Attention has begun to focus on the pulmonary delivery of antifungal agents for invasive fungal infections as inhalation of the fungal spores is often the initial step in the pathogenesis of many of these infections. Invasive fungal infection in the lungs in immunocompromised patients has high mortality rates despite current systemic (oral or intravenous) therapies. However, drug delivery of antifungal agents directly to the lungs could potentially result in high concentrations of drug in the lungs, a quicker onset of action, and reduction of systemic side effects. Voriconazole (VRC) is a second, generation triazole antifungal agent with increased potency, a broad spectrum of antifungal activity, and a fairly poor aqueous solubility. It is the recommended therapeutic agent for the treatment of Invasive Pulmonary Aspergillosis (IPA), and its use has improved therapeutic outcomes in immunocompromised patients with IPA. Still, systemic administration by oral or intravenous delivery is limited by high inter- and intra-patient pharmacokinetic variability, many potential drug interactions, and a narrow therapeutic index with many adverse effects, leading to clinical failures. Therefore, development of novel particulate formulations containing VRC for targeted drug delivery to the lungs is critical to improving therapeutic outcomes in patients with invasive fungal infections in the lungs. Within the framework of this dissertation, two particle engineering processes, thin film freezing (TFF) and advanced evaporative precipitation into aqueous solution (AEPAS), were investigated. The goal was to investigate microcrystalline VRC, nanocrystalline VRC, and nanostructured amorphous VRC formulations suitable for pulmonary delivery and to determine the effect of morphology on the in vivo deposition and distribution of inhaled particulate VRC formulations. TFF process parameters significantly affected the solid state properties and aerodynamic performance of the dry powder formulations containing VRC. Following dry powder insufflation into the lungs of mice, microstructured crystalline TFF-VRC achieved higher and more prolonged concentrations of VRC in the lungs with slightly lower systemic bioavailability than nanostructured amorphous TFF-VRC-PVP K25. AEPAS and TFF of template nanoemulsions did not lead to production of crystalline nanoparticles, as predicted. In particular, VRC proved to be a difficult molecule to stabilize in the nanocrystalline and nanostructured amorphous states. Ultimately, this body of work demonstrated that the particle engineering process, TFF, could be used to develop voriconazole formulations suitable for dry powder inhalation with more favorable pharmacokinetic parameters compared to inhaled voriconazole solution. / text

Este estudo avaliou a incidência e distribuição de Candida spp. isoladas de casos de infecção hospitalar no período entre 2005 to 2007, em um hospital público infantil, em São Paulo. Brasil. As amostras foram isoladas de sangue, urina e outros materiais biológicos (36,6%, 37,12% e 26,52%, respectivamente). As análises micromorfológicas e bioquímicas revelaram que a distribuição por espécie foi 62,12% Candida albicans, 37,88% não-albicans. Uma maior incidência de amostras de C. albicans foi observada em casos de candidúria (53,62%) enquanto espécies não-albicans foram mais prevalentes em candidemia (71,43%) (p < 0.05). A respeito da produção enzimática, 68,94%, 47,73%, 65,91% e 66,67% foram positivas para proteínase, fosfolipase, lipase e hemolisina. Em relação aos antifúngicos, para Anfotericina B, 96,97% dos isolados mostraram e para 5- fluorcitosina foi observado o maior índice de resistência. Os fatores de risco identificados para candidemia hospitalar foram as doenças pré-existentes, terapia antibiótica de largo espectro e a presença de cateter venoso central. / This study evaluated the incidence and distribution of Candida spp. identified from cases of nosocomial infection in the period from 2005 to 2007, in a public children\'s hospital in São Paulo, Brazil. The strains were isolated from the blood, urine and other biological specimens (36,6%, 37,12% and 26,52%, respectively). Micromorphological and biochemical analyses revealed that the overall distribution by species was 62,12% Candida albicans, 37,88% non-albicans. A higher incidence of C. albicans strains was observed in cases of candiduria (53,62%) while non-albicans species were more prevalent in cases of candidemia (71,43%) (p < 0.05). Concernig the production of enzymes, 68,94%, 47,73%, 65,91% and 66,67% presented positive proteinase, phospholipase, lipase and hemolin activity. In relation to antifungal for the Amphotericin B, 96,97% of isolates showed sensitivity and for 5- fluorocytosine was observed the biggest index of resistence. The Risk factors identified for nosocomial candidemia was underlying disease, therapy with broad-spectrum antibiotics and the presence of a central venous catheter.

