The thesis introduces two developed methods to quantify extracellular vesicles (EVs) isolated from cancer cell lines and healthy human saliva by using capillary zone electrophoresis. In the first chapter, the importance of EVs, as well as the existing EV isolation, characterization, and quantification methods, are described. The general principle of capillary electrophoresis (CE) is explained for a better understanding of these two methods. Chapter II describes the idea and concepts of Extracellular Vesicles quantitative Capillary Electrophoresis (EVqCE). The method evolved from the previous study carried out in our research group for the quantification of viruses. After the isolation of EVs from different cell lines, the characterization and quantification of EVs were performed using nanoparticle tracking analysis (NTA) and flow cytometry. EVqCE consists of four steps for EV quantification. In this study, EVqCE was employed to know the concentrations of EVs in unknown samples, followed by calculation of the average mass of the RNA present in EVs. In the next chapter, one of the human body fluids, i.e., saliva, was chosen for the quantification of EVs. Salivary EVqCE was developed in a similar way as EVqCE for cell lines since the general theories and procedures are practically the same. However, human saliva contains an abundant amount of viscous proteins and ribonuclease (RNase), that were the major obstacles for salivary EVs detection and quantification. The method for the isolation of EVs from the saliva was optimized, and the quantification was performed successfully. The average mass of RNA in saliva EVs was also calculated and analyzed. The concentration of saliva EVs in unknown samples were compared with the results from NTA and flow cytometry to validate the salivary EVqCE. In the last chapter, I described the application of EVqCE to study the quality control of EVs. The method calculates the degradation level of EVs samples under different conditions, providing a potential way for real-time monitoring of the EVs status in the body fluid sample.

Indiana University-Purdue University Indianapolis (IUPUI) / Secretory immunity is believed to play a role in natural resistance to dental caries. Although dental caries has dramatically decreased in children in the United States, there remains a population of caries-susceptible children even in fluoridated communities. Previous studies have shown a positive correlation between salivary immunoglobulin A (sIgA) antibody levels to Streptococcus mutans and caries resistance in adults. In the present study, an enzyme-linked immunosorbent assay (ELISA) was used to compare IgA antibody levels to S. mutans in saliva from 20 caries susceptible (DMFS greater than 5) and 20 caries-resistant (DMFS less than or equal to 1) children (ages 7-11). All subjects resided in fluoridated communities. Salivary S. mutans numbers were significantly higher (p ≤ 0.05) in the caries susceptible (31.2 percent of total streptococci) group than in the caries resistant (1.6 percent of total streptococci) group. Whole saliva from caries-resistant children had significantly higher (p = 0.05) levels of IgA antibodies to S. mutans than saliva from caries-susceptible children. However, whole saliva from caries-resistant children had similar levels of IgA1 or IgA2 antibodies against S. mutans to saliva from caries-susceptible children. These results suggest that IgA antibody to S. mutans may play a role in natural protection from dental caries in children and confirm previous reports indicating a role for salivary IgA antibodies to S. mutans in mediation of caries.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Introduction: According to the World Health Organization, dental cavities are the number one chronic disease in children. Saliva coats the teeth all day and can serve many functions to maintain and protect teeth. Saliva has many proteins that can be both detrimental and essential to the preservation of tooth enamel. The purpose of this study is to determine if a correlation exists between the total protein concentration in saliva and the prevalence of cavities in the mouth. We hypothesized that there would be a positive correlation with total salivary protein concentration and the prevalence of cavities in the participant. Methods: Saliva samples were taken from the patient during their comprehensive exam at the University of Central Florida (UCF) Dental Center and were analyzed using the DC assay to determine the protein concentration in the saliva. These results were compared to the number of cavities found in the mouth of each patient to determine if a correlation exists between protein concentration and cavity number. Results: The correlation between the variables was fairly weak, indicating that the data from this study does not support a correlation between salivary protein concentration and cavity number. Discussion: Future research should look at specific salivary proteins, control the time of day of collection, and take into account more variables in order to get a more precise study.

No description available.

