Periodontal disease is a chronic inflammation of the gingiva and periodontium that leads to progressive destruction and irreversible damage to the supportive structures of the teeth. It affects nearly half of the United States population and is a particular risk factor in adults older than 65 years of age. Oral microorganisms assemble in plaque as a polymicrobial biofilm and Porphyromonas gingivalis, an important secondary colonizer in oral biofilms, has been implicated in periodontal disease. Although the protective functions of various salivary molecules such as antimicrobial proteins have been delineated, lipids present in saliva and on the oral mucosa have been largely ignored and there is growing evidence that the role of lipids in innate immunity is more important than previously realized. In fact, recent studies suggest that sphingoid bases and fatty acids, which exhibit potent broad spectrum antimicrobial activity against a variety of bacteria and fungi, are likely important innate immune molecules involved in the defense against oral bacterial and fungal infections. However little is known about their spectrum of activity or mechanisms of action. In addition, the effects of these lipids that are endogenous to the oral cavity have not been explored against oral bacteria. In this study I hypothesized that oral mucosal and salivary lipids exhibit dose-dependent antimicrobial activity against P. gingivalis and alter cell morphology and metabolic events. To test this hypothesis, I first examined the effects of two fatty acids: sapienic acid and lauric acid, and three sphingoid bases: sphingosine, dihydrosphingosine, and phytosphingosine, against a variety of gram-positive and gram-negative bacteria including P. gingivalis. Using broth microdilution assays to determine minimum inhibitory and minimum bactericidal concentrations, I show that antimicrobial activity against bacteria is dose-dependent, lipid specific, and microorganism specific. Kill kinetics were also variable across each bacteria-lipid combination. Upon examination of select bacteria-lipid combinations via scanning and transmission electron microscopy, different morphologies were evident across all treatments, demonstrating differential activity of each lipid for a particular bacterium as well as for each bacterium across different lipids. In addition, all sphingoid bases and fatty acids were taken up and retained in association with P. gingivalis cells and could be extracted along with bacterial lipids and separated using thin layer chromatography. Using a combination of two-dimensional in-gel electrophoresis and Western blots followed by mass spectroscopy and n-terminus degradation sequencing, I show that sapienic-acid treatment induces a unique stress response in P. gingivalis, as evidenced by the ability of P. gingivalis to upregulate a set of proteins involved in fatty acid biosynthesis metabolism and energy production, protein processing, cell adhesion, and virulence. Finally, utilizing flow cytometry and confocal microscopy, I assessed the effects of oral antimicrobial lipids against a representative host cell and describe oral lipid concentrations that are both antimicrobial to P. gingivalis cells and non-cytotoxic to the representative host cells tested. Combined, these data strongly suggest that sphingoid bases and fatty acids found within the saliva and on oral mucosa likely do contribute to the innate antimicrobial activity of saliva, mucosal surfaces, and skin and this dose-dependent activity is both lipid specific and bacteria specific. This information adds to current knowledge of the innate functions of endogenous lipids in the oral cavity. With bacterial resistance to current antibiotics increasing, the exploration of new antimicrobial agents is important and these lipid treatments may be beneficial for prophylactic treatments or therapeutic intervention of infection by supplementing the natural immune function of endogenous lipids on skin and other mucosal membranes.

