Orientador: Carla Raquel Fontana Mendonça / Resumo: O presente estudo tem como objetivo avaliar a fototoxicidade do azul de metileno (AM), clorina-e6 (Ce-6) e curcumina (CUR) associados a irradiações com diferentes intensidades de luz para a terapia fotodinâmica (TFD) contra cepas de Enterococcus faecalis e Cutibacterium acnes, em suspensão e em biofilme. As fontes de luz utilizadas na TFD foram LEDs no comprimento de onda azul (450 nm) e vermelho (660 nm) de acordo com cada fotossensibilizador testado. Como resultado observamos que, em E. faecalis a TFDa mediada por AM e Ce6 foi mais eficiente quando exposta a intensidade de luz de 153 mW/cm², porém com a CUR os melhores resultados foram obtidos com a intensidade de luz de 103 mW/cm². Para C. acnes em suspensão, a Ce6 e a CUR submetidas às diferentes intensidades de luz, causaram reduções bacterianas similares. Em biofilme, a Ce6 associada a intensidade de 122 mW/cm² de luz foi mais eficiente, no entanto, a CUR não proporcionou morte de biofilmes de C. acnes independente das intensidades testadas. O AM, independente da fase, apresentou os melhores resultados com a intensidade de luz de 153 mW/cm². Considerando a quantificação de espécies reativas de oxigênio, a concentração de AM foi determinante na produção de EROs. Na concentração de 100 μg/mL de AM na dose de 120 J/cm2 radicais hidroxila foram mais evidentes em relação a quantidade de oxigênio singleto formado. Na concentração de 50 μg/mL, a intensidade de 61 mW/cm² gerou similarmente as espécies testadas, quanto que a int... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Prostatacancer är den vanligaste cancerformen hos män i Sverige, och det är även den cancerform som flest män dör utav. Detta till trots så är mekanismerna bakom canceretiologin till stor del okända. Uppskattningsvis 20% av alla canceruppkomster i människor tros vara länkade till infektioner från patogener och de inflammationer som de orsakar. Tidigare studier har visat indikationer på att kronisk inflammation av prostatan kan hjälpa tumörer att undvika immunförsvaret. Bakterien Cutibacterium acnes förekommer i stor utsträckning i prostatavävnad hos patienter som lider av prostatacancer vilket kan peka på en länk mellan mikroorganismen och cancerformen. Tumörassocierade makrofager kan producera immunhämmande proteiner, däribland liganden PD-L1, ett protein som kan hjälpa tumörcellerna att undgå immunförsvaret. Studien ämnade därför att undersöka huruvida makrofager som infekterats med typ I eller typ II C. acnes producerar en högre mängd av PD-L1 än obehandlade makrofager och i detta syfte utfördes ett ELISA-test som bestämde koncentrationen av PD-L1 i odlingsmediumet. Genom en statistisk analys konstaterades att makrofager som infekterats med bakterien ökade produktionen av PD-L1 jämfört med obehandlade makrofager. Resultatet av studien visar indikationer på att C. acnes kan spela en roll i spridningen av tumörceller i kroppen, dock krävs vidare studier för att bekräfta hypotesen. / Prostate cancer is the most common form of cancer among men in Sweden, and the form of cancer that most men die from. In spite of this, the mechanisms behind the etiology of the cancer are largely unknown. Approximately 20% of all cancer etiologies are believed to be linked to infection by pathogens, and the inflammatory response they cause within the body. Earlier studies have shown indications that chronic inflammation of the prostate might create conditions that are favourable for the tumour cells. The bacterium Cutibacterium acnes is prevalent in prostate tissue from patients suffering from prostate cancer, which might indicate a link between the microorganism and this form of cancer. Tumour associated macrophages produce immunosuppressive proteins, such as the ligand PD-L1, a protein that might provide immunoevasive qualities for the tumour cells. The aim of the study was to examine whether macrophages that have been infected with either type I or type II C. acnes would produce a greater amount of PD-L1 than untreated macrophages. For this purpose, an ELISA test was performed to determine the concentrations of PD-L1 in the culture media. I found that macrophages that had been infected with the bacterium produced greater amounts of PD-L1 than untreated macrophages. The result of this study shows some indication that C. acnes could play a role in the proliferation of tumour cells and thus the spread of cancer through immunosuppresive mechanisms, although further studies are needed to confirm this hypothesis.