The purpose of the present study was to study differences between cancer patients and noncancer patients in taste acuity and in salivation to food and stimuli associated with food. Subjects were twenty male cancer patients and eighteen patients hospitalized for noncancer-related illnesses. All cancer patients were tested prior to chemotherapy or radiation therapy. The study was conducted on two consecutive days. On Day 1 taste acuity was measured to bitter, sweet, sour and salty flavors using the forced choice three-stimulus drop technique on concentration from 6-2000 mm/l. Subjects completed a questionnaire on appetite difficulties, the Multiple Adjective Affect Check List (MAACL), and rated a list of snacks on a 5-point scale. On Day 2 salivary responding (using the Strongin-Hinsie Peck Test) was measured after subjects were told to expect food, after the presentation of food and after ingestion. For each subject, testing occurred in the morning and in the afternoon to high and low preferred foods. Cancer patients were significantly more likely than noncancer patients to report appetite difficulties which included premature satiety, decreased appetite, and changes in food preference. Cancer and noncancer patients did not differ reliably on the MAACL or in taste acuity. In salivation testing, the presentation of food increased salivation in noncancer patients but decreased salivation in cancer patients. However, the differences between cancer and noncancer patients was not reliable. The interaction between illness condition and test trials during the presentation of food did approach significance. The lack of reliable effects for illness condition may have occurred because the interval of food deprivation was too short to elicit reliable increases in salivation and external and social cues which normally accompany mealtime were not present during testing. / Ph. D.

Os mosquitos representam o grupo mais importante de vetores de doenças infecciosas para o homem. Para que possam ter sucesso no repasto sanguíneo e sejam capazes de adquirir os nutrientes necessários para a maturação dos ovos, a saliva desses vetores possui atividades anti-hemostáticas e imunomoduladoras. Sabendo-se da importância da saliva dos artrópodes hematófagos para sua alimentação e para a transmissão de patógenos causadores de doenças o objetivo deste estudo foi caracterizar as atividades imunomoduladoras de um peptídeo descrito no salivoma de A. aegypti, presente somente em glândulas salivares de mosquitos fêmeas e cujo papel biológico ainda é desconhecido. Observou-se que o peptídeo denominado AeMOPE-1 inibiu a produção de mediadores pró-inflamatórios por macrófagos ativados, assim como induziu a resposta imune para o perfil Th2. O tratamento com o AeMOPE-1 reduziu os sintomas clínicos da colite experimental. Essa é a primeira descrição de um peptídeo salivar de A. aegypti com atividade imunomoduladora em macrófagos e com potencial ação terapêutica. / Mosquitoes are the most important vectors of pathogens that causing infectious to humans. To female succeed in their hematophagous habit and thus acquire the nutrients necessary for the maturation of their eggs, their saliva has components with anti-hemostatic and immunomodulatory activities. Thus, the aim of this project was to study the role of a novel salivary peptide described in the A. aegypti sialome, present only in female salivay glands, whose biological functions are still unknown. It was observed that the peptide named AeMOPE-1 inhibited the production of pro-inflammatory mediators by activated macrophages, as well as induced the immune response to the Th2 profile. Treatment with AeMOPE-1 reduced the clinical symptoms of experimental colitis. This works presents the first salivary peptide from A. aegypti with potent activity on macrophage biology and potential clinical application.

