Orientador: Maria Leticia Cintra / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T17:26:59Z (GMT). No. of bitstreams: 1 Pimentel_VanessaNascimento_M.pdf: 2864833 bytes, checksum: 3ee9e69bb5f58fae7867132642bdf6e6 (MD5) Previous issue date: 2010 / Resumo: O líquen plano oral (LPO) e o acometimento oral da doença do enxerto contra o hospedeiro crônica (DECHc) apresentam características clínicas e histológicas semelhantes, apesar da etiologia distinta. A apoptose, induzida por linfócitos T citotóxicos, tem sido proposta como o tipo de morte dos ceratinocitos, em ambas as doenças. A citotoxidade celular é mediada, dentre outros mecanismos, por grânulos contendo granzima B e perforina. Poucos trabalhos foram desenvolvidos demonstrando o papel destas moléculas no líquen plano; dentre estes, alguns estudaram a interação das células que expressam estas moléculas com os demais componentes do infiltrado. Contudo, há raros estudos sobre este tema na DECHc. Considerando que as características em comum podem refletir similaridades nos mecanismos imunológicos, o objetivo do nosso estudo foi correlacionar os achados morfológicos e imuno-histoquímicos do LPO e da DECHc oral, na tentativa de compreender melhor a patogênese destas doenças. Foram analisadas 29 amostras de LPO e 27 de DECHc oral, coletadas no período de 1994 a 2007, de pacientes atendidos no Hospital das Clínicas e na Unidade de Transplante de Medula Óssea da UNICAMP. Novos cortes foram corados por H&E e pela técnica imuno-histoquímica para CD4, CD8, MAC 387, ICAM-1, granzima B e perforina. O número de células CD4-positivas foi estatisticamente maior no LPO do que na DECHc (p<0,0001), enquanto as médias totais (do epitélio + do tecido conjuntivo) de células positivas para granzima B e perforina foram maiores na DECHc que no LPO (p<0,05). Foi observado também que, quanto maior o número de células perforina+, maior era o número de células granzima B+, tanto no epitélio como no tecido conjuntivo, nos dois grupos de doenças (p<0,05). No LPO, o número de corpos apoptóticos isolados mostrou uma correlação positiva com a granzima B e negativa com a perforina do tecido conjuntivo (p<0,01). Inversamente, nas lesões orais da DECHc, o número de corpos apoptóticos agrupados apresentou uma correlação positiva com a perforina do tecido conjuntivo. Não houve diferença entre o LPO e a DECHc oral com relação ao número de corpos apoptóticos (isolados ou agrupados), nem quanto à extensão da degeneração hidrópica da camada basal, e nem mesmo com relação ao número de células imunomarcadas para CD8, ICAM-1 e MAC 387. Estes achados indicam que a apoptose, no LPO, parece correlacionar-se com a ação da granzima B, enquanto que, na DECHc oral, a perforina parece ser mais atuante. Embora o LPO e a DECHc oral apresentem similaridades clínicas e histológicas, parece haver diferenças na patogênese destas doenças. Os resultados encontrados podem ser úteis para aprimorar a compreensão dos mecanismos imunológicos, bem como podem embasar o desenvolvimento de novas estratégias terapêuticas para o controle da apoptose e redução da morbidade associada a estas doenças. / Abstract: Oral lesions in lichen planus (OLP) and chronic graft-versus-host disease (cGVHD) have similar clinical and histological features, but distinct etiology. Apoptosis induced by cytotoxic T lymphocyte has been proposed as the form of keratinocytes death. The cell cytotoxicity is mediated, among other mechanisms, by granules containing granzyme B and perforin. Few studies have been published showing the role of those molecules in lichen planus, as well as their relationship with other inflammatory infiltrate components. However, rare works were reported about this theme in cGVHD. Since common features can reflect similarities in immunological mechanisms, the purpose of our study was to correlate the morphological and immunohistochemical findings of OLP and cGVHD to better understand the pathogenesis of these diseases. We analyzed 29 samples of OLP and 27 of oral cGVHD collected in the period between 1994 and 2007, from patients treated at the University Hospital and Bone Marrow Transplant Unit of UNICAMP. Additional sections were obtained and stained for H&E and immunohistochemical technique targeting CD4, CD8, MAC 387, ICAM-1, perforin and granzyme B. The number of CD4-positive cells number was significantly higher in OLP than in cGVHD (p<0,0001), while the total means (epithelium plus connective tissue number) of the granzyme B- and perforin-positive cells were significantly higher in cGVHD (p<0,05). Also, it was found that the higher the number of perforin+ cells, the higher the number of granzyme-B + cells in the epithelium and in the connective tissue for both groups (p<0.05). In OLP, the number of single apoptotic bodies had a positive correlation with connective tissue granzyme B immunostaining and a negative correlation with perforin (p<0.01). On the contrary, in oral cGVHD, the number of apoptotic body clusters presented a positive correlation with connective tissue perforin (p<0.01). Our findings indicate that apoptosis in OLP seems to