RESUMO: O complexo Irre Recognition Module (IRM) é um importante subgrupo de proteínas transmembranares da superfamília das imunoglobulinas que desempenham função de adesão celular e participam de diversos processos do desenvolvimento de Drosophila melanogaster, tais como: direcionamento axonal, fusão de mioblastos, espaçamento dos órgãos sensoriais, autofagia das glândulas salivares, eliminação de células suplementares e especificação de destino celular na retina pupal. A família IRM é composta por quatro membros bem caracterizados: roughest (rst; irregular-chiasmC); kin-of-irre (kirre); hibris (hbs) e sticksand-stones (sns). Uma característica marcante entre dois membros deste grupo, kirre e rst, é a ocorrência de redundância funcional durante o processo de fusão de mioblastos na musculatura somática embrionária e na fase de \"cell sorting\" das células interomatidiais na etapa final de padronização da retina pupal de Drosophila. Neste contexto, foi observado que kirre pode suprir a ausência ou diminuição nos níveis de expressão de mRNA de rst para manter o fenótipo selvagem destes tecidos. Em glândulas salivares larvais de Drosophila, modelo amplamente utilizado para estudo da morte celular programada (MCP), mutantes rstD, um alelo semidominante de rst, apresentaram fenótipo de persistência deste tecido mesmo após 12 horas à sua eliminação observada em animais selvagens. Considerando o importante processo de redundância funcional promovido por membros do grupo IRM, durante o desenvolvimento de tecidos de Drosophila melanogaster, avaliamos a correlação entre os 4 membros deste grupo durante o processo de autofagia da glândula salivar larval. Para tanto, os níveis de transcrição de genes do complexo foram analisados por RT-qPCR após a formação do pupário até o desfecho do processo de histólise. Verificamos uma diminuição a nível transcricional de rst após o pico no título de ecdisona que leva a histólise da glândula em animais selvagens, e níveis alterados dos mRNAs dos membros do complexo em animais mutantes rstD. Contudo, a superexpressão de rst não foi suficiente para gerar glândulas persistentes. Além disso, descrevemos a localização espacial, por imunohistoquímica, dos membros do complexo, enfatizando a colocalização de rst e kirre, contrariamente aos membros sns e hbs. / ABSTRACT: The complex Irre Recognition Module (IRM) is an important subgroup of transmembrane proteins of the immunoglobulin superfamily that play a role in cell adhesion and participates in several processes of development of Drosophila melanogaster, such as: axonal targeting, fusion of myoblasts, spacing of sensory organs , autophagy of the salivary glands, elimination of supplementary cells and specification of cellular target in the pupal retina. The IRM family is composed of four well-characterized members: roughest (rst; irregular-chiasmC); kin-of-irre (kirre); hibris (hbs) and sticks-and-stones (sns). A striking feature of this group is the occurrence of functional redundancy during the process of fusion of myoblasts in the embryonic somatic musculature and in the phase of cell sorting of the interomatid cells in the final step of standardization of the pupal retina of Drosophila. In this context, it can be concluded that it can not provide an absence or decrease the mRNA expression levels of maintaining the wild tissue phenotype. In larval salivary glands of Drosophila, a widely used model for the study of programmed cell death (MCP), rstD mutants, a semidominant rst allele, showed persistence phenotype of this tissue after 12 hours for its observed elimination in wild animals. During the development of Drosophila melanogaster tissues, we evaluated a correlation between the 4 members of this group during the autophagy process of the salivary gland. To that end, transcription levels of complex genes were analyzed by RT-qPCR after patient formation until the end of the histolysis process. We have seen a transcriptional decrease in the first unpronounced postdoc peak of ecdysone leading to histolysis of the gland in wild animals, and altered levels of mRNAs of the complex members in rstD mutant animals. However, a first overexpression was not enough to generate persistent glands. In addition, we describe a spatial location, by immunohistochemistry, of two members of the complex, emphasizing a first and second hand colocalization, unlike the sns and hbs members.

