followed by a recovery posttreatment period of 24, 48 or 72 hours. High cytotoxicity (dose-response and time-response) was observed in HeLa, SiHa, C33A and MRC-5 cell lines. Although the styrylpyrone cytotoxicity was not significantly different among the cell lines tested, the citotoxicity was apparently higher in HeLa and C33A than MRC-5 and SiHa in the case of treatments for 24 or 48 hours. Moreover, HeLa and SiHa were able to recover their prolifetative status, which were directly proportional to the posttreatment recovery time. On the other hand, C33A did not demonstrate a similar posttreatment recovery. Despite the posttreatment recovery time, the expression of the apoptotic proteins bcl-2 and bak seems not to be modulated by the treatment.

Orientador: Altair Antoninha Del Bel Cury / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-17T11:17:25Z (GMT). No. of bitstreams: 1 Fernandes_FredericoSilvadeFreitas_D.pdf: 739091 bytes, checksum: 727731c2061dd20f60d28b6f01753ccf (MD5) Previous issue date: 2011 / Resumo: Os limpadores químicos de prótese têm sido bastante indicados para o controle do biofilme formado sobre próteses removíveis de pacientes com comprometimento motor. Apesar de estudos prévios terem mostrado que uma única imersão nesses agentes é capaz de reduzir os níveis de Candida albicans do biofilme formado sobre próteses removíveis, pouco se sabe sobre o efeito do uso diário desses limpadores sobre o biofilme residual de Candida. Assim, o objetivo desse estudo foi avaliar a eficácia do uso diário de um limpador químico enzimático sobre o biofilme de C. albicans formado sobre a superfície de materiais para confecção de próteses removíveis; bem como a atividade enzimática das células de Candida desse biofilme após exposições diárias a esse limpador de prótese. Foram confeccionados espécimes de resina de polimetilmetacrilato (PMMA) e resina de poliamida, nos quais foi realizada, inicialmente, a padronização da rugosidade de superfície (0,34 ± 0,02 ?m). Após a formação da película adquirida, os espécimes foram divididos aleatoriamente em 12 grupos (n=9) para formação do biofilme de C. albicans por 72 horas. Após esse período, os espécimes foram tratados por 1, 4 ou 7 dias, sendo realizado um tratamento por dia, com um limpador químico enzimático (Polident 3 Minutes) ou com água destilada (controle negativo). Após os respectivos períodos de tratamento, os microrganismos remanescentes foram removidos da superfície dos espécimes por meio de ondas ultra-sônicas (7W por 30s). Em seguida, as unidades formadoras de colônia (UFC) foram calculadas e a atividade enzimática das células remanescentes foi avaliada. Os dados foram submetidos à ANOVA um fator ou dois fatores, seguido do teste de Tukey-Kramer. O biofilme de Candida albicans formado sobre a resina de poliamida apresentou maiores níveis de Candida e uma maior atividade fosfolipásica que o biofilme formado sobre a resina de PMMA (p<0,001). O limpador químico enzimático reduziu significantemente os níveis de Candida albicans em todos os períodos avaliados (p<0,001), entretanto os níveis desse microrganismo aumentaram com o tempo, sendo observada diferença estatisticamente significante entre os períodos avaliados (p<0,001). As exposições diárias a esse limpador químico aumentaram a virulência das células de Candida, no que diz respeito à atividade fosfolipásica. Nas condições desse estudo, conclui-se que o uso diário do limpador químico enzimático não foi capaz de impedir a proliferação de Candida albicans no biofilme residual, apesar de ter interferido no crescimento desse biofilme. / Abstract: Chemical cleansing with immersion in denture cleansers has been indicated for denture biofilm control in patients with limited motor capacity. Although previous studies have shown that a single immersion in those agents is able to substantially reduce Candida albicans biofilm levels, the effect of the routine use of denture cleansers on the Candida residual biofilm is poorly understood. This study evaluated the efficacy of daily use of an enzymatic denture cleanser on C. albicans biofilm formed on denture base materials; and the enzymatic activities of Candida biofilm cells after daily exposure to this cleanser agent. Polymethyl methacrylate (PMMA) and polyamide resins specimens were prepared (n=54), and their surface roughness was standardized (0.34 ±0.02 ?m). Saliva-coated specimens were randomly divided by lottery into 12 groups (n=9) for biofilm assay. C. albicans biofilm was formed for 72 hours, and then specimens were treated for 1, 4 or 7 days, once a day, with an enzymatic cleanser (Polident 3 Minutes), or distilled water (negative control). Remaining adherent microorganisms were removed from the treated specimens by ultrasonic waves at 7 watts for 30 seconds, and then colony-forming units (CFU) were calculated and remaining cells enzymatic activities were determined. Data were analyzed by 1-way or 2-way ANOVA followed by the Tukey-Kramer test. C. albicans biofilm formed on polyamide resin showed significantly higher Candida levels and phospholipase activity (p<0.001) than biofilm formed on PMMA resin. The enzymatic cleanser significantly reduced C. albicans levels in all evaluated periods (p<0.001); however, the number of this microorganism increased with time, showing statistical difference among the treatment periods (p<0.001). The daily exposure to the denture cleanser increased Candida cells virulence, with regard to phospholipase activity. Our study has shown that the enzymatic cleanser daily use did not prevent C. albicans proliferation in residual biofilm; however, this agent reduced this fungus rate of growth. / Doutorado / Protese Dental / Doutor em Clínica Odontológica

Le développement de nouvelles compositions propulsives, pour applications stratégique et spatiale par exemple, conduit à élaborer des charges énergétiques de plus en plus puissantes... / The development of new compositions of propellants, for strategic and space applications for instance, leads to synthesis of more and more powerful energetic materials...