be correlated with granzyme B release, while in oral cGVHD, perforin seems to be more important. There were no significant differences between OLP and cGVHD oral regarding the following features: the amount of apoptotic bodies (isolated o clusters), the extension of hydropic basal cell degeneration, and the number of positive cells for CD8, ICAM-1 and MAC-387. These results indicate that apoptosis, in OLP, seems to correlate with granzyme B release while, in oral cGVHD, perforin is preponderant. Although OLP and oral cGVHD present clinical and histological similarities, differences seem to exist in the athogenesis of these diseases. Our results might help to better understand the immunological mechanisms of these two conditions, as well as supporting the development of new therapeutic strategies for controlling apoptosis and reducing morbidity associated with them. / Mestrado / Anatomia Patologica / Mestre em Ciências Médicas

Orientadores: Sergio Marangoni, Luis Alberto Ponce-Soto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T18:20:05Z (GMT). No. of bitstreams: 1 Bonfim_VeraLucia_D.pdf: 7120787 bytes, checksum: 3839207f36a378b463b63406202ef7e4 (MD5) Previous issue date: 2008 / Resumo: O envenenamento ofídico é um evento comum nos países pobres da área tropical e subtropical e as conseqüências deste, muito graves. Nestes países, pequena têm sido a valorização destas conseqüências, sendo pouco registradas estatisticamente como casos de saúde pública e muitas vezes tratados com procedimentos pouco efetivos ou ultrapassados. Neste trabalho foram utilizados metodologias refinadas e protocolos otimizados de purificação em HPLC com o intuito de se estudar mais detalhadamente a composição do veneno de duas serpentes de importância médica e epidemiológica, Bothrops jararacussu e Calloselasma rhodostoma, e analisar a relação estrutura função de PLA2 D49 (6-1, 6-2 e Cr-IV1) e PLA2 homólogas K49 (Bj-VII e Cr 5). A Bothrops jararacussu, é uma serpente encontrada nas Américas, desde o México até a Argentina sendo que no Brasil cerca de 90% dos acidentes ofídicos são causados pelo gênero Bothrops. A Calloselasma rhodostoma, é uma serpente amplamente distribuída no sudeste da Ásia e é a causa mais comum de acidentes ofídicos na Malásia e Tailândia. As novas toxinas foram obtidas com alto grau de pureza e homogeneidade molecular, como mostram os perfis cromatográficos, eletroforéticos e de espectrometria de massa por MALDi-TOF. A nova PLA2 D49 foi purificada do veneno de Calloselasma rhodostoma após dois passos cromatográficos: exclusão molecular em uma coluna Protein Pack SW 300 (0,78 cm x 3,30 cm), eluída com 0,25M de bicarbonato de amônio, pH 7,9, a um fluxo de 0,3 ml/min e, fase reversa em HPLC usando uma coluna µ-Bondapack C-18. Esta apresentou atividade fosfolipásica de 14,25±0,36 nmol/mg/min na presença de substrato sintético cromogênico (4-nitro-3-(octanoyloxy) benzoic acid), massa molecular de 14,029 Da determinada através de espectrometria de massa por MALDI-TOF, valor calculado de pI igual a 8.55, caráter básico e grande homologia seqüencial evidenciada pela estrutura primária quando esta é comparada com outras PLA2 D49 provenientes de veneno de serpente. A outra proteína, denominada Cr 5, uma PLA2 homóloga K49 foi isolada do veneno de Calloselasma rhodostoma em um único passo cromatográfico de fase reversa em HPLC (µ-Bondapack C-18). A massa molecular de 13,965 Da foi determinada através de espectrometria de massa por MALDI-TOF. A composição de aminoácido mostrou que a Cr 5 tem alto conteúdo de Lys, Tyr, Gly, Pro e 14 cisteínas, resíduos típicos de uma PLA2 básica. A completa seqüência de aminoácidos da Cr 5 contém 120 resíduos e mostra alta identidade quando comparada a outras PLA2 K49 isoladas de venenos de serpentes da família viperidae. Com relação à caracterização biológica, esta foi realizada também com as isoformas 6-1 e 6-2 (provenientes da fração BthTX-II de Botrhops jararacussu) e com a PLA2 K49 Bj-VII, estas purificadas e parcialmente caracterizadas em trabalho anterior. As PLA2 6-1, 6-2 e Cr-IV 1, exibiram miotoxicidade local e na dosagem de 40 µg/150 µl a 6-1 causou 83,06% de citotoxicidade em miotubos e 93,78% em mioblastos, já a 6-2 75,12 % em miotubos e 86,26% em mioblastos e a Cr-IV 1, 91,50% e 88,40% de citotoxicidade em miotubos e mioblastos, respectivamente. O efeito citotóxico também foi visto em fibroblastos mostrando na dosagem de 5 µM (linhagem 3T3) 85,63%, 86% e 91,34% de citotoxicidade na presença de 6-1, 6-2 e Cr-IV 1 respectivamente, e na linhagem COS7, 95,42%, 95,07% e 96,42% respectivamente, além de atividade inflamatória, sendo esta mais pronunciada na presença da Cr-IV 1. As PLA2s K49 homólogas, Bj-VII e Cr 5, evidenciaram miotoxicidade local e na dosagem de 40 ug/150 µl, a Bj-VII causou 87±50% de lise em mioblastos e 84±85% em miotubos e 91±25% de lise em mioblastos e 91±0.8 % em miotubos quando em contato com a Cr 5, além de atividade inflamatória evidenciada pela indução de edema. No caso destas proteínas, todos os efeitos biológicos induzidos por elas ocorreram na ausência de atividade fosfolipásica