Este estudo avaliou a incidência e distribuição de Candida spp. isoladas de casos de infecção hospitalar no período entre 2005 to 2007, em um hospital público infantil, em São Paulo. Brasil. As amostras foram isoladas de sangue, urina e outros materiais biológicos (36,6%, 37,12% e 26,52%, respectivamente). As análises micromorfológicas e bioquímicas revelaram que a distribuição por espécie foi 62,12% Candida albicans, 37,88% não-albicans. Uma maior incidência de amostras de C. albicans foi observada em casos de candidúria (53,62%) enquanto espécies não-albicans foram mais prevalentes em candidemia (71,43%) (p < 0.05). A respeito da produção enzimática, 68,94%, 47,73%, 65,91% e 66,67% foram positivas para proteínase, fosfolipase, lipase e hemolisina. Em relação aos antifúngicos, para Anfotericina B, 96,97% dos isolados mostraram e para 5- fluorcitosina foi observado o maior índice de resistência. Os fatores de risco identificados para candidemia hospitalar foram as doenças pré-existentes, terapia antibiótica de largo espectro e a presença de cateter venoso central. / This study evaluated the incidence and distribution of Candida spp. identified from cases of nosocomial infection in the period from 2005 to 2007, in a public children\'s hospital in São Paulo, Brazil. The strains were isolated from the blood, urine and other biological specimens (36,6%, 37,12% and 26,52%, respectively). Micromorphological and biochemical analyses revealed that the overall distribution by species was 62,12% Candida albicans, 37,88% non-albicans. A higher incidence of C. albicans strains was observed in cases of candiduria (53,62%) while non-albicans species were more prevalent in cases of candidemia (71,43%) (p < 0.05). Concernig the production of enzymes, 68,94%, 47,73%, 65,91% and 66,67% presented positive proteinase, phospholipase, lipase and hemolin activity. In relation to antifungal for the Amphotericin B, 96,97% of isolates showed sensitivity and for 5- fluorocytosine was observed the biggest index of resistence. The Risk factors identified for nosocomial candidemia was underlying disease, therapy with broad-spectrum antibiotics and the presence of a central venous catheter.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-27T18:53:59Z No. of bitstreams: 1 daniellecristinazimmermannfranco.pdf: 1980224 bytes, checksum: 136850704bd6cd949245b93374380f44 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-27T19:17:49Z (GMT) No. of bitstreams: 1 daniellecristinazimmermannfranco.pdf: 1980224 bytes, checksum: 136850704bd6cd949245b93374380f44 (MD5) / Made available in DSpace on 2016-06-27T19:17:49Z (GMT). No. of bitstreams: 1 daniellecristinazimmermannfranco.pdf: 1980224 bytes, checksum: 136850704bd6cd949245b93374380f44 (MD5) Previous issue date: 2012-07-30 / As dermatofitoses são micoses que afetam os tecidos queratinizados, causadas por fungos pertencentes ao grupo dos dermatófitos e que afetam o homem e os animais. O óleo resina de copaíba possui diversas ações farmacológicas, incluindo antimicrobiana. Assim, pode ser uma alternativa para o tratamento farmacológico das dermatofitoses. O objetivo deste estudo foi determinar o perfil químico do óleo resina de Copaifera langsdorffii Desf. e investigar sua atividade antifúngica frente aos principais fungos causadores de dermatofitoses: Microsporum spp e Tricophyton spp. Foram analisados o perfil químico do óleo resina de copaíba por cromatografia gasosa de alta resolução (CG-AR) e a atividade antifúngica do óleo por meio da determinação de sua concentração inibitória mínima (CIM) e a concentração fungicida mínima (CFM). Os fármacos de referência, cetoconazol e terbinafina, foram empregados como controles positivos. Por meio de microscopia eletrônica de varredura (MEV), foram investigadas as alterações morfológicas causadas nos fungos expostos aos tratamentos propostos. O ácido copálico foi identificado na amostra, confirmando a originalidade do óleo resina de copaíba e o β-cariofileno foi encontrado como componente majoritário (31,4%). O óleo resina de copaíba, por meio do teste de triagem da atividade antifúngica, mostrou-se inativo frente a M. canis ATCC 32903, M. gypseum ATCC 14683, T. mentagrophytes ATCC 9533 e T. mentagrophytes ATCC 11480, apresentando atividade fungicida frente às linhagens de T. mentagrophytes ATCC 11481, com valores de CIM e CFM iguais a 31,25 μg de β-cariofileno.mL-1; e de T. rubrum com valores de CIM igual a 250 μg de β-cariofileno.mL-1 e de CFM igual a 500 μg de β-cariofileno.mL-1. Os fungos expostos a ação do óleo e dos fármacos de referência apresentaram deformações