As neoplasias de glândula salivar (NGS) são tumores raros que despertam interesse por sua diversidade histopatológica e comportamento clínico. A compreensão da patobiologia assim como, dos mecanismos envolvidos no comportamento invasivo destas lesões é necessária para melhor entender a biologia das NGS e posteriormente delinear novas estratégias terapêuticas. A presente tese foi dividida em dois artigos. O objetivo do primeiro estudo foi descrever os dados demográficos, clinicopatológicos e de prognóstico das NGS diagnosticados em um centro de atenção terciário. Para tal, foi realizada uma análise retrospectiva utilizando os dados de arquivos e de prontuários. Foram identificados 109 casos de NGS cuja média de idade dos pacientes foi de 46.47 anos e a relação homens:mulheres foi de 0.94:1. As glândulas salivares maiores foram mais acometidas (75.2%) e os tumores benignos os mais prevalentes (75.2%) sendo o adenoma pleomórfico o tumor benigno mais comum e o carcinoma adenóide cístico o principal maligno. O objetivo do segundo estudo foi analizar o padrão de expressão da via de sinalização do HGF/c-Me/PI3K em NGS e correlacionar com o perfi proliferativo e desfechos clínicos das lesões. Foram construídos microarranjos de tecido (TMAs) de 93 casos de NGs e as lâminas foram submetidas a análise imunoistoquímica para HGF, p-Met, p-Akt e Ki67. Foi observada maior expressão de HGF nos tumores benignos (p=0.04), enquanto que as protínas p-Met (p=0.03), p-Akt (p=0.00) e Ki-67 (p=0.00) foram mais expressas nos tumores malignos. Nas neoplasias malignas houve maior ativação da via HGF observada pela maior expressão do seu receptor fosforilado (p-Met) bem como, maior ativação da via do PI3k pela fosforilação de Akt (p-Akt) resultando em um maior perfil proliferativo. Pode-se concluir que a via de sinalização do HGF/c-Met/PI3k parece estar ativa nas NGS regulando a proliferação especialmente nas neoplasias malignas. / Salivary gland tumors (SGT) are rare yet interesting neoplasms due to their histopatological diversity and clinical behavior. Understanding the pathobiology as well as the mechanisms involved in the invasive behavior of these lesions is needed to better comprehend the biology of SGT and further delineate new therapeutic strategies. This thesis was divided in two papers. The aim of the first study was to describe the demographic, clincopathological and prognostic data of SGT diagnosed in a tertiary care center. For this purpose, a retrospective analysis using data from the archives and records was performed. One hundred and nine cases of SGT were identified. The patients mean age was 46.47 years and the male:female ratio was 0.91:1. The major salivary glands were the most affected (75.2%) and the benign SGT were more prevalent (78%) being pleomorphic adenoma the most common benign tumor and adenoid cystic carcinoma the most common malignant tumor. The objective of the second study was to analyze the expression pattern of HGF/c-Met/PI3K signaling pathway in SGT and correlate the findings with the proliferative profile and clinical outcomes of cases. Tissue microarrays (TMAs) of 93 cases of SGT were constructed; the slides were submitted to immunohistochemical analysis for HGF, p-Met, p-Akt and Ki-67. Increased expression of HGF was observed in benign tumors (p = 0.04), while p-Met (P = 0.03), p-Akt (p = 0:00) and Ki-67 (p = 0:00) were most expressed in malignant tumors. In salivary glands carcinomas there was a higher activation of the HGF pathway observed by the higher expression of its phosporylated receptor (p-Met) as well as the higher activation of PI3k pathway through Akt (p-Akt) phosphorilation, resulting in a higher proliferative profile. It can be concluded that HGF/c-Met/PI3K signaling pathway appears to be active in SGT regulating the proliferation specially in malignant tumors.

Orientador: Albina Messias de Almeida Milani Altemani / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T00:26:10Z (GMT). No. of bitstreams: 1 Neves_CatarinadeOliveira_D.pdf: 1050550 bytes, checksum: fc3ca7c67f2a52e8450896158a67f0d6 (MD5) Previous issue date: 2009 / Resumo: CD10 e Podoplanina (D2-40), além de expressos nas células mioepiteliais, estão envolvidos na progressão tumoral e podem ser utilizados como marcadores prognósticos. Em 79 neoplasias salivares com diferenciação mioepitelial (44 malignas e 35 benignas), analisamos a expressão dessas proteínas nas células neoplásicas, na reação desmoplásica tumoral e, nos carcinomas adenóides císticos (CAC), verificamos possível correlação com fatores prognósticos. CD10 foi negativo nas células epiteliais em 100% dos casos. Nas mioepiteliais, foi positivo em 25,71% das lesões benignas e em 27,27% das malignas, sendo esses resultados significantemente inferiores àqueles da a-SMA (60% e 88,64%, respectivamente). CD10 foi positivo em 83,33%, 30%, 27,7% e 40% dos carcinomas epiteliais-mioepiteliais (CEME), adenomas pleomórficos, mioepiteliomas e carcinomas mioepiteliais, respectivamente, e negativo em 100% dos CAC, adenocarcinomas polimórficos de baixo grau (APBG) e adenomas de células basais. No estroma tumoral, a expressão do CD10 (38,64%) foi