mensurável in vitro, suportando a idéia proposta pela literatura de mecanismos catalíticos e farmacológicos independentes. Apesar de todas apresentarem algumas diferenças biológicas, elas possuem um comportamento semelhante na estrutura-função, sugerindo uma ancestralidade comum embora sejam proteínas provenientes de venenos de serpentes separadas geograficamente / Abstract: Snake poisoning is a common event in poor countries of tropical and subtropical areas and the consequences of this is very serious. In these countries, there is the valorization of these consequences, due to few registers of published health cases and a lot of times treated with procedures a little effective or outdated. In this work we refined methodologies and optimized purification protocols used in HPLC with the intention to study the composition of the venom of two serpents of epidemic importance, Bothrops jararacussu and Calloselasma rhodostoma, and to analyze the relationship of structures function of PLA2 D49 (6-1, 6-2 and Cr-IV1) and homologous PLA2 K49 (Bj-VII and Cr 5). Bothrops jararacussu is a serpent found in America, from Mexico to Argentina being in Brazil, about 90% of the snake accidents caused by Bothrops genus. Calloselasma rhodostoma is a serpent thoroughly distributed at the Southwest of Asia and it is the most common cause of snake accidents in Malaysia and Thailand. The new toxins were obtained with high degree of purity and molecular homogeneity, like showed the chromatographics profiles, electrophoresis and MALDi-tof mass spectrometric (MS) analysis. A new D49 PLA2 was purified from the venom of Calloselasma rhodostoma after two chromatographic steps; molecular exclusion was loaded onto a Protein-Pack 300 SW column (0.78 cm 3 30 cm) and eluted with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min and reverse phase HPLC on µ-Bondapack C-18 and showed phospholipasic activity of 14.25±0.36 nmol/mg/min in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy) benzoic acid), a molecular mass of 14.029 Da was determined by MALDI-TOF mass spectrometry, calculated pI value 8.55, basic character and great sequential homology evidenced by the primary structure when this was compared with other PLA2 from D49 snake venom. The other protein, denominated Cr 5 PLA2 homologous (K49) was isolated from Calloselasma rhodostoma venom in one chromatographic step in reverse phase HPLC (RP-HPLC) (on l-Bondapack C-18). A molecular mass of 13.965 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that Cr 5 had a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical residues of a basic PLA2. The complete amino acid sequence of Cr 5 PLA2 contains 120 residues and this sequence shows high identity values when compared to other K49 PLA2s isolated from the venoms of viperid snakes. In relation with biological characterization, this was also done with the isoforms 6-1 and 6-2 (from fraction BthTX-II of Botrhops jararacussu) and with PLA2 K49 Bj-VII, these purified and partially characterized in The Masters. PLA2 6-1, 6-2 and Cr-IV 1 exhibited in mice local myonecrosis and edema upon intramuscular and intravenous injections, respectively. In dosage of 40 ug/150 ml, 6-1 showed 83.06% of citotoxicity in myotubes and 93.78% in myoblasts, 6-2 showed 75,12 % in myotubes and 86,26% in myoblasts, to Cr-IV 1 91.50% and 88.40% of citotoxicity in myoblasts and myotubes respectively. The citotoxic effect also in fibroblasts (cell line 3T3 and COS7) and in dosage de 5 µM (cell line 3T3) 85.63%, 85% and 91.34% of citotoxicity in the presence of 6-1, 6-2 and Cr-IV 1 respectively, and lineage COS7, 95.42%, 95.07 and 96.42% respectively, besides inflammatory activity, being this one more pronounced in the presence of the Cr-IV 1. PLA2 homologue K49, Bj-VII and Cr 5, evidenced local myotoxicity and in dosage of 40 ug/150 ml, showed 87±50 of lise in myoblasts and 84±85 in myotubes and 91±25% and 91±0.8 % when in contact with Bj-VII and Cr 5, respectivally besides of inflammatory activity evidenced by the edema induction. In the case of PLA2 K49 (Bj-VII and Cr 5), all of the biological effects induced by them happened in the absence of activity phospholipase measurable in vitro, supporting the idea proposed by the literature of catalytic and pharmacological mechanisms independent. Even though all show some kind of biologic differences all of them have a similar behave in function and structure which suggests a common ascendance of course we know they are proteins coming from different snakes geographic separated. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Submitted by Lúcia Brandão (lucia.elaine@live.com) on 2015-12-09T18:22:03Z No. of bitstreams: 1 Dissertação - Fernanda Torlania Alves Gomes.pdf: 2631196 bytes, checksum: 54e703f505ae72013581a2180b57e832 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-01-19T19:42:05Z (GMT) No. of bitstreams: 1 Dissertação - Fernanda Torlania Alves Gomes.pdf: 2631196 bytes, checksum: 54e703f505ae72013581a2180b57e832 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-01-19T19:43:37Z (GMT) No. of bitstreams: 1 Dissertação - Fernanda Torlania Alves Gomes.pdf: 2631196 bytes, checksum: 54e703f505ae72013581a2180b57e832 (MD5) / Made available in DSpace on 2016-01-19T19:43:37Z (GMT). No. of bitstreams: 1 Dissertação - Fernanda Torlania Alves Gomes.pdf: 2631196 bytes, checksum: 54e703f505ae72013581a2180b57e832 (MD5) Previous issue date: 2014-04-14 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / There is a growing demand of using plants from the Amazon to the development of drugs and cosmetics. The Eperua genus belongs to the family Fabaceae and consists of 14 species distributed from Central Amazonia. The oil-resin extracted from the trunk of Eperua has been used in popular medicine similarly to the copaiba oil as healing, antibacterial and antifungal. Very few studies have been conducted in order to test the biological activities of the oil-resin of these species. This study aimed to investigate the effects of the acid fraction of E.oleifera oil-resin on cell proliferation, collagen production in human fibroblasts, inhibition of metalloproteinases and cytotoxicity against malignant cell lines. It was evaluated the effects of the oil-resin on the morphology, proliferation and collagen production in fibroblast cells MRC-5 and FPH. There was a reduction of cell proliferation of fibroblast cells treated with the oil-resin at a concentration of 5 µg/mL. The induction of collagen production in fibroblasts was significant in the treatments performed above 2.5 µg/mL relative to untreated cells. Additionally, the cytotoxicity of the oil was evaluated on three lines and six neoplastic and three non-neoplastic cell line. The results showed significant cytotoxicity against all lines, with IC50 values between (13 - 50 µg/mL). The evaluation of the hemolytic potential of the oil-resin showed high toxicity in erythrocytes of Swiss mice Mus musculus, with IC50 = 38.29 µg/mL. According to the comet assay, the acid fraction of E. oleifera oil-resin induced genotoxicity in MRC-5 cells with DNA damage index between (40 - 60 %) when compared to the negative controls (0-20%). Inhibitory action of the oil-resin was observed in the concentration-dependent manner on collagenase activity, with IC50 = 46.64 µg/mL. The zymography assay testing the activity of MMP - 2 and MMP – 9 from HaCaT cells, showed little significant enzymatic inhibition, because of its not viable treatment in high concentrations. Results showed that the acid fraction of E. oleifera oil-resin showed cytotoxic, genotoxic and hemolytic effects, and collagenase inhibition. Taken together, our data suggests that E. oleifera could be considered as a source of new therapeutic drug, by isolating the active compounds and possibly decreasing the undesirable toxic side effects. / É crescente a demanda do uso de plantas da Amazônia para o desenvolvimento de fármacos e cosméticos. O gênero Eperua, pertencente à família Fabaceae, é constituído por 14 espécies distribuídas pela Amazônia Central. O óleo-resina extraído do tronco das árvores deste gênero tem sido utilizado na medicina popular de modo similar ao da copaíba, como cicatrizante, antifúngico e bactericida. Pouquíssimos estudos foram realizados com o propósito de testar as atividades biológicas do óleo-resina destas espécies. O presente trabalho objetivou investigar os efeitos da fração ácida do óleo-resina de Eperua oleifera na proliferação celular e produção de colágeno em fibroblastos humanos, inibição de metaloproteinases e citotoxicidade contra linhagens celulares tumorais e não tumorais. Foram avaliados os efeitos do óleo na morfologia, proliferação e produção de colágeno em fibroblastos das linhagens FPH e MRC-5. Foi avaliada a citotoxicidade da fração ácida do óleo-resina sobre seis linhagens neoplásicas e três linhagens não neoplásicas. Houve significativa citotoxicidade do óleo contra todas as linhagens avaliadas, com valores de CI50 entre (13 - 50 µg/mL). A indução da produção de colágeno em fibroblastos foi significativa nos tratamentos realizados a partir de 2,5 µg/mL. Houve redução da proliferação celular nos tratamentos com o óleoresina na concentração de 5 µg/mL. A avaliação do potencial hemolítico do óleo indicou toxicidade em eritrócitos de camundongos suiços Mus musculus, com valor de CI50 = 38,29 µg/mL. O teste do cometa indicou que o óleo induziu genotoxicidade nas células MRC-5 com índice de dano no DNA entre (40 – 60%) nas concentrações de 7,5 - 30 µg/mL. Foi observada ação inibitória do óleo-resina na atividade de colagenases de forma concentraçãodependente, com CI50 = 46,64 µg/mL. Ensaios zimográficos da atividade de MMP-2 e MMP-9 de células HaCaT não indicaram importante inibição enzimática quando avaliadas em baixas concentrações do óleo-resina. Os resultados obtidos evidenciaram que a fração ácida do óleoresina de E. oleifera apresentou efeito citotóxico, genotóxico, hemolítico, e inibidor de colagenases. Face ao exposto, nossos dados sugerem que E. oleifera pode vir a ser considerada uma fonte de novos agentes quimioterapêuticos, por meio do isolamento dos compostos ativos e, possível diminuição dos efeitos tóxicos indesejáveis.

