Salivary glands are affected during radiotherapy in the head and neck region, leading to reduction in salivary flow and changing saliva composition. In this thesis, comprising 6 articles, we evaluated changes in the proteomic profile of the acquired enamel pellicle (AEP) and saliva in patients with head and neck cancer (HNC) submitted to radiotherapy, to search for therapeutic strategies and prognosis biomarkers. In the first two articles the protocols for standardization of proteomic analysis of saliva and collection of AEP are described. In articles 3, 4 and 5, HNC patients had their AEP and saliva (unstimulated and stimulated saliva) collected before (BRT), during (2-5 weeks; DRT) and after (3-4 months; ART) radiotherapy. Saliva and AEP were also collected from healthy patients (control; C). Proteins were extracted and processed for label-free proteomics. Salivary flows were also evaluated. In total, 1,055 proteins were identified in the unstimulated saliva, among which 47 were common to all groups, while 86, 86, 286 and 395 were exclusively found in C, BRT, DRT and ART, respectively. Remarkably, alpha-enolase was increased 35-fold DRT compared with BRT, while proline-rich proteins (PRPs) were decreased. ART there was a 16-fold increase in scaffold attachment factor-B1 and a 3-fold decrease in alpha-enolase and cystatins. When compared with C, salivary proteins of BRT patients showed increases in cystatin-C, lysozyme C, histatin-1 and PRPs (article 3). Significant differences were observed between stimulated and unstimulated salivary flows for C and BRT (p>0.001), but not for DRT and ART. Proteins involved with apoptosis and antibacterial function were decreased in stimulated saliva in comparison to unstimulated saliva DRT and ART (article 4). In the AEP, statherin was increased more than 9-fold and hemoglobins were increased more than 5-fold DRT compared to BRT, while lactotransferrin, PRPs cystatins, neutrophil defensins and histatin-1 were decreased. ART, lactotransferrin and isoforms of histones were increased, while statherin and alpha-amylase were decreased. MOAP-1 was exclusively found ART compared to BRT. When compared to Control, AEP of patients BRT showed an increase in proteins related to the perception of bitter taste, mucin-7 and alpha-amylases, while cystatin-S was decreased (article 5). In article 6, we evaluated the protective effect AEP formed in vitro for different times on enamel, as well as its engineering with statherin peptide (StatpSpS), against initial erosion. AEP provided almost instant protection, with formation times as short as 1 min protecting the native enamel against erosion, and longer formation times did not improve the protection. StatpSpS by itself provided similar protection as the AEP. In conclusion, both HNC and radiotherapy remarkably alter the proteome of saliva and AEP. The unstimulated salivary flow is the best alternative to search for biomarkers. Monitoring alpha-enolase levels in unstimulated saliva DRT and MOAP-1 in AEP ART might be possible strategies to predict the efficacy of radiotherapy. Our results provide important information for designing more effective dental products for these patients and contribute for a better understanding of the progressive changes in salivary proteins induced by radiotherapy and the protective roles of the AEP proteins DRT.

As glândulas salivares são afetadas durante a radioterapia na região da cabeça e pescoço, levando à redução do fluxo salivar e alterando a composição da saliva. Nesta tese, composta de 6 artigos, avaliamos as alterações no perfil proteômico da película adquirida do esmalte (PAE) e da saliva em pacientes com câncer de cabeça e pescoço (CCP) submetidos à radioterapia, para procurar estratégias terapêuticas e biomarcadores prognósticos. Nos primeiros 2 artigos, os protocolos para padronização da análise proteômica da saliva e coleta da PAE são descritos. Nos artigos 3, 4 e 5, pacientes com CCP tiveram suas PAEs e salivas coletados antes (BRT), durante (2-5 semanas; DRT) e após (3-4 meses; ART) radioterapia. As salivas e PAEs também foram coletadas de pacientes saudáveis (controle; C). As proteínas foram extraídas e processadas para proteômica. Os fluxos salivares também foram avaliados. No total, 1.055 proteínas foram identificadas na saliva não-estimulada, sendo 47 comuns a todos os grupos, enquanto 86, 86, 286 e 395 foram encontradas exclusivamente C, BRT, DRT e ART, respectivamente. Notavelmente, alfa-enolase foi aumentada 35X DRT em comparação com BRT, enquanto proteínas ricas em prolina (PRPs) diminuíram. ART houve aumento de 16X da scaffold attachment factor-B1 e diminuição de 3X da alfa-enolase e cistatinas. Em comparação ao C, nos pacientes BRT houve aumento de cistatina-C, lisozima C, histatina-1 e PRPs na saliva (artigo 3). Diferenças significativas foram observadas entre fluxos salivares estimulados e não-estimulados para C e BRT (p>0,001), mas não para DRT e ART. Proteínas envolvidas com apoptose e resistência antibacteriana foram diminuídas na saliva estimulada comparada com a não-estimulada DRT e ART (artigo 4). Na PAE, Statherin aumentou mais de 9X e hemoglobinas aumentaram mais de 5X DRT comparado com BRT, enquanto lactotransferrina, proteínas ricas em prolina, cistatinas, neutrófilo-defensinas e histatina-1 diminuíram. ART, lactotransferrina e histonas aumentaram, porém Statherin e alfa-amilases diminuíram. MOAP-1 foi encontrada exclusivamente ART comparada com BRT. Quando comparado ao Controle, PAE dos pacientes BRT apresentou aumento das proteínas relacionadas à percepção do sabor amargo, mucina-7 e alfa-amilases, enquanto cistatina-S diminuiu (artigo 5). No artigo 6, avaliou-se o efeito protetor da PAE formada in vitro no esmalte por diferentes tempos, bem como sua engenharia com peptídeo da estaterina (StatpSpS), contra erosão inicial. A PAE protegeu quase instantaneamente esmalte natural contra a erosão, mesmo com tempos de formação tão curtos quanto 1 min. Tempos de formação mais longos não aumentaram a proteção. O StatpSpS sozinho conferiu proteção similar àquela da PAE. Em conclusão, tanto o CCP quanto a radioterapia alteram profundamente o proteoma da saliva e da PAE. O fluxo salivar não-estimulado é a melhor alternativa na busca por biomarcadores. Monitorar níveis de alfa-enolase na saliva não-estimulada DRT e MOAP-1 na PAE ART podem ser estratégias possíveis para predizer a eficácia da radioterapia. Nossos resultados fornecem importantes informações para o desenvolvimento de produtos odontológicos mais eficazes para estes pacientes e contribuem para um melhor entendimento do papel protetor da PAE e das alterações progressivas que ocorrem nas proteínas salivares em consequência da radioterapia.