significantemente maior (p=0.007) que a da a-SMA (11,36%). Entretanto, a expressão do CD10 não apresentou correlação com os fatores prognósticos do CAC. O D2-40 foi negativo, nas células epiteliais e estromais, e positivo nas mioepiteliais em 59% dos carcinomas e em 42,86% das lesões benignas. Concluímos que, ao contrário do D2-40, o CD10 tem pouca utilidade para identificar células mioepiteliais malignas, exceto no CEME onde pode ser útil no diagnóstico diferencial com a variante tubular do CAC. Sua expressão estromal ocorre em células de fenótipo distinto dos miofibroblastos, está associada com invasão tumoral e não se correlaciona com fatores prognósticos do CAC. / AbstrAct: CD10 and Podoplanin (D2-40) are expressed in myoepithelial cells and, in addition, are involved in tumoral progression and can be utilized as prognostic markers. In a series of 79 salivary neoplasias with myoepithelial differentiation (44 malignant and 35 benign), the expression of these proteins was analyzed in tumor cells as well as in tumor-associated stromal cells and it was correlated with prognostic factors in a select group of lesions (adenoid cystic carcinomas). In epithelial cells, CD10 was negative in 100% of the cases. In myoepithelial cells, CD10 was positive in 25.71% of the benign neoplasias and in 27.27% of the malignant ones and this expressions was significantly lower in comparison to that of a-smooth muscle actin (a-SMA) (60% and 88.64%, respectively). In neoplasias classified according to histological subtype, CD10 was positive in 83.33%, 30%, 27.27% and 40% of epithelial-myoepithelial carcinomas (EMC), pleomorphic adenomas, myoepitheliomas and myoepithelial carcinomas, respectively and negative in 100% of adenoid cystic carcinomas (ACC), polymorphic low-grade adenocarcinomas (PLGA) and basal adenomas. In tumor-associated stromal cells, CD10 expression was significantly higher (p=0.007) than that of a-SMA (38.64% versus 11.36%). However, no correlation was detected between CD10 expression and prognostic factors in ACC. D2-40 was negative in epithelial cells and in tumorassociated stromal cells as well and positive in myoepithelial cells of carcinomas (59%) and benign lesions (42.86%). This reactivity in myoepithelial cells did not differ significantly of that using a-SMA as a marker, except for PLGA where D2-40 was negative. In conclusion, CD10 differs of D2-40 once it shows low utility to detect neoplastic myoepithelial cells. However, EMC is an exception and in this tumor, CD10 could be useful to separate this lesion from tubular variant of ACC. In the tumor-associated stroma, CD10 expression in non-myofibroblast cells seems to be associated with tumor invasion but it does not show correlation with prognostic factors in ACC. / Doutorado / Anatomia Patologica / Doutor em Ciências Médicas

Considerando que o diabetes experimental altera a morfologia e função das glândulas salivares de ratos, o objetivo deste estudo foi analisar o efeito da irradiação com laser em baixa intensidade no metabolismo das glândulas parótidas (GP) e submandibulares (GSM) de ratas diabéticas, assim como analisar a glicemia destes animais. Ratas Wistar com aproximadamente 195 g foram divididas em diabéticas (D) e controles (C), e subdivididas de acordo com a dose de irradiação recebida: C-D0 (0 J/cm2), C-D5 (5 J/cm2), C-D10 (10 J/cm2), C-D20 (20 J/cm2). O diabetes foi induzido por injeção de estreptozotocina 60 mg/kg p.c., e confirmado após 72 h, através da glicemia. Vinte e nove dias após a indução, as ratas receberam a irradiação de acordo com o grupo a qual pertenciam. O laser de diodo (Ecco Fibras®), 660nm, foi utilizado nas áreas correspondentes às GP e GSM. Após 24 h, as ratas foram eutanasiadas e as glândulas removidas para posterior análise iônica, histológica e bioquímica de concentração total de proteína, atividade das enzimas catalase, peroxidase e amilase. Além disso, análise glicêmica também foi realizada. Análise de variância e teste de Tukey revelaram diminuição da glicemia nos grupos D5 e D20 (p<0,05), assim como diminuição na atividade da enzima catalase, nestas mesmas dosagens, nas GP e GSM para valores similares às ratas não-diabéticas (p>0,05), sendo que para as GSM, a dose de 10 J/cm2 também se mostrou efetiva. Análise histológica demonstrou acúmulo de lipídeos nas GP dos animais diabéticos, sendo que para os grupos que receberam irradiação, este acúmulo foi reduzido. Com base nos resultados, podemos concluir que a irradiação com laser vermelho nas GP e GSM altera a atividade enzimática da catalase, assim como altera a concentração de glicose sangüínea e o acúmulo de lipídeos. Com isto, mais estudos devem ser realizados objetivando continuar as análises de parâmetros antioxidantes, assim como, análise glicêmica, uma vez que a hiperglicemia e alterações no sistema antioxidante são diretamente relacionadas às complicações do diabetes. / Considering that experimental diabetes alters rat salivary gland morphology and function, the aim of this study was to evaluate the effect of laser irradiation (LI) on the glycaemic state and some biochemical parameters of the parotid glands (PG) and