Os osteoblastos são células de origem mesenquimal envolvidas na formação óssea. Essas células podem sofrer alterações decorrentes de traumas, intervenções, e infecções. As infecções podem ser minimizadas a partir do uso de antimicrobianos. O poli(L-lactídeo) ou PLLA, é um polímero sintético que se destaca por sua biocompatibilidade e absorção, o qual pode ser utilizado como um liberador farmacológico local, como alternativa à terapêutica antimicrobiana sistêmica. Esse polímero também é empregado como matriz de suporte celular na engenharia de tecidos ósseos, por auxiliar na reparação e regeneração. A incorporação de partículas nesse polímero pode gerar reações adversas, portanto, devemos nos certificar que o dispositivo polimérico incorporado com antimicrobianos não seja citotóxico. Proposição: Analisar a estrutura e a citotoxidade em osteoblastos de dispositivos poliméricos de PLLA incorporados com antimicrobianos, sendo eles: Amoxicilina ou Azitromicina ou Clindamicina ou Metronidazol para uso local. Metodologia: Foram confeccionados 270 dispositivos poliméricos com 6mm de diâmetro composto de PLLA com a incorporação de antimicrobianos a 20%, Amoxicilina (AM) ou Azitromicina (AZ) ou Clindamicina (CL) ou Metronidazol (ME) sendo confeccionados através de dois métodos: eletrofiação (malhas) ou deposição (filmes). Posteriormente, foi realizado o teste de citotoxicidade direta MTT nesses dispositivos com a cultura de osteoblastos em 24, 48 e 72h de experimento. Para análise da estrutura do dispositivo, foram feitas análises macroscópicas através de fotografias digitais e microscópicas com Microscópio Eletrônico de Varredura (MEV). Resultados: A reação de citotoxidade mostrou que malhas e filmes incorporados à antimicrobianos são compatíveis com a cultura de osteoblastos, não apresentando citotoxicidade em nenhum momento do estudo (p<0.05). Na fotografia pudemos observar que os dispositivos apresentam coloração semelhante em relação às malhas e coloração diferenciada para filmes dependendo do tipo de antimicrobiano incorporado. No MEV, através da análise dos dispositivos pudemos notar que houve diferença no aspecto das superfícies dos filmes, sendo que os filmes de AM apresentaram aspecto irregular e poroso, enquanto AZ aparece liso com alguns grânulos, os de CL e ME possui superfícies ásperas e os de PLLA apresentaram superfície lisa. Quanto às malhas, notamos que todas as amostras apresentaram microfibras e poros que imitam a matriz extracelular, diferenciando-se apenas na espessura das fibras. Houve a presença de osteoblastos em todos os filmes confeccionados, mas os filmes de AM não induziram a proliferação, aparecendo apenas células isoladas. Enquanto nas malhas só foram observados osteoblastos em malhas de AM, ME e PLLA. Conclusão: Os dispositivos poliméricos confeccionados com PLLA incorporados com antimicrobianos podem ser usados na reparação e regeneração óssea uma vez que não apresentaram citotoxicidade em osteoblastos. / Osteoblasts are mesenquima originated cells, which are involved in the bone formation. These cells may suffer alterations due to traumas, interventions and infeccions. The infections can be minimized by the handling of antimicrobials. Poly (L-lactide) or PLLA is a synthetic polymer known for its biocompatibility and absorption, which can be used as a local pharmacological releaser, as an alternative to the systemic antimicrobial therapy. This polymer also can be frequently used as a supporting structure to cellular matrix in the bone tissue engineering as it can be used for support in repair and regeneration. The particle incorporation in this polymer can create side effects, therefore, we need to certificate that the polymeric device incorporated with antimicrobials are not cytotoxic. Proposition: Analyse the structure and cytotoxicity in osteoblasts of PLLA polymeric devices associated with antimicrobials, being them: Amoxicillin, Azithromycin, Clindamycin and Metronidazole. Methods: For this study 270 polymerical devices were manufactured with 6mm diameter of PLLA with a 20% antimicrobials incorporation of Amoxicillin (AM), Azithromycin (AZ), Clindamycin (CL) and Metronidazole (ME) that have been produced through two methods: eletrospinning (mesh) or casting (film). Afterwards a MTT cytotoxicity test was made over the periods of 24, 48 and 72 hours of experiment. To make a structural analysis of the device a macroscopic analysis was performed through photographs and microscopic imaging with scanning electron microscope (SEM). Results: The cytotoxicity reaction exhibited that meshes and films incorporated with antimicrobials are comparable with the osteoblasts culture, indicating that there was no cytotoxicity in any moment (p < 0.05). In the phothograph we could observe that the devices showed a similar coloration among the meshes and different coloration for the films depending on the incorporated antimicrobial. The SEM analysis displayed a difference in the surface appearance of the films. The AM films displayed an irregular and porous appearance, meanwhile, AZ looked smooth with few grains, the CL and ME have rough surfaces and PLLA presents smooth surfaces. As for the meshes, we noticed that all the samples had microfibers and pores that mimic the extracellular matrix, differing only in the thickness of the fibers. Osteoblasts were present in all films but AM did not induce proliferation, with only isolated cells emerging. In meshes osteoblasts were only found in AM, ME and PLLA. Conclusion: Polymeric devices made with PLLA incorporated with antimicrobials can be used in bone repair and regeneration given that they did not offer cytotoxicity for osteoblasts.