Sphingoid bases (sphingosine, dihydrosphingosine and phytosphingosine) have been recently found in the oral cavity where they may serve to fortify innate immunity against commensals and periodontal pathogens. In fact, sphingoid bases have potent antimicrobial activity against Gram- positive and Gram- negative bacteria including oral pathogens like Porphyromonas gingivalis. It is not known whether these lipids are cytotoxic or alter the chemokine and cytokine responses of human dendritic cells, a finding important to their future potential as a therapeutic for treatment of periodontal disease. Objectives: The objective of this study was to determine the effects of sphingoid bases on the cytotoxicity and cytokine responses of human myeloid dendritic cells. Methods: Dendritic cells were treated with sphingoid bases (0.2-80.0 μM) for 16 hours in the presence or absence of 0.02 μM hemagglutinin B, a nonfimbrial adhesin of P. gingivalis used as a pro-inflammatory stimulus. The cytotoxicity of the inocula and its ability to induce the production of chemokines and pro-inflammatory cytokines was determined after 16 hours. Results: Higher concentrations of sphingoid bases were cytotoxic (e.g., 40.0-80.0 μM), but physiologic concentrations of sphingoid bases (e.g., 0.2-20.0 μM) were not. At 5, 10, or 20 μM, sphingosine did not enhance or attenuate any HagB-induced IL-8, GM-CSF, MIP-1α, MIP-1β, or TNFα response of human myeloid dendritic cells. At 5 or 10 μM, neither phytosphingosine nor dihydrosphingosine enhanced or attenuated any HagB- induced IL-8, GM-CSF, MIP-1α, MIP-1β, or TNFα response of human myeloid dendritic cells. Conclusion: Sphingoid bases exhibit dose-dependent cytotoxicity and cytokine responses against human myeloid dendritic cells. But at physiologic concentrations sphingoid bases appear to be safe and efficacious at the doses needed to prevent or treat microbial infections in the oral cavity.

Orientador: : Carlos Roque Duarte Correia / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-24T05:38:03Z (GMT). No. of bitstreams: 1 Martinez_PabloDavidGrigol_D.pdf: 10635816 bytes, checksum: b6d50b746463694bb1ef50352d36d1e3 (MD5) Previous issue date: 2013 / Resumo: O emprego de enecarbamatos endocíclicos como substratos na organocatálise foi avaliado na primeira parte deste trabalho. Explorou-se a ação catalítica de ácidos fosfóricos na reação de Friedel-Crafts envolvendo enecarbamatos di e trissubstiuídos como eletrófilos. Foram empregados indóis, furanos, naftol e sesamol como nucleófílos neste tipo de reação. Foi possível desenvolver um protocolo operacional simples e em condições brandas capaz de fornecer bons rendimentos. A diastereosseletividade observada com enecarbamatos trissubstituídos favoreceu o produto trans numa faixa de 2,7:1 até 95:5. Usando ácidos fosfóricos quirais, a enantiosseletividade também foi verificada, atingindo-se excesso enantiomérico de 20% até o momento, fato que demonstra a potencialidade da metodologia. Posteriormente, realizou-se um estudo sobre a preparação de aminoálcoois a pela hidrogenólise de 2-aril-3-epoxipirrolidinas. Esses substratos foram obtidos pela arilação de Heck-Matsuda em enecarbamatos derivados do ácido L-piroglutâmico como etapa chave. Observou-se que a estereoquímica dos substituintes da pirrolidina apresentou decisiva influência para as condições reacionais e rendimento da hidrogenólise, permitindo em alguns casos a clivagem de até duas ligações numa única reação. Os aminoálcoois obtidos a partir desta metodologia foram utilizados na síntese concisa da 2-epi-bulgecinina bem como de bases esfingóides exemplificada na síntese de análogo da esfingosina / Abstract: In the first part of this work, the use of endocyclic enecarbamates as substrates in organocatalysis was evaluated. The effect of phosphoric acids as catalysts in promoting Friedel-Crafts reactions involving di and trisubstituted enecarbamates as electrophiles was explored. Different arenes were employed as nucleophiles like indols, furans, naftol and sesamol. It was possible to develop a simple protocol with mild conditions and good yields. The diastereoselectivity observed with trisubstituted enecarbamates favored the trans product in a range of 2.7:1 to 95:5. By using chiral phosphoric acids, the enantioselectivity achieved, so far, was 20% ee, evidencing the potentiality of this methodology. Later, hydrogenolysis reactions over 2-aryl-3-epoxypyrrolidines were performed to prepare aminoalcohols. The key step to synthesize the former pyrrolidines was the Heck-Matsuda reaction of enecarbamates obtained from L-pyroglutamic acid. The stereochemistry of the substituents in the hydrogenolytic step did influence the experimental conditions and yields, allowing in some cases two bonds cleavage in one pot. The aminoalcohols prepared in this methodology were used in a short synthesis of 2-epibulgecinine and in preparation of sphingoid bases exemplified in the synthesis of sphingosine analogue / Doutorado / Quimica Organica / Doutor em Ciências