submandibulary glands (SMG) in diabetic and non diabetic rats, which were divided into 8 groups: D0/5/10/20 and C0/5/10/20, respectively. Diabetes was induced by administration of streptozotocin (60 mg/kg) and confirmed later by the glycaemia results. Twenty-nine (29) days after the induction, the PG and SMG of groups D-C5, D-C10 and D-C20, were irradiated with 5, 10 and 20 J/cm2 of laser diode (660nm/100mW) respectively. On the following day, the rats were euthanized and the blood glucose determined. The PG and SMG were removed and total protein concentration and amylase, peroxidase and catalase activities were performed, as well as, ionic and histological analysis. The analysis of the results (ANOVA and Tukey tests) revealed a decrease in the glycaemia level for groups D5 and D20 (p<0.05). In addition, diabetic rats showed higher catalase and peroxidase activities than non diabetic groups, without radiation. The doses of 5 and 20 J/cm2 decreased catalase activity, of PG and SMG, of diabetic groups to non diabetic values (p<0.05). Moreover, the dose of 10 J/cm2 also was effective to SMGs. Histological analysis showed accumulation of lipids droplets on parotid glands of diabetic animals. However, the lipid droplets quantity was lower in comparison with that observed to non irradiated diabetic animals. The results of the present study suggest that the classical organs which control glycaemia should also be analyzed after being irradiated with LI, once hyperglycaemia and alterations of the antioxidant system are the main causes of diabetes complications.

O objetivo do presente estudo foi o de analisar o efeito da irradiação com laser em baixa intensidade no sistema antioxidante enzimático de glândulas salivares submandibular (GSM) e parótida (GP) de ratas diabéticas induzidas por estreptozotocina. As ratas foram inicialmente divididas em grupos não-diabéticas (C) e diabéticas (D) e mantidas pelo período experimental de 30 dias. No vigésimo nono dia as ratas foram subdivididas em seis grupos, sendo três grupos de animais não diabéticos (C0, C5 e C20) e três de animais diabéticos (D0, D5 e D20), de acordo com a dose de irradiação laser que cada grupo recebeu (0, 5 e 20 J/cm2 respectivamente). Para a indução do diabetes foi realizada a injeção intraperitoneal de estreptozotocina (60 mg/kg de peso corporal) dissolvida em tampão citrato de sódio 0,1 M, pH 4,5. Os animais pertencentes aos grupos C (não diabéticos) receberam a injeção somente do veículo. As glicemias foram verificadas 72 horas após a indução do diabetes, para a confirmação do estado diabético nos grupos D. Foram considerados diabéticos os animais que apresentaram glicemia superior a 250mg de glicose/dl de sangue. A irradiação com laser em baixa intensidade seguiu a metodologia determinada pelo método de Simões et. al. (2009). Os animais foram eutanasiados 24 horas após a irradiação, sendo imediatamente removidas as glândulas salivares para a realização das análises bioquímicas. Foram determinados através de espectrofotometria, os valores de total antioxidante (TAS) e as atividades das enzimas superóxido dismutase (SOD), catalase (CAT) e glutationa peroxidase (GPx). Através da análise dos resultados podemos concluir que em GSM de ratas diabéticas o laser com dose de 20J/cm2 causou aumento na atividade da enzima SOD. E independente da dose, causou aumento nos valores de TAS e atividade da enzima CAT. Já em GSM de ratas não diabéticas a dose de 20J/cm2 causou diminuição dos valores de TAS. E independente da dose a irradiação com laser levou a um aumento da atividade da enzima CAT. Nas parótidas de ratas diabéticas, independente da dose, a irradiação com laser causou diminuição da atividade da enzima CAT. E em GP de ratas não diabéticas a dose de 5J/cm2 causou diminuição da atividade da enzima CAT. / The aim of the present study was to analize the effect of low-power laser irradiation in the antioxidant enzymatic system of submandibular (GSM) and parotid (GP) salivary glands of diabetic rats induced by streptozotocin. The rats were initially divided into non-diabetic animals (C) and diabetic-animals (D) and maintained by the experimental period of thirty days. Twenty-nine days after diabetes induction, the animals were randomly divided into six groups: three diabetic groups (D0, D5 and D20) and three non-diabetic groups (C0, C5 and C20), according with laser irradiation dose that each one received (0, 5 and 20J/cm2, respectively). For diabetes induction an intraperitoneal injection of streptozotocin (STZ) (60mg/Kg body weight), dissolved in 0.1M sodium citrate buffer, pH 4.5 was performed. In non-diabetic animals, only the citrate buffer was used. The diabetes condition was confirmed seventy-two hours after animals have received the STZ injection. Rats with blood glucose level higher than 14mM (250 mg/100ml) were considered diabetic. The laser irradiation was performed according to Simões et.al. method (2009). Twenty-four hours after the irradiation rats were euthanized. Then, immediately after the euthanasia, salivary glands were removed for biochemical analysis. The total antioxidant values (TAS) and the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GPx) enzymes were determined in spectrophotometer. Analyzing the results we can conclude that in GSM of diabetic rats the laser irradiation with 20J/cm2 increased the SOD activity. With the two different doses, increased the TAS values and CAT activity. However, in GSM of non-diabetic rats, laser irradiation with 20J/cm2, decreased the TAS values and led to an increase in CAT activity, regardless of the dose. In parotid glands laser irradiation decreased the CAT activity with either dose of 5J/cm2 or 20 J/cm2 and the laser irradiation with dose of 5J/cm2, decreased the CAT activity in parotid glands of non-diabetic rats.