Thesis submitted in fulfilment of the requirements for the Degree of Master's in Chemistry, Durban University of Technology, 2017. / Quinoline and its derivatives represent an important class of nitrogen-containing heterocylces as they are useful intermediates in organic synthesis and possess a broad spectrum of biological activities, such as anti-asthmatic, anti-inflammatory and anti-malarial activity. Hence, synthesis of novel compounds with potent biological activities is important in medicine. Significant research is directed into the development of new quinoline based structures and new methods for their preparations. In the past, synthesis of complex molecules was accomplished by step-wise reaction. This was time consuming and yield was generally low. Nowadays, multi-component reactions (MCRs) are being used since three or more substrates can be reacted in a one-pot reaction. Therefore yields are higher and the reaction is more efficient. In this research investigation novel quinoline derivatives, using the multi-component reaction protocol, were synthesized. After characterization of the product by several spectroscopic techniques, the biological potential of these compounds were assessed using lung cancer cell lines, bacteria and molecular modeling in an enzymatic system. In the synthetic part of this study, the first step was the preparation of the starting compound 2- chloro-3-formyl quinoline for which the Vilsmeier-Haack cyclisation protocol was used. The cyclisation was carried out by combining DMF and POCl3 at 5°C to form an electrophile which then reacted in situ with N-phenylacetamide at 100ºC to afford 2-chloro-3-formyl quinoline in high yield (95%). This was followed by the synthesis of a series of novel quinoline derivatives in a MCR system comprising 2- chloro-3-formyl quinoline, malononitrile, aromatic amines and dimethyl acetylenedicarboxylate in the presence of a catalytic amount of triethylamine. Valuable features of this routine included high yields, extensive substrate range and straight forward procedures. Eight novel poly-functionalised dihydropyridine quinoline derivatives were synthesized, purified and characterized. The outline for the synthesis of poly-functionalised dihydropyridine quinoline derivatives is presented graphically in Scheme 1. Scheme 2 shows the eight compounds synthesized and used subsequently for further studies. . Step 1 CH3 a N O H CHO N Cl Step 2 CHO CN N Cl CN NH2 R O OCH3 b OCH3 O MeO2C MeO2C N Cl CN N NH2 R = m-CH3, o-OCH3, p-Cl, m,p-Cl, o-F, m-F, p-F R Reaction Conditions: a. DMF, POCl3 b. Et3N, EtOH Scheme 1: Graphical representation for the synthesis of poly-functionalised dihydropyridine quinoline derivatives The novel eight compounds were screened for their potential activity in lung cancer cell lines. A549 cells were incubated for 24 hours with a range of concentrations of each compound, in triplicate, in a micro-titre plate together with an untreated control. Each experiment was conducted twice on separate occasions; the results from the first set matched the repeated experiment. The cells were then incubated (37ºC, 5% CO2) with the MTT substrate for 4 hours. Thereafter all supernatants were aspirated and DMSO was added to the wells. Finally the optical density was measured at 570 nm at a reference wavelength of 690 nm with an ELISA plate reader. The net MTT dependant absorbance (optical density) of each sample was calculated by subtracting the average absorbance of the blank from the average absorbance of each sample. Data were represented as mean optical density plus or minus the standard deviation. Four of the synthesized compounds (A1-A8) were evaluated for their cytotoxicity activities. The anti-cancer assay indicated that poly-functionalised dihydropyridine quinoline compounds, A2, A3 and A4 have good potential as anti-cancer drugs. Among them, A2 and A4 proved to be dose dependent with A4 having the highest toxicity at 250 µM and A8 having the highest toxicity at 125, 250 and 500 µM, whereas A1, A5, A6 and A7 were not cytotoxic. O H3CO H3CO O N Cl CN NH2 O H3CO H3CO O N Cl CN N NH2 OCH3 O H3CO H3CO O N Cl CN N NH2 O H3CO H3CO O N Cl CN NH2 CH3 Cl A1 A2 A3 A4 O H3CO H3CO O N Cl CN N NH2 F O H3CO H3CO O N Cl CN N NH2 O H3CO H3CO O N Cl CN NH2 O H3CO H3CO O N Cl CN N NH2 F Cl F Cl A5 A6 A7 A8 Scheme 2: Structures of novel poly-functionalised dihydropyridine quinoline derivatives by MCRs Since molecular docking is a key tool in structural molecular biology and computer-assisted drug design, these compounds were subjected to molecular docking and the binding mode for the compounds, within the active site of the protein, was analyzed. Docking of A1 to Human mdm2 protein provided insights into the binding regions. Three hydrogen bonds were formed between GLU 25 (2.7 Å distance), LEU 27 (3.2 Å distance) and LEU 54 (3.2 Å distance) atoms with binding energy of -8.91 kcal/mol. Docking of A1 with Human mdm2 indicated the lowest binding energy thereby showing strong affinity of the ligand molecule with the receptor which has been stabilized by strong hydrogen bond interactions in the binding pocket. This confirms that A1 is a better inhibitor for E3 ubiquitin-protein ligase mdm2 than all the other compounds tested (A2-A8). Further, the eight novel poly-functionalised dihydropyridine quinoline derivatives were evaluated for their antibacterial activity. This was performed using the MABA method against three strains i.e. Gram negative; Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922) and Gram positive; Staphylococcus aureus (ATCC 29213) using the broth micro dilution method. Standard antibiotics (ciprofloxacin and nalidixic acid) were used as positive controls and DMSO was used as a negative control. The results obtained from the anti-bacterial assay showed that compounds A4, A7 and A8 have high activity, whereas A2 and A3 showed poor activity against all the tested bacterial strains. Compound A6 showed no activity against S. aureus and E. coli. / M