L’objectif de ces travaux de thèse a été de contribuer à la compréhension de la physiopathologie des infections sur matériel ostéo-articulaire (IMOA) à C. acnes. Dans un premier temps, nous avons typé par MLST 108 isolats de C. acnes responsables de 34 cas d’IMOAs monomicrobiennes et corrélé les résultats de typage aux données clinico-biologiques. Nous avons ainsi montré que les IMOAs à C. acnes correspondent à 2 entités cliniques : i) les cas « homotypiques » qui sont des vraies infections dues à un clone de C. acnes responsable d’une réponse inflammatoire de l’hôte ii) les cas hétérotypiques qui sont une colonisation ou contamination itérative du matériel ostéo-articulaire en l’absence de réponse inflammatoire de l’hôte. Ces données de typage ont souligné les limites de la définition microbiologique actuelle d’une IMOA quand il s’agit de C. acnes et la nécessité d’intégrer un outil moléculaire fiable dans le diagnostic microbiologique de routine de ces infections. Nous avons par conséquent évalué la technique SLST développée pour un typage rapide et optimal de C. acnes en routine. Le pouvoir discriminant de cette technique était insuffisant suggérant que seule la mise en place du NGS en temps réel dans les laboratoires de microbiologie pourrait répondre au besoin de typage moléculaire pour améliorer le diagnostic microbiologique de ces infections.Nos résultats de typage ont montré que la clonalité de l’infection plutôt que le complexe clonal (CC) est le principal facteur déterminant du processus physiopathologique et inflammatoire de l’IMOA à C. acnes. Cette conclusion est concordante avec les résultats que nous avons obtenus dans un modèle in vitro d’infection de macrophages THP-1 par C. acnes. Grâce à ce modèle, nous avons pu montrer que la réponse inflammatoire in vitro de nos isolats est souche –et non CC– dépendante. De plus, les réponses inflammatoires in vitro et in vivo n’étaient pas corrélées. Ceci souligne les limites des modèles in vitro dans l’étude de la physiopathologie des IMOAs à C. acnes dans lesquels l’adaptation de la bactérie à l’hôte est complètement négligée lors de l’étude de la réponse immune. Dans la dernière partie de ce travail, nous avons confirmé l’importance de l’environnement dans le conditionnement du comportement de la bactérie. Nous avons mené une étude comparative des caractéristiques culturales ex vivo et ex vitro de nos isolats de C. acnes. Nous avons montré l’impact du CC et des conditions environnementales, par extension la pression que peut exercer l’hôte, sur le profil cultural de C. acnes. En conclusion, nos travaux montrent qu’une approche multifactorielle, intégrant à la fois la génomique et la réponse de l’hôte, est nécessaire pour comprendre la physiopathologie des IMOAs à C. acnes. / The aim of this thesis was to contribute to the understanding of the physiopathology of C. acnes orthopedic-device related infections (ODRI). Firstly, we typed by MLST 108 C. acnes isolates responsible for 34 cases of monomicrobial ODRIs and correlated typing results with bio-clinical data. We have shown that C. acnes ODRIs correspond to two clinical entities: i) "homotypic" cases corresponding to true infections with a single pathogenic clone of C. acnes eliciting an inflammatory response ii) heterotypic cases corresponding to colonization or iterative contamination of the implant without systemic inflammatory response. These typing data highlighted the limitations of the current microbiological definition of ODRI when it comes to C. acnes and the need to incorporate a reliable molecular tool into the routine microbiological diagnosis of these infections. We therefore evaluated the SLST technique developed for a rapid and optimal typing of C. acnes. The discriminating power of this technique was insufficient suggesting that only the establishment of real-time NGS in microbiology laboratories could improve the microbiological diagnosis. Our typing results showed that the clonal status of the infection and not CC is the main determining factor in the physiopathological and inflammatory process of C. acnes ODRI. This conclusion is consistent with our results obtained on an in vitro model of a macrophage THP-1 infection. Using this model, we have shown that the in vitro inflammatory response of our isolates is strain- and non-CC-dependent. In addition, the in vitro and in vivo inflammatory responses were not correlated. This underscores the limitations of in vitro models in the study of C. acnes ODRIs in which the adaptation of the bacteria to the host is completely neglected during the study of the immune response. Finally, we confirmed the importance of the environment in the conditioning of the behavior of the bacterium. We conducted a comparative study of the ex vivo and ex vitro growth characteristics of our C.acnes isolates. We have shown the impact of CC and environmental conditions, by extension the pressure that can exert the host, on the cultural profile of C. acnes. In conclusion, our work shows that a multifactorial approach, integrating both genomics and host response, is needed to understand the physiopathology of C. acnes ODRI.

Submitted by Maria Lúcia Frade (marifrad@hotmail.com) on 2018-06-25T15:04:46Z No. of bitstreams: 1 Dissertação com artigo versão de arquivamento 25_06_2018.pdf: 5490840 bytes, checksum: 7f04a23e5fbc4a98d7cc0bd23f546892 (MD5) / Approved for entry into archive by Maria Irani Coito null (irani@fcfar.unesp.br) on 2018-06-25T19:35:56Z (GMT) No. of bitstreams: 1 frade_ml_me_arafcf_int.pdf: 5490840 bytes, checksum: 7f04a23e5fbc4a98d7cc0bd23f546892 (MD5) / Made available in DSpace on 2018-06-25T19:35:56Z (GMT). No. of bitstreams: 1 frade_ml_me_arafcf_int.pdf: 5490840 bytes, checksum: 7f04a23e5fbc4a98d7cc0bd23f546892 (MD5) Previous issue date: 2018-05-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A acne vulgar é uma das dermatoses mais frequentes em jovens e a infecção causada pela bactéria Cutibacterium acnes (antigo Propionibacterium acnes) tem importante papel na patogênese da acne. Existem várias opções de tratamento que variam de acordo com a gravidade da doença, porém são associados com reações indesejadas e resistência aos antimicrobianos. A terapia fotodinâmica antimicrobiana (TFDa) então é proposta como tratamento alternativo para inativar C. acnes juntamente com a incorporação do fotossensibilizador (FS) em uma formulação tópica para otimizar a terapia. Este trabalho objetivou avaliar a eficácia in vitro da TFDa mediada pelo azul de metileno (AM) incorporado em hidrogel de quitosana e poloxamer (HG-AM) contra C. acnes em fase planctônica e biofilme. A viabilidade celular após TFDa foi avaliada através da quantificação das colônias formadas por mililitro de amostra (UFC/mL). Para caracterizar estruturalmente os HGs foram realizadas as análises oscilatórias e de comportamento de fluxo em reômetro de estresse controlado com geometria placa-cone e a bioadesão em pele de orelha de porco utilizando um texturômetro. A análise estatística dos dados feita foi a Análise de Variância (one way ANOVA) com pós-teste de Tukey. Os resultados deste estudo mostrou que a TFDa contra C. acnes foi eficaz utilizando HG-AM em fase plactônica. A concentração bactericida mínima foi de 12,5 µg/mL associada a uma fluência de 90 J/cm2. O biofilme de C. acnes não foi totalmente eliminado pela TFDa com HG-AM nas condições testadas. A máxima redução microbiana alcançada com o HG-AM foi de 1,9 log10 com 75 µg/mL do FS utilizando 150 J/cm2 e 30 minutos de pré-incubação do biofilme com o HG-AM. O HG a 0,25% de quitosana (HG1) mostrou-se adequado para aplicação na superfície da pele pois concentrações maiores de quitosana aumentam a elasticidade do hidrogel, podendo atrasar a liberação do FS. A incorporação do AM não afetou suas características reológicas e bioadesivas de forma significativa. Foi possível concluir que a TFDa mediada por HG-AM foi mais eficiente do que AM em solução para a redução total da carga microbiana em fase planctônica nas mesmas condições. / Acne vulgaris is one of the most frequent dermatosis in young people and the infection caused by the bacteria Cutibacterium acnes (formerly Propionibacterium acnes) plays an important role in the pathogenesis of acne. There are several treatments options that vary according to the severity of the disease, but are associated with unwanted reactions and antimicrobial resistance. Antimicrobial photodynamic therapy (aPDT) is then proposed