Orientadores: Albina Messias De Almeida Milani Altemani, Elizabeth Ferreira Martinez / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T03:06:25Z (GMT). No. of bitstreams: 1 Montalli_VictorAngeloMartins_D.pdf: 4723030 bytes, checksum: 1764455ec3666d02c7e74ee7c326caed (MD5) Previous issue date: 2014 / Resumo: As lesões salivares tumorais compostas por dupla população celular (epitelial e mioepitelial) são consideradas originárias do ducto intercalado e estas lesões são subdivididas em diversas entidades que apresentam sobreposição morfológica, com delimitações entre elas nem sempre nítidas. Entre as neoplasias benignas estão o adenoma pleomórfico (AP) e o adenoma de células basais (ACB). Recentemente foi descrita uma nova entidade tumoral benigna, com composição epitelial e mioepitelial, denominada de lesão do ducto intercalado (LDI). Diante disso, o nosso primeiro objetivo foi analisar os perfis morfológicos e imuno-histoquímicos de LDIs e ACBs classificados em tubulares (ACB-T) e não tubulares (ACB-NT) para verificar se as LDIs e ACB-Ts representam entidades distintas. Ainda, dado o papel crítico da célula mioepitelial na morfogênese das lesões tumorais salivares histogeneticamente relacionadas ao ducto intercalado, nosso segundo objetivo foi avaliar in vitro a influência de fatores do microambiente tumoral (proteínas da matriz extracelular e fatores de crescimento) sobre a morfologia, viabilidade e proliferação de células mioepiteliais advindas de AP. Para a análise morfológica e imuno-histoquímica, foram estudados oito casos de LDIs, nove ACBs-T e 19 ACBs-NT. Todos os ACB-T continham áreas LDI-like, enquanto nos ACB-NT estas eram raras e escassas. As células luminais das LDIs e ACBs-T exibiram positividade para CK7, lisozima, S100 e DOG1. No grupo ACB-NT, poucas células luminais mostraram tal expressão, sendo principalmente positivas para CK14. As células mioepiteliais das LDIs, ACB-T e ACB-NT foram positivas para CK14, calponina, AML e p63, mas essas eram mais numerosas nos ACBs. No estudo in vitro, a morfologia e diferenciação das células mioepiteliais foram avaliadas qualitativamente por imunofluorescência indireta (expressão da vimentina e AML, respectivamente). As células mioepiteliais exibiram morfologia poliédrica em todas as matrizes, independentemente da suplementação do fator de crescimento. AML foi imunoexpressa de forma heterogênea nas células mioepiteliais, porém houve aumento da expressão desta proteína quando acrescentado o TGF- ?1, independentemente do tipo de matriz usada. TGF- ?1 também aumentou significantemente a viabilidade das células mioepiteliais cultivadas na matriz fibronectina. Conclusões: as LDI, ACB-T e ACB-NT formam um continuum de lesões onde as LDIs estão estreitamente relacionadas com o ACB-T, visto que em ambos o imunofenótipo das células luminais e mioepiteliais é semelhante àquele observado nos ductos intercalados. A principal diferença entre LDI e ACB-T é a quantidade de células mioepiteliais, que é maior no último. Além disso, nossos resultados indicam que pelo menos alguns ACBs podem surgir via LDI. Os estudos em cultura de células sugerem que as diferentes matrizes celulares não influenciam a morfologia e diferenciação da célula mioepitelial. Dentre os fatores de crescimento estudados apenas TGF- ?1 associou-se com aumento da expressão de AML (diferenciação celular) e aumentou significantemente a viabilidade celular associado à matriz fibronectina / Abstract: Salivary tumor lesions composed of dual cell population (epithelial and myoepithelial) are considered to originate from the intercalated duct. These lesions are subdivided into several entities that share morphological features. Among the benign tumors are pleomorphic adenomas (PA) and basal cell adenoma (BCA). Recently, a new entity was described that is a benign tumor with epithelial and myoepithelial composition, called intercalated duct lesion (IDL). Our first objective was to analyze the morphological and immunohistochemical profiles of IDLs and BCAs classified into tubular (T-BCA) and non-tubular subtypes (NT-BCA), to determine whether or not IDL and tubular BCA represent distinct entities. Also, given the critical role of myoepithelial cells in the morphogenesis of the salivary tumor lesions histogenetically related to the intercalated duct, our second objective was to evaluate in vitro the influence of tumor microenvironment factors (extracellular matrix proteins and growth factors) on the morphology, viability and proliferation of myoepithelial cells arisen from PA . Eight IDLs, nine tubular BCAs and 19 non-tubular BCAs were studied by immunohistochemical technique. All tubular BCAs contained IDL-like areas, which represented 20-70% of the tumor. In non-tubular BCA, IDL-like areas were occasional and small (<5%). One patient presented IDLs, tubular BCAs and IDL/tubular BCA combined lesions. Luminal ductal cells of IDLs and tubular BCAs exhibited positivity for CK7, lysozyme, S100 and DOG1. In the non-tubular BCA group, few luminal cells exhibited such immunoprofile; they were mainly CK14-positive. Basal/myoepithelial cells of IDLs, tubular BCAs and non-tubular BCAs were positive for CK14, calponin, ?