em suas estruturas. Os resultados estimulam a realização de ensaios in vivo para aplicação do óleo resina de copaíba no tratamento das dermatofitoses humanas e animais causadas por fungos do gênero Tricophyton spp. / The dermatophytosis are mycosis that affect the keratinized tissues caused by fungi of the dermatophytes group and affect the humans and the animals. The oleoresin extracted from copaiba has numerous pharmacological actions, including antimicrobial activity. Thus, it may be an alternative for pharmacological treatments of the dermatophytosis. The aim of this study was to determine the chemical profile of the oleoresin extracted from Copaifera langsdorffii Desf. and investigate him antifungic activity against the main fungi that cause the dermatophytosis: Microsporum spp and Tricophyton spp. The chemical profile of the oleoresin extracted from copaiba by high resolution gas chromatography (HR-GC) and the oleoresin’s antifungical activity by determination of him minimal inhibitory concentration (MIC) and minimal fungidal concentration (MFC) were analyzed. The reference drugs, ketoconazole and terbinafine, were used as positive control. The scanning electron microscopy (SEM) allowed to investigate the morfological alterations caused in the fungi exposed to the proposed treatments. The copalic acid was identified confirming the originality of the oleoresin from copaiba and the β-caryophyllene was the majority compound (31.4%) found. The oleoresin extracted from copaiba showed no active against M. canis ATCC 32903, M. gypseum ATCC 14683, T. mentagrophytes ATCC 9533 and T. mentagrophytes ATCC 11480. This oleoresin showed fungicidal activity against the T. mentagrophytes ATCC 11481, with the MIC and CFM values equal to 31.25 μg of β-caryophyllene.mL-1; and T. rubrum, with MIC equal to 250 μg of β-caryophyllene.mL-1 and MFC equal to 500 μg of β-caryophyllene.mL-1. The fungi exposed to the action of the oleoresin and drugs reference presented damage on its structures. The results stimulate the achievement of in vivo assays to confirm the benefits from the application of the oleoresin extracted from copaiba in the treatment of dermatophytoses in humans and animal caused by fungi of the Tricophyton spp gender.

Orientador: Altair Antoninha Del Bel Cury / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-21T23:47:13Z (GMT). No. of bitstreams: 1 RicominiFilho_AntonioPedro_D.pdf: 2535934 bytes, checksum: eff4f940490cd7e063a65eb2cce8dcec (MD5) Previous issue date: 2013 / Resumo: A colonização de diferentes substratos presentes na cavidade oral por micro-organismos e o desenvolvimento de biofilme são fatores etiológicos da maioria das doenças orais. Além dos dentes, materiais como titânio e polimetilmetacrilato são comumente encontrados neste ambiente e o papel que estes substratos desempenham na prevalência de populações bacteriana e fúngica em biofilmes orais são pouco compreendidas. Além disso, o comportamento da população microbiana de biofilmes orais multiespécies na presença de antimicrobianos liberados na saliva permanece desconhecido. Assim, o objetivo deste estudo foi (i) avaliar o efeito de diferentes substratos na prevalência de micro-organismos em biofilmes orais multiespécies e (ii) o efeito de antimicrobianos liberados na saliva na população microbiana de biofilmes multiespécies. Para o primeiro estudo, discos de hidroxiapatita, titânio e polimetilmetacrilato (PMMA) foram utilizados como substrato para o desenvolvimento do biofilme mimetizando esmalte dental, implantes dentários e base de prótese, respectivamente. O modelo de biofilme multiespécies foi composto por cinco bactérias (Streptococcus oralis, Streptococcus mutans, Actinomyces naeslundii, Veillonella dispar e Fusobacterium nucleatum) e um fungo (Candida albicans). Biofilmes maduros (64,5 h de desenvolvimento) foram removidos por ondas ultrassônicas, plaqueados em meio ágar e as contagens de UFC de cada micro-organismo foram calculadas. A microscopia eletrônica de varredura foi utilizada para visualizar a superfície dos materiais. Os dados foram analisados por ANOVA um critério. Para o segundo estudo o mesmo modelo de biofilme multiespécies foi utilizado. Dois antibióticos, azitromicina e metronidazol, e um antifúngico, fluconazol, foram avaliados. Biofilmes maduros (64,5 h de desenvolvimento) foram expostos a azitromicina, metronidazol ou fluconazol em concentrações encontrada na saliva de 2,12 ug/mL, 15,15 ug/mL e 2,56 ug/mL, respectivamente, por 24h. Após este período, o biofilme foi removido por ondas ultrassônicas, plaqueados em meio ágar e