as an alternative treatment to inactivate C. acnes together with the incorporation of the photosensitizer (PS) into a topical formulation to optimize therapy. This work aimed to evaluate the in vitro efficacy of methylene blue (MB) mediated TFDa incorporated in chitosan and poloxamer (HG-MB) hydrogels against planktonic and biofilm C. acnes. Cell viability after aPDT was evaluated by quantifying the colonies formed per milliliter of sample (CFU / mL). To characterize structurally the HG were performed the oscillatory and behavior of flow in a controlled stress rheometer with cone-plate geometry and bioadhesion in pig ear skin using a texture analyser. The statistical analysis of the data was the Analysis of Variance (one way ANOVA) with post-test of Tukey. The results of this study showed that aPDT was effective using HG-MB in planktonic phase. The minimum bactericidal concentration was 12.5 μg/mL associated with a fluence of 90 J/cm2. The C. acnes biofilm was not totally eliminated by the aPDT with HG-MB under the conditions tested. The maximum microbial reduction achieved with HG-MB was 1.9 log10 with 75 μg/mL of the PS using 150 J/cm2 and 30 minutes biofilm incubation with HG-MB. HG at 0.25% of chitosan (HG1) was adequate for application to the skin surface because higher concentrations of chitosan increase the elasticity of the hydrogel and may delay PS release. The incorporation of MB did not significantly affect its rheological and bioadhesive characteristics. It was possible to conclude that HG-MB mediated aPDT was more efficient than MB solution for the total reduction of planktonic microbial load under the same conditions. / 133456/2016-1

Prostatacancer är en av världens vanligaste cancerformer. Varje år diagnostiseras ungefär 1,3 miljoner män världen över med prostatacancer och trots det vet man inte de bakomliggande orsakerna. Det finns flera studier som visar att det finns en koppling mellan infektion och olika typer av cancer och Cutibacterium acnes återfinns i hög utsträckning i prostatacancervävnad. Det har därför föreslagits att det finns ett samband mellan infektion av C. acnes och prostatacancer. Närvaro av tumörassocierade makrofager har visats sig vara gynnande för olika typer av cancer och från dessa makrofager frisätts kemokiner, bland annat CCL22. CCL22 kan vara inblandad i en lokal hämning av immunförsvaret som ofta förknippas med tillväxt av cancer. I detta arbete odlas makrofager från blodgivare med C. acnes för att ta reda på om makrofagerna ökar sitt uttryck av CCL22 mRNA. Genom att analysera resultaten från en kvantitativ realtids-PCR indikeras det att makrofager som behandlats med C. acnes signifikant ökar sitt uttryck av CCL22 mRNA. Sammanfattningsvis bedöms resultaten från detta arbete styrka uppfattningen att det kan finnas en koppling mellan C. acnes och orsakerna bakom prostatacancer men mer arbete återstår för att kunna klargöra dess relevans. / Prostate cancer is one of world´s most common forms of cancer. Every year about 1.3 million men around the world are diagnosed with prostate cancer and even so we cannot fully explain the etiology. There have been several studies indicating that there is a correlation between infection and different forms of cancer. Cutibacterium acnes (C.acnes) can be found to a large extent in prostate cancer tissue and a correlation between infection of C. acnes and prostate cancer has therefore been suggested. The presence of tumor-associated macrophages has been shown to favor various types of cancer. Several chemokines including CCL22 are released from these macrophages. CCL22 may be involved in a local inhibition of the immune system that is often associated with cancer growth. In this work, macrophages from a blood donor are grown with C. acnes to find out if the macrophages increase their expression of CCL22 mRNA after this exposure. By analyzing the results of a quantitative real-time PCR for CCL22 mRNA, it was demonstrated that macrophages treated with C. acnes significantly increase their expression of CCL22 mRNA. In conclusion, the results of this work indicate that there could be a link between C. acnes and the causes of prostate cancer, but more work remains to be able to clarify its relevance.