-SMA and p63; they were more numerous in BCA lesions. The in vitro study analyzed morphology and differentiation of myoepithelial cells by vimentin and SMA expressions, respectively, which were qualitatively assessed using indirect immunofluorescence. Myoepithelial cells showed polyhedral morphology in all extra cellular matrixes regardless of the supplementation of growth factors. These cells expressed SMA heterogeneously but when TGF- ?1 was added such expression increased. This modification did not show relationship with the type of extracellular matix. The viability of myoepithelial cells cultured on fibronectin matrix increased significantly with addition of TGF - ?1. Conclusions: IDL, tubular BCA and non-tubular BCA form a continuum of lesions in which IDLs are related closely to tubular BCA. In both, the immunoprofile of luminal and myoepithelial cells recapitulates the normal intercalated duct. The difference between the adenoma-like subset of IDLs and tubular BCA rests mainly on the larger numbers of myoepithelial cells in the latter. Our findings indicate that at least some BCAs can arise via IDLs. The cell culture studies suggest that the different matrixes do not influence the morphology and differentiation of myoepithelial cells. Among the growth factors studied, only TGF - ?1 was associated with an increased expression of SMA (cell differentiation) and a significant increase of the cellular viability associated with the fibronectin matrix / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Sabendo que o conhecimento do sistema estomatognático é de fundamental importância para que os clínicos e cirurgiões cheguem a um diagnóstico preciso e instituam o tratamento adequado, propôs-se o presente estudo, que consistiu na dissecção da cabeça de quatro cadáveres caninos e quatro felinos. O objetivo foi a descrição e documentação das estruturas musculares e tegumentares detalhando a origem e inserção, inervação e função de cada estrutura dos tecidos moles bem como a descrição completa das glândulas salivares para a aplicação clínico-cirúrgica na prática odontológica de cães e gatos. / Concerned about the knowledge of the stomatognatic system as of fundamental importance to the practitioner and surgeons to reach a precise diagnosis and institute the appropriate treatment, the present study was proposed, consisting in the dissection of the head of four canine and four feline cadavers. The objective was the description and doccumentation of the muscular structures and tegument, detailing the origin and insertion, inervation and function of each structure of the soft tissue as well as the complete description of the salivary glands for a clinical-surgical application in dentistry.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / C. Michael Smith / The tools for arthropods with sucking-mouth parts to attack hosts are mainly in the saliva. For plant-sucking insects, these salivary secretions are primarily produced in the salivary glands. Secreted proteins (also referred to as salivary gland secretomes) are among the important components in the saliva of sucking insects. Gall midges (Cecidomyiidae), a large family of plant-sucking insects, apparently secrete proteins (some of them are effector proteins) into host tissues, inducing various forms of plant outgrowth (galls). Three major insect pest species in the genera Mayetiola, the stem gall midges, are known to produce saliva that can reprogram plant cells and manipulate the host plant growth, causing serious damage to the plants of small grains. The three pest species are the Hessian fly (Mayetiola destructor), the barley midge (Mayetiola hordei), and the oat midge (Mayetiola avenae). Another economically important species of this gall midge family is the wheat midge (Sitodiplosis mosellana). It is a major insect pest of spring wheat and feeds on wheat heads, causing damage to the developing wheat seeds. A global analysis of the salivary gland secretome of first instar larvae of the Hessian fly, (a member of Mayetiola and) a model species for studying insect-plant interactions, has previously revealed a large number of genes encoding Secreted Salivary Gland Proteins, so called SSGPs. For comparison, we conducted analyses on transcripts encoding SSGPs from salivary glands of the first instar larvae of the wheat midge, barley midge, and oat midge. In the first chapter, a transcriptomic analysis of wheat midge has been conducted. In