Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-11-07T17:53:28Z No. of bitstreams: 1 StellaMarisdeFreitasLimaTese2018.pdf: 12066835 bytes, checksum: 9ad384b4f040333154c3598b59852583 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-11-07T17:53:52Z (GMT) No. of bitstreams: 1 StellaMarisdeFreitasLimaTese2018.pdf: 12066835 bytes, checksum: 9ad384b4f040333154c3598b59852583 (MD5) / Made available in DSpace on 2018-11-07T17:53:52Z (GMT). No. of bitstreams: 1 StellaMarisdeFreitasLimaTese2018.pdf: 12066835 bytes, checksum: 9ad384b4f040333154c3598b59852583 (MD5) Previous issue date: 2018-08-21 / Failure of endodontic therapy is mainly based on the persistence of the microorganism inside the root canal system, perpetuating periradicular pathologies. The need for endodontic reintervention reduces the success rate and turns the development of new therapies into a necessity. Host defense peptides (HDPs) have a therapeutic potential because of its characteristics as broad spectrum antimicrobial activity, immunomodulatory capacity and repair induction. The present study aimed to demonstrate HDPs HHC-10, LL-37 and synoeca-MP activities compared to the commonly used chemical agents Ca(OH)2, NaOCl and chlorhexidine (CHX) in in vitro and in vivo environments. In vitro analysis involved (1) antimicrobial assays for MIC/MBC/MFC determination, antibiofilm and synergistic activities (against Candida albicans, Enterococcus faecalis and Staphylococcus aureus), (2) hemolytic assay and determination of therapeutic index, (3) immunomodulation assays (from RAW 264.7 monocytes) and (4) osteoclastogenesis assays (from mouse bone marrow cells). In vivo assays were based on periradicular lesions induced and treated in rats with subsequent analysis by radiographic, tomographic and histological examinations. Results demonstrated effective antimicrobial activity of HHC-10 (except antibiofilm activity) and synoeca-MP besides NaOCl and CHX. Higher therapeutic index was observed for peptides (HHC-10 and synoeca-MP) with low or no hemolytic activity. Synergism analysis demonstrated better interaction between synoeca-MP and CHX. Immune assays from monocyte cultures demonstrated a pro-inflammatory activity of LL-37 and synoeca-MP and a pro- and anti-inflammatory effect of HHC-10 and Ca(OH)2. However, none of the agents tested reduced osteoclastogenesis in vitro. In in vivo endodontic treatment conditions, HHC-10, synoeca-MP and Ca(OH)2 significantly reduced periapical lesions, although only peptides were able to reduce inflammation in histological analysis. Therefore, HHC-10 and synoeca-MP demonstrated in vitro and in vivo potential for application in endodontic therapy. However, further researches are needed regarding the mechanism of action of HDPs and the possible therapeutic formulations for future analysis in animal models. / O insucesso da terapia endodôntica está baseado na persistência de microrganismos principalmente no interior do sistema de canais radiculares, perpetuando as patologias perirradiculares associadas à tal infecção. A necessidade de reintervenção endodôntica reduz o percentual de sucesso e, portanto, o surgimento de novas terapias é necessário. Dessa forma, peptídeos de defesa do hospedeiro (PDHs) possuem potencial terapêutico associado às suas características de atividade antimicrobiana de amplo espectro, capacidade imunomodulatória e indução de reparo. O presente estudo objetivou demonstrar a atividade dos PDHs HHC-10, LL-37 e synoeca-MP comparado aos agentes químicos comumente utilizados Ca(OH)2, NaOCl e clorexidina (CHX) em ambientes in vitro e in vivo. Para tanto, foram realizadas análises in vitro envolvendo: (1) ensaios antimicrobianos com determinação de MIC/MBC/MFC, atividade antibiofilme e atividade sinérgica (contra Candida albicans, Enterococcus faecalis e Staphylococcus aureus), (2) ensaio hemolítico e determinação do índice terapêutico, (3) ensaios de imunomodulação (a partir de monócitos RAW 264.7) e (4) ensaios osteoclastogênese (a partir de células da medula óssea de camundongos). Os ensaios in vivo foram conduzidos a partir das lesões perirradiculares induzidas e tratadas em ratos com subsequente análise por exames radiográficos, tomográficos e histológicos. Assim, os resultados demonstraram efetiva atividade antimicrobiana dos peptídeos HHC-10 (exceto atividade antibiofilme) e synoeca-MP além do NaOCl e CHX. Observou-se maiores índices terapêuticos para os peptídeos (HHC-10 e synoeca-MP) com baixa ou nenhuma atividade hemolítica. As análises de interação demonstraram combinação sinérgica necessitando de menor concentração a partir da interação entre synoeca-MP e CHX. A partir da cultura de monócitos, observou-se uma atividade pró-inflamatória a partir dos peptídeos LL-37 e synoeca-MP e um efeito pró- e anti-inflamatório do peptídeo HHC-10 e do Ca(OH)2. No entanto, nenhum dos agentes testados reduziram a osteoclastogênese in vitro. Já em condições de tratamento endodôntico in vivo, os PDHs HHC-10, synoeca-MP e o Ca(OH)2 reduziram significativamente lesões periapicais, apesar de somente os peptídeos serem capazes de reduzir inflamação em análises histológicas. Portanto, os peptídeos HHC-10 e synoeca-MP demonstraram potencial in vitro e in vivo para aplicação na terapia endodôntica. No entanto, faz-se necessário maiores investigações sobre o mecanismo de ação dos mesmos e possíveis formulações terapêuticas para futuros testes em modelos animais.