Certaines espèces de phytoplanctons produisent des métabolites secondaires hautement toxiques appelés phycotoxines. Ces phycotoxines peuvent contaminer les fruits et mer et poser un risque sanitaire pour l'homme. Elles sont responsables d'effets néfastes chez l'homme et peuvent provoquer des diarrhées, des nausées et des douleurs abdominales. Pour améliorer l'évaluation du risque sanitaire lié à la présence de phycotoxines dans les coquillages, une meilleure caractérisation du potentiel toxique est indispensable. Aussi, l'objectif de ce doctorat a été d'étudier les effets cytotoxiques des phycotoxines lipophiles seules et en mélange à l'aide de nouvelles approches in vitro en toxicologie. Nos études ont montrées que si les analogues de l'acide okadaïque (AO) induisaient tous l'apoptose, la génotoxicité, l'inflammation cellulaire et la perturbation du cycle cellulaire, sur les lignées intestinales Caco-2 et HT29-MTX, ces effets se produisaient à des concentrations différentes en fonction des toxines. Ensuite, nos recherches sur la lignée hépatique humaine HepaRG ont montrées l'implication des enzymes de phase I dans le processus de détoxication de l'AO et de ses analogues les dinophysistoxines (DTX), et de la pectenotoxin-2 (PTX-2). Pour finir, l'utilisation de la méthode des index de combinaison pour l'étude de la toxicité des mélanges binaires de toxines sur des lignées intestinales a mis en évidence des effets synergiques du mélange Azaspiracide-1/ Yessotoxine. / Some species of phytoplankton produce highly toxic secondary metabolites called phycotoxins. Phycotoxins can contaminate seafood and pose a health risk to humans. They can cause several adverse effects in humans, including diarrhea, nausea and abdominal pain. To improve risk assessment associated with the presence of lipophilic phycotoxins in shellfish, a better characterization of the toxic potential is essential. The aim of this PhD was to study the cytotoxic effects of single and mixed lipophilic shellfish toxins using new approaches in in vitro toxicology. Our studies have shown that, analogs of okadaic acid (OA) induced apoptosis, genotoxicity, cellular inflammation and disturbance of the cell cycle, on intestinal cell lines Caco-2 and HT29-MTX, and that this effects occurred at different concentrations depending on the toxin. Then, our researches on human hepatic lineage HepaRG have shown the involvement of phase I enzymes in the detoxification of the pectenotoxin-2 (PTX-2), and OA and its analogues the dinophysistoxins (DTX). Finally, the use of the combination index method for studying the toxicity of binary mixtures of phycotoxins on intestinal cell lines showed synergistic effects of azaspiracid-1 / yessotoxin mixture.

Background: Silica is the common name for silicon dioxide (SiO2) materials and exists in both crystalline and amorphous forms. While crystalline silica is known for its severe health effects, amorphous silica has been considered safe and applied in many areas. However, some recent studies have showed evidence of their toxicity, raising concerns about its use as nanomaterial for biomedical applications. When nanomaterials enter the body, they are enveloped in biological fluids rich in biomolecules, which compete for binding to the nanomaterial. Such effect could alter their surface chemistry and therefore affect their bio-distribution and interaction with cells. Aim and objectives: As part of the EU-funded NANODRUG network programme, the aim of this project was the in vitro toxicity assessment of commercially-sourced fumed and colloidal amorphous silica particles in relation to their physico-chemical properties and potential application as carriers for drug delivery. The objectives were 1) characterization of silica particles hydrodynamic (Hd) size and dispersity in different cell culture media; 2) in vitro toxicological assessment of silica particles in human skin cells; 3) delineation of toxicity mechanisms in relation to their size; 4) assessment of the influence of Foetal Bovine Serum (FBS) on particle Hd size and toxicity; and 5) contributing to the overall objective of the NANODRUG programme - development of safe nanodrugs for skin application - through collaborations with different partners.

The viral determinants of African horsesickness virus (AHSV) cytopathology are not well understood. Several AHSV proteins may play a role, including non-structural protein NS3, a cytotoxic membrane protein that localises to sites of virus release and plasma membrane disorganisation in infected cells. AHSV NS3 is highly variable and clusters into three phylogenetic groups, termed α, β and γ. In chapter 2 we examined the role of NS3 in determining the phenotypic characteristics observed during AHSV infection of cells. Three AHSV strains, AHSV-2 (γ NS3), AHSV-3 (β NS3) and AHSV-4 (α NS3), were shown to have quantitatively different phenotypes in Vero cells. To investigate the contribution of NS3 to these differences, reassortants were generated between these strains in which the S10 genome segment encoding NS3 was exchanged, alone or in combination with other segments. Exchange of NS3 resulted in changes in virus release and membrane permeability, indicating an important role for NS3 in these viral properties. The cytopathic effect and decreased viability of infected cells was not associated with NS3 alone and it is likely that a number of viral and host factors contribute to these complex phenotypes. In chapter 3 the cytolytic effect of the NS3 proteins of the orbiviruses AHSV, bluetongue virus (BTV) and equine encephalosis virus (EEV) were compared. Inducible expression in Escherichia coli (E. coli) showed differences in cytotoxicity, with EEV NS3 having a greater lytic effect than than AHSV and BTV NS3. Cytotoxicity was linked to increased membrane permeability of the cells as confirmed by an increased uptake of membrane impermeant compounds. When expressed in insect cells however all three NS3 proteins caused a marked but equivalent decrease in cell viability. Although the orbivirus NS3 proteins have similar predicted secondary structures, differences could lie in structural stability and association with membranes of specific cell types, which impacts on cytotoxicity. To determine the regions within AHSV NS3 that mediate cytotoxicity, a series of truncated mutants of NS3 where constructed and expressed in E. coli. The combined presence of both hydrophobic domains of AHSV NS3 was found to be critical for membrane permeabilisation and cytotoxicity. In