A saliva é um fluido biológico com importante papel nos fenômenos que ocorrem na cavidade bucal. O efeito da sua composição sobre as perdas de estruturas dentais de origem não cariosa, como o desgaste dental, tem sido estudado. No entanto, há conflitos nos resultados apresentados por esses estudos, mostrando uma dificuldade em identificar os fatores salivares que possam proteger ou intensificar a evolução do processo. Assim, o presente estudo se propôs a analisar as características clínicas, comportamentais e de alguns parâmetros salivares em dois grupos experimentais: pacientes que apresentam lesões cervicais não cariosas (LCNCs) (n=20) e um grupo controle (n=20). Foram coletados dados clínicos e comportamentais através de um exame clínico e de uma entrevista, a seguir amostras de saliva estimulada e não-estimulada foram coletadas e analisados: pH, capacidade tampão, fluxo salivar, concentração de proteínas totais, atividade da amilase salivar, concentração de ureia salivar e a concentração dos íons sódio, fósforo, potássio, magnésio e cálcio. A capacidade tampão foi medida pela titulação da saliva com uma solução de HCL 0,01N; o fluxo salivar se deu pela relação entre o volume de saliva e o tempo de coleta (ml/min); as concentrações de proteínas totais, ureia e a atividade da amilase foram determinadas por método colorimétrico; as concentrações dos íons cálcio, fósforo, magnésio, potássio e sódio foram determinadas por espectrometria de emissão óptica com plasma acoplado indutivamente (ICP-OES). Os resultados foram submetidos aos testes Qui-quadrado, teste t e Mann-Whitney (p<0,05). As características relacionadas aos hábitos de higiene dental, dieta ácida, hábitos parafuncionais, presença de distúrbios gástricos, secura bucal e prévio tratamento periodontal, não mostraram relação com a presença de lesões cervicais não cariosas. Os pacientes portadores de LCNCs se queixaram mais de sensibilidade dental (p=0,0014). Foi observado um maior número de lesões cervicais de pequena profundidade (79%), em formato de cunha (72%), apresentando hipersensibilidade dentinária (HD) (86%), localizados nos dentes posteriores (88,18%) e na maxila (66,14%), sendo os pré-molares os dentes mais afetados (56,69%). Os níveis de cálcio na saliva não-estimulada do grupo de pacientes com LCNCs foi significativamente maior em relação ao controle (p=0,041). A concentração de potássio na saliva estimulada foi significativamente maior no grupo controle (p=0,028). As variáveis fluxo salivar, pH, capacidade tampão, concentração de proteínas totais, ureia, amilase, sódio, magnésio e fósforo não mostraram diferenças estatisticamente significativas entre os dois grupos. Conclui-se que os fatores comportamentais não interferiram no aparecimento das lesões cervicais não cariosas. As LCNCs são pouco profundas, em formato de cunha, acometem mais dentes superiores e pré-molares e são acompanhadas de HD. As concentrações de cálcio e potássio podem interferir na formação das LCNCs. / Saliva is a biological fluid with an important role in the phenomena that occur in the oral cavity. The effect of salivary composition in loss of tooth structure with no carious origin, such as tooth wear, has been studied. However, there are conflicts in the results presented by studies showing a difficulty in identifying the salivary factors which may protect or intensify the development of tooth wear process. The aim of this study was to analyze the clinical, behavioral and some salivary parameters into two groups: patients with non-carious cervical lesions (NCCLs) (n=20) and a control group (n=20). Clinical and behavioral data were collected through a clinical examination and an interview. Saliva samples stimulated and unstimulated were collected and analyzed: pH, buffer capacity, flow rate, total protein, amylase activity, urea, sodium, phosphorus, potassium, magnesium and calcium. The buffer capacity was measured by titration of saliva samples using 0.01N HCl solution; flow rate was the relationship between the volume of saliva and the collection time (ml/min); the concentrations of total protein, urea and amylase activity were determined by colorimetric method; concentrations of calcium, phosphorus, magnesium, potassium and sodium were determined by inductively coupled plasma optical emission spectrometry (ICP-OES). The results were tested to the chi-square test, t-test and Mann-Whitney U test (p<0.05). The characteristics related to dental hygiene, acidic diet, parafunctional habits, presence of gastric disorders, dry mouth, and previous periodontal treatment did not show relationship to the presence of non-carious cervical lesions. Patients with non-carious cervical lesions complained over tooth sensitivity (p=0.0014). It observed an increased number of cervical lesions small depth (79%), wedge-shaped (72%), with dentin hypersensitivity (86%), located the posterior teeth (88,18%) and maxilla (66,14%), the premolars being the most affected teeth (56,69%). The concentration of calcium in unstimulated saliva of patients with NCCLs was significantly higher compared to control (p=0.041). The concentration of potassium was significantly higher in stimulated saliva in the control group (p=0.028). The results flow rate, pH, buffer capacity, total protein, urea, amylase, sodium, magnesium and phosphorus showed no significant differences between the two groups. It is concluded that behavioral factors do not interfere in the presence of non-carious cervical lesions. The NCCLs are shallow, wedge-shaped, affecting more maxilla and premolars and are accompanied by DH. The calcium and potassium concentrations may interfere with the formation of NCCLs.