Doctor of Philosophy / Department of Anatomy and Physiology / Chris R. Ross / The immediate antiviral defense residing in the innate immune system of multicellular organisms critically determines the outcome of viral infection. This dissertation presents a study of the "effectors" and "receptors" of porcine innate immunity in infection caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is the most devastating pathogen impacting the swine industry. In the first investigation, eleven novel porcine host defense peptides (HDPs), [Beta]-defensins (pBDs), were identified and characterized. All of these peptides have a consensus [Beta]-defensin motif and phylogenetically are similar to orthologs from other species. A differential expression pattern for these 11 newly identified genes was found. For example, pBD-2 and pBD-3 were expressed in bone marrow, lung, skin and other lymphoid tissues. pBD-2 and pBD-3 were further characterized for their gene structure, and antimicrobial activity of synthetic peptides. The second study was conducted to evaluate PRRSV-induced differential expression of porcine HDPs and direct antiviral activity of selected HDPs against PRRSV. In vitro incubation of PRRSV with synthetic pBD-3 or protegrin-4 (PG-4) significantly inhibited viral infectivity. Using nine protegrin-derived peptides, it was determined that cyclization of PG-4 increased anti-PRRSV activity and mutation of some residues in PG-4 diminished some of the activity. These findings suggest the potential role of porcine HDPs as a group of innate antiviral effectors. In the third and fourth investigations, porcine Toll-like receptor (TLR) 3 and TLR7 were identified and functionally expressed. Increased expression of TLR3 was observed in PRRSV-infected porcine lungs. Stimulation of porcine alovelar macrophages with poly (I:C), a synthetic TLR3 ligand, increased expression of interferon-[Beta] and suppressed PRRSV infectivity. Activation of porcine TLR3 overexpressed in a PRRSV-sensitive cell line, elicited antiviral responses to PRRSV infection. Partial silencing of TLR3 in PAMs resulted in increased PRRSV infection. In summary, these data provide molecular information on porcine TLR3 and TLR7, and their involvement in PRRSV pathogenesis, which may elicit new strategies to prevent this costly swine disease.

Doctor of Philosophy / Department of Human Nutrition / Tonatiuh Melgarejo / Background and Objective: Crohn’s disease (CD) patients express low levels of host defense peptides (HDPs) especially β-defensins, which may compromise intestinal barrier function. Antibiotic treatment for bacterial infections in CD is limited and rarely curative, making it necessary to find alternative therapeutic approaches. We therefore investigated to what extent an essential amino acid; L-isoleucine (L-ILE) might induce the expression of human β-defensins (HBDs) in colonic epithelial cells as an alternative approach to help patients with CD. Antimicrobial activity of HBD2 was also assessed against four bacterial isolates which can cause secondary infections in CD. Methods: HTB-37 Caco-2 cells were stimulated with L-ILE at a concentration of 0 - 500µg/ml for 6 hours. Total RNA was extracted using RNeasy Micro Kit (QIAGEN). Reverse transcription was carried out with Superscript ®III First-Strand Synthesis System. The cDNA was amplified using specific primers for HBD1-3. Antimicrobial activity of HBD2 was determined using the broth dilution assay. Results: HBD1 was constitutively expressed under all conditions. HBD2 was expressed in HTB-37 cells after stimulation with L-ILE. Below 25µg/ml L- ILE stimulation, no expression of HBD2 was observed. HBD2 exhibited antimicrobial activity against bacterial isolates tested, with a MIC of 32, 64 and 128 µg/ml for both strains of E. coli, S. aureus and P. aeruginosa respectively. Conclusions: Our results indicate that L-ILE stimulation of HTB-37 Caco-2 cells can induce HBD2 expression. Data collected from our in vitro studies might have major implications for modifying the intestinal microbiota towards a healthier state in CD patients. Promoting the expression of HBD2 by colonic cells may lead to a lower rate of infection in these patients. Future in vivo studies are warranted to determine the potential clinical use of intra colonic administration of L-ILE in CD patients. The observed antimicrobial activity of HBD2 against bacterial isolates provides evidence that it is a crucial component of mucosal epithelial defense against infections which can complicate disease symptoms in CD.