chapter 4 the AHSV-2, AHSV-3 and AHSV-4 NS3 proteins (from the γ, β and α NS3 clades)were compared to examine the impact of sequence variation in NS3 on structure and function. The proteins were expressed in the baculovirus expression system as both wild-type proteins and C-terminal eGFP (enhanced green fluorescent protein) fusions. Exogenous addition of the baculovirus expressed proteins to Vero cells resulted in different permeabilisation levels that could be linked to that induced by the AHSV strains. Cell viability and membrane association assays in insect cells showed that all three proteins were equivalently cytotoxic and membrane associated. The subcellular localisation of the eGFP-NS3 fusion proteins was examined by confocal fluorescen imaging of live cells. NS3 localised to the plasma membrane, and as distinct punctuate foci in the perinuclear region. This suggests localisation to the internal membrane systems of cells and has important implications for the function of this membrane permeabilising protein. / Thesis (PhD)--University of Pretoria, 2011. / Genetics / unrestricted

Heterotrophic plate counts (HPCs) are commonly used to assess the general microbiological quality of drinking water. Drinking water quality specifications world-wide recommend HPC limits from 100 to 500 cfu.m1-1. However, a number of recent studies revealed evidence that commonly used indicator bacteria may not be as harmless as generally accepted. It appears that immuno-compromised individuals, which represent increasing components of many consumer populations, are particularly at risk. This would include the very young and very old, patients with diseases such as AIDS, and patients on therapy after organ transplantations and cancer treatment. Since, epidemiological and animal infectivity studies are complex and difficult to control, attempts have been made by researchers to examine HPCs directly in order to assess health risks. These analyses included: cytotoxicity, invasiveness, enzyme analyses, antibiotic susceptibility and identification. In this study, 339 bacterial colonies were isolated at random from selected drinking water supplies in South Africa using heterotrophic plate count tests. In a first step to screen for potentially pathogenic properties, 188 (55.5%) of the isolates showed α- and β-haemolysis on human- and horse-blood agar media. Subsequent analysis of the haemolytic isolates for enzymatic properties associated with pathogenicity revealed the presence of chondroitinase in 5.3% of the isolates, coagulase in 16.0%, DNase in 60.6%, elastase in 33 .0%, fibrinolysin in 53.7%, gelatinase in 62.2%, hyaluronidase in 21. 3 %, lecithinase in 47.9%, lipase in 54.8%, and proteinase in 64.4%. Fluorescein and pyocyanin were not produced by any of the isolates. The Kirby-Bauer quality controlled disc diffusion method was applied in the demonstration of antibiotic resistance by the HPC isolates. Among the haemolytic isolates 77.7% were resistant to oxacillin (1 µg), 59.6% to penicillin G (2 units), 47.3% to penicillin G (10 units), 54.3% to ampicillin (10 µg) and 43.1% to ampicillin (25 µg). Cell culture studies revealed that 96% of haemolytic isolates were cytotoxic to HEp-2 cells and 98.9% of the 181 cytotoxic isolates adhered to HEp-2 or Caco-2 cells. Gram-negative isolates tended to adhere in larger numbers than gram-positive isolates. The average index of adherence for Gram-negative bacteria was 20-30 bacteria per HEp-2 cell, compared to 3-7 for Gram-positive bacteria. HEp-2 cells were invaded by 43.6% and Caco-2 cells by 49.7% of the 181 cytotoxic isolates. The invasion index on HEp-2 cells was 1.9xlO-1 to 8.9xl0-6, compared to 7.7xl0-2 to 8.3xlO-6 on Caco-2 cells. The most commonly isolated genera showing potentially pathogenic features were: Aeromonas, Acinetobacter, Aureobacterium, Bacillus, Chryseobacterium, Corynebacterium, Klebsiella, Moraxella, Pseudomonas, Staphylococcus, Tsukamurella and Vibrio. All these genera are known to contain opportunistic pathogens. Our results support earlier findings on potentially pathogenic features of bacteria detected by heterotrophic plate counts on drinking water. These findings seem to be in agreement with some epidemiological studies, which indicated an association between HPCs of drinking water and the incidence of gastroenteritis in consumers. However, the extent of the health risk concerned needs to be defined in detail for meaningful revision of quality guidelines for HPCs in drinking water. / Dissertation (MSc (Medical Virology))--University of Pretoria, 2005. / Medical Virology / unrestricted

Acrolein is an electrophilic α, β-unsaturated aldehyde. Additionally, acrolein is a metabolite of the antineoplastic alkylating agent cyclophosphamide and is implicated in off-target effects, including to bladder hemorrhagic cystitis and cyclophosphamide-induced cardiotoxicity, both of which have led to serious secondary iatrogenic injury during and following chemotherapy. At low concentrations acrolein inhibits cell proliferation without inducing apoptosis, while at high concentrations may result in secondary apoptosis promotion. This investigation assessed the role of the enzyme poly (ADP-ribose) polymerase (PARP) in acrolein induced toxicity using the established toxicological H9c2 (2-1) cardiomyoblast in vitro model. H9c2 (2-1) cells were plated in 24-well plates at 75,000 cells per well three days prior to testing, followed by acrolein dosing at concentrations between 10 µM and 1000µM for either 30 or 55 minutes. PARP activity was quantitatively measured in total cell lysates using a biotin-avidin-conjugated horseradish peroxidase-TMB reporter system in a 96-well microplate formate. The lowest effective dose of toxicity at 30 minute dosing was found at 25 μM (PARP Activity 1.65-fold control) which returned to baseline at 100 μM; concentrations at or above 250 μM results in significant PARP activity reductions (≤ 0.46-fold control). Biomarkers were further characterized for cytotoxicity (AST presence), and viability (MTT reduction) in order to facilitate mechanistic characterization of PARP-mediated acrolein cardiotoxicity. Investigation of a PARP inhibitor was assessed to explore the intervention for acrolein induced cardiac tissue damage.