O objetivo do trabalho foi determinar o efeito do pH de armazenamento (pH 3,5; 7,0 e 10,0) nos parâmetros de crescimento subcrítico (n e ? f0) de duas porcelanas odontológicas com diferentes microestruturas (com e sem leucita). A hipótese testada foi a de que o pH do meio afeta os parâmetros de crescimento subcrítico dos dois materiais. Duas porcelanas feldspáticas foram avaliadas: 1) Ultropaline Dentina (UD), JenDental/Ucrânia; 2) Veneer Material 7 (VM7), Vita/Alemanha. A porcelana UD é composta de matriz vítrea com dispersão de partículas de segunda fase de leucita, enquanto a porcelana VM7 apresenta exclusivamente fase vítrea. Espécimes em forma de disco com características específicas (diâmetro e espessura) foram sinterizados com os dois materiais seguindo o ciclo proposto pelos fabricantes. Os espécimes foram armazenados em diferentes soluções com pH específico (3,5; 7,0 e 10,0) por 10 dias antes de serem testados. Os parâmetros de fadiga foram obtidos pelo método de fadiga dinâmica utilizando o ensaio de resistência à flexão biaxial (“piston on three balls") com cinco taxas constantes de carregamento (0,01; 0,1; 1; 10 e 100 MPa/s). A tenacidade à fratura e a dureza foram avaliadas pelo método da fratura por indentação (IF). A microestrutura dos materiais foi analisada por meio de microscopia eletrônica de varredura, difração de raios X e análise química por fluorescência de raios X. O efeito do pH no coeficiente de susceptibilidade ao crescimento subcrítico (n) foi diferente para os dois materiais estudados. Os resultados mostraram que, para a porcelana vítrea (VM7), o valor de n não variou significativamente em função do pH. Para a porcelana com leucita (UD), houve uma diminuição do valor de n quando a armazenagem foi feita em pH 3,5. O efeito do pH no parâmetro escalar (? f0) também foi diferente para os dois materiais. Para a porcelana VM7, tanto o pH ácido como o básico resultaram em menores valores de resistência em relação ao pH neutro. Para a porcelana UD, somente a armazenagem em pH básico resultou em queda do valor de ? f0. Os valores de dureza dos dois materiais diminuíram conforme o pH foi aumentado, porém, somente a diferença entre o grupo armazenado a seco e o grupo armazenado em pH 10 foi estatisticamente significativa para as duas porcelanas. Com relação à tenacidade, o pH não influenciou significativamente nas médias obtidas para os dois materiais. Com base nos resultados foi possível concluir que o efeito pH do meio de armazenamento no crescimento subcrítico de porcelanas dontológicas varia de acordo com a microestrutura do material. / The objective of the study was to determine the effect of the pH of the storage medium (pH 3,5; 7,0 e 10,0) on the fatigue parameters (n and ? f0) of two dental porcelains with different microstructures (with and without leucite). The hypothesis tested was that the pH affects the fatigue parameters of both materials. The dental porcelains tested were: 1) Ultropaline Dentin (UD), JenDental/Ucraine; 2) Veneer Material 7 (VM7), Vita/Germany. UD contains leucite and VM7 is only vitreous. Disc specimens were produced with both materials following manufacturers’ firing cycles. The specimens were stored in water solutions with different pHs (3,5; 7,0 e 10,0) for 10 days and after that the fatigue parameters were obtained by means of the dynamic fatigue test. Hardness and fracture toughness were determined by means of the indentation fracture technique. Microstructural analysis of the materials was also carried out. The effect of the pH in the fatigue parameters was different for the two materials tested. The results showed that, for VM7, the n value did not vary as a function of the pH. For porcelain UD, the n value was lower when the specimens where stored in pH 3,5. The parameter ? f0 was also affected by the pH. For both materials, the storage in basic pH resulted in a decrease in the ? f0 value. The hardness of both materials decreased with the increase in the pH of the storage media, and fracture toughness was not affect by the pH. Based on the results, it was possible to conclude that the effect of the pH (of the storage media) on the fatigue parameters of the dental porcelains studied varied according to the porcelain’s microstructure.