as contagens de UFC de cada micro-organismo foram calculadas. Microscópio eletrônico de varredura e microscópio a laser de varredura confocal com células coradas por hibridização in situ por fluorescência (FISH) foram utilizados para avaliar a estrutura do biofilme. Os dados foram analisados por teste t para amostras independentes e testes não paramétricos de Mann-Whitney. O primeiro estudo não mostrou diferença na população para cada micro-organismo no biofilme entre os entre três materiais avaliados (p>0,05). No segundo estudo, todos os antimicrobianos avaliados foram capazes de alterar a população microbiana (p<0,05), no entanto nenhum dos agentes antimicrobianos foi capaz de eliminar completamente um micro-organismo específico do biofilme. Azitromicina reduziu as populações de A. naeslundii e V. dispar enquanto aumentou C. albicans (p<0,05). Metronidazol reduziu todos os micro-organismos avaliados, com uma grande redução para V. dispar e F. nucleatum (p<0,001). Fluconazol reduziu populações de C. albicans e F. nucleatum e aumentou as contagens de S. oralis e V. dispar (p<0,05). Pode concluir-se que os substratos não foram capazes de interferir na formação dos biofilmes multiespécies e que os antimicrobianos em concentrações semelhantes às liberadas na saliva alteraram a população microbiana / Abstract: The colonization of different substrata present in the oral cavity by microorganisms and the biofilm development are the etiological factors of the majority of oral diseases. Besides the teeth, materials such as titanium and polymethylmetacrylate are commonly found in this environment and the role these substrata play on the prevalence of bacterial and fungal population in oral biofilms are poorly understood. In addition, the behavior of microbial population of multispecies oral biofilms in the presence of antimicrobials released in saliva remains unknown. Thus, the aim of this study was (i) to evaluate the effect of different substrata on the prevalence of microorganisms in an oral multispecies biofilms and (ii) the effect of antimicrobials released in saliva on the microbial population of a multispecies biofilms. For the first study hydroxyapatite, titanium and polymethylmetacrylate (PMMA) discs were used as substrata for biofilm development mimicking tooth enamel, dental implant and denture base, respectively. The multispecies biofilm model was composed by five bacteria (Streptococcus oralis, Streptococcus mutans, Actinomyces naeslundii, Veillonella dispar and Fusobacterium nucleatum) and one yeast (Candida albicans). Mature biofilms (64.5 h of development) were removed by ultrasonic waves, plated on agar media and CFU counts of each microorganism were calculated. Scanning electron microscopy was used to visualize the materials' surface. Data were analyzed by one-way ANOVA. For the second study the same multispecies biofilm model was used. Two antibiotics, azithromycin and metronidazole, and one antifungal, fluconazole, were evaluated. Mature biofilms (64.5 h development) were exposed to azithromycin, metronidazole or fluconazole at concentrations found in saliva of 2.12 ug/mL, 15.15 ug/mL and 2.56 ug/ml, respectively, for 24h. After this period, the biofilm was removed by ultrasonic waves, plated on agar media and CFU counts of each microorganism were calculated. Scanning electron microscopy and confocal scanning laser microscopy with cells stained by fluorescent in situ hybridization (FISH) technique were used to assess the biofilm structure. Data were analyzed by independent-samples t-test and Mann-Whitney nonparametric test. The first study showed no difference in the biofilm population for each microorganism among the three materials evaluated (p>0.05). In the second study, all antimicrobials evaluated were able to change microbial population (p<0.05), however none of the antimicrobials was able to completely eliminate a specific microorganism from the biofilm. Azithromycin reduced A. naeslundii and V. dispar population while increased C. albicans (p<0.05). Metronidazole reduced all the microorganisms evaluated, with a great reduction for V. dispar and F. nucleatum (p<0.001). Fluconazole reduced C. albicans and F. nucleatum population and increased S. oralis and V. dispar counts (p<0.05). It can be concluded that the substrata were not able to interfere with the formation of multispecies biofilms and antimicrobials in concentrations similar to those released in the saliva changed microbial population, however they were not able to eliminate microorganisms / Doutorado / Protese Dental / Doutor em Clínica Odontológica