this analysis, a total of 3,500 cDNA clones were sequenced, and 1,301 high quality sequences were obtained and approximately 25% of the cDNAs (with high quality sequences) encoded SSGPs. The SSGPs were grouped into 97 groups based on sequence homology. Among the SSGP-encoding transcripts, 206 encoded unique proteins with no sequence similarity to any known protein and 29 encoded proteins similar to known proteins including proteases, serpines, thioesterases, ankryins, and feritins. The compositions of SSGP transcripts from the wheat midge were then compared with that of Hessian fly. The analyses have identified many common characteristics between the species. Despite these commonalities, no sequence similarity was found between SSGPs from wheat midge and those from Hessian fly, suggesting that SSGPs from these two insect species perform different functions to manipulate host plants. The second chapter contains results of comparative transcriptomic analyses on the barley and oat midges. A total of 2570 cDNA clones were sequenced from the barley midge, and 743 were high quality cDNA sequences, and the analysis identified 458 cDNA clones encoding SSGPs, of these, 178 encoded unique proteins (also called unigenes). Transcripts encoding SSGPs were grouped into 51 groups based on sequence homology. A total of 3226 cDNA clones were sequenced from oat midge, and 718 cDNA sequences were high quality and used for further analysis. The analysis identified 450 cDNA clones encoding SSGPs. Among the SSGP-encoding transcripts, 194 are unigenes, which were placed into 50 groups. The compositions of SSGP transcripts from the barley and oat midges were then compared with that of Hessian fly. The analysis identified five groups containing 102 (57.3%) unigenes from barley midges and seven groups containing 107 (55.1%) unigenes from oat midges which encode SSGPs that are conserved among the three species. The SSGPs conserved among the three midges are from family one (SSGP-1), family 4 (SSGP-4), family 11 (SSGP-11), and family 71 (SSGP-71). The SSGPs conserved among the three species indicate conserved functions such as a role in plant manipulation. Some SSGP unigenes were found to be conserved between only two species. Specifically, there were eight gene groups which are conserved between two species. Within these eight groups 19 (10.7%) unigenes from the barley midge and 25 (12.9%) unigenes from the oat midge were found to be conserved between only the barley and oat midges, whereas no homologues have been found in the Hessian fly. The remaining unigenes encode SSGPs that are unique to different midge species. The highly divergent SSGP groups that have been identified with no homology among the three midges indicate potential roles of these SSGPs in host specification. Due to the important roles of effector proteins in insect-plant interactions for gall midge species and since no insect effector protein have been identified directly from infested plant tissues so far, I have chosen one of the SSGP family, SSGP-1, which are conserved among all three gall midge species, for further analysis in chapter 4. Members in family SSGP-1 are also the most abundantly expressed at the transcript level. Based on Hessian fly data, family 1 contains seven genes and are named SSGP-1A1, SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1C2, SSGP-1D1, and SSGP-1E1. To detect the presence of these proteins in the infested wheat tissues, and to identify probable targets from wheat that interact with the SSGPs in the feeding site, we have generated and purified recombinant proteins for five of the seven proteins, namely SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1D1, and SSGP-1E1 (since SSGP-1A1 and SSGP-1C2 are very similar to SSGP-1A2 and SSGP-1C1, respectively). Antibodies were produced for the recombinant proteins for western blot analyses and indirect immunostaining. Immunostaining on dissected tissues including salivary glands, guts, and Malpighian tubules from 3-day old larvae, was conducted with antibodies against the five SSGPs, and detected a specific localization of all proteins in salivary glands except SSGP-1E1, which exhibited a weak signal in the foregut, in addition to localization in salivary glands. Western blot analyses demonstrated that these five proteins were expressed in larvae at all stages. The continuous production of these proteins suggests that they play roles in initiation and maintenance in Hessian fly infestation. Consistent with their effector functions, these five proteins were detected for the first time in infested wheat tissues based on western blot analyses. To identify possible target proteins from host plants that interact with SSGP-1 family proteins, in vitro pull-down assays were performed. Putative interacting targets for SSGP-1A2, SSGP-1B1, and SSGP-1C1 have been identified by LC-MS/MS. These putative interaction target proteins included uncharacterized proteins, ribosomal proteins, a lipoxygenase, and a tubulin. Identification of these putative targets provided a base for further confirmation of their interaction with Hessian fly effectors in the future.