Orientador: Ary Fernandes Junior / Resumo: Os óleos essenciais (OE) são produtos do metabolismo secundário da planta e são conhecidos pelas suas propriedades antimicrobianas. O principal componente do OE da palmarosa é o geraniol (70-80%). A incorporação de compostos da biodiversidade agrega valor as formulações dermocosméticas. OEs são uma boa opção devido à semelhança estrutural com o equilíbrio hidro-lipídico da pele. Assim, o objetivo do estudo foi avaliar a atividade antibacteriana desses compostos e o desenvolvimento de protótipos de dermocosméticos com nanopartículas para o tratamento da acne vulgar. Nanoemulsões com OE e geraniol foram preparadas de acordo com os valores do equilíbrio hidrofílico-lipofílico (EHL) e a caracterização das nanoemulsões seguiram os parâmetros da ANVISA (2004) para testes preliminares de estabilidade. Foram preparadas dez formulações. Nanoemulsões especiais contendo nanopartículas de prata (AgNP) foram preparadas e testadas contra C. acnes. Os ensaios de microtitulação com resazurina (REMA) foram realizados para determinar a Concentração Inibitória Mínima (CIM) e a Concentração Bactericida Mínima (CBM) para o OE de palmarosa e geraniol contra cepas American Type Culture Collection (ATCC) de Cutibacterium acnes 11827, 11828 e 6919. A curva de sobrevivência (CS) foi realizada para analisar o sinergismo entre os compostos e a clindamicina contra C. acnes, cepa 6919. As formulações foram estáveis ao pH, condutividade elétrica, densidade, centrifugação e análise de stress térmico. Anális... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Essential oils (EO) are secondary metabolism products of plant and they are known for antimicrobial properties.The major component of palmarosa EO is geraniol (70-80%). Incorporation of compounds of biodiversity guarantee value to dermocosmetic formulations. EOs are good option due to structural similarity with skin hydro-lipid balance. Thus, the desire was to evaluate the antibacterial activity to these compounds and the development of dermocosmetic prototypes with nanoparticles to treat acne vulgaris. Nanoemulsion with EO and geraniol were prepared according to values of lipophilic-hydrophilic balance (LHB) and nanoemulsions characterization followed the ANVISA (2004) parameters for preliminary stability tests. Ten formulations were prepared. Special nanoemulsion containing silver nanoparticles (AgNP) was prepared and tested against C. acnes. Resazurin Microtiter Assays (REMA) was performed to determine the Minimum inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for palmarosa EO and geraniol against American Type Culture Collection (ATCC) strains of Cutibacterium acnes 11827, 11828 and 6919. The target of Time Kill Curve (TKC) test was determinate the synergism between compounds and clindamycin to C. acnes strain 6919. The formulations were stable to pH, electrical conductivity, density, centrifugation and thermal stress analysis. Analysis of size, zeta potential (ZP) and polydispersity index (PDI) were between 60 and 165 d.nm; -16.6 and -34.8 mv... (Complete abstract click electronic access below) / Mestre