Orientador: Pablo Agustin Vargas / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-26T11:33:31Z (GMT). No. of bitstreams: 1 Fonseca_FelipePaiva_D.pdf: 1634857 bytes, checksum: 43da6be9627bec5197103495826aa6d6 (MD5) Previous issue date: 2015 / Resumo: Tumores de glândulas salivares correspondem a aproximadamente 3% de todas as neoplasias de cabeça e pescoço e as neoplasias malignas derivadas destas estruturas anatômicas ainda representam um grande desafio para a oncologia de cabeça e pescoço devido a sua difícil abordagem cirúrgica e pobre resposta às outras abordagens terapêuticas. Um melhor entendimento do seu perfil molecular contribuiria significativamente para um melhor manejo terapêutico futuro e o estudo do potencial angiogênico dos tumores de glândulas salivares representa um interessante alvo de investigação. Tem sido demonstrado que as semaforinas induzem a apoptose de células tumorais, modulam a migração celular neoplásica e inibem a angiogênese em diferentes neoplasias humanas, competindo com o fator de crescimento endotelial vascular (VEGF) pela ligação aos seus principais receptores, as neuropilinas-1 e -2, desta forma inibindo os efeitos mitogênicos e pró-angiogênicos de VEGF. Assim, o objetivo deste estudo é investigar a expressão das semaforinas de classe 3 A e B (Sema3A e Sema3B), e dos seus receptores neuropilinas-1 e -2 (Np-1 e Np-2) em tumores de glândulas salivares, determinando seus significados clínicos. Duzentos e quarenta e oito tumores benignos e malignos de glândulas salivares selecionados de quatro instituições brasileiras foram organizados em blocos de parafina em microarranjo tecidual em matriz e submetidos a reações de imunoistoquímica contra CD34, Sema3A, Sema3B, Np-1 e Np-2. As imunoreações foram quantificadas utilizando algoritmos digitais e os resultados foram correlacionados com parâmetros clinicopatológicos e índices de sobrevida. Tumores malignos apresentaram uma maior densidade vascular, porém uma menor área vascular do que sua contraparte benigna. Em glândulas salivares normais a expressão de Np-1 e -2 esteve restrita às células ductais, enquanto que Sema3A e Sema3B estiveram principalmente no componente acinar. Tumores benignos e malignos revelaram uma expressão similar de todos os marcadores e a co-expressão de Np-1/Np-2 correlacionou-se significativamente com a ocorrência de parestesias e estágios mais avançados dos tumores. Apesar de não ser estatisticamente significativa, a sobre-expressão simultânea de ambos os receptores também indicou uma menor taxa de sobrevida. Desta forma, Sema3A, Sema3B, Np-1 e Np-2 devem estar envolvidas no desenvolvimento das glândulas salivares normais e na patogênese das neoplasias benignas e malignas derivadas destas estruturas; entretanto, a expressão destas proteínas não apresentou um potencial prognóstico estatisticamente significativo no presente estudo / Abstract: Salivary gland tumors correspond to approximately 3% of all head and neck neoplasms and the malignant neoplasias derived from these anatomic structures still represent a major pitfall in head and neck oncology because of their difficult surgical approach and poor response to other therapies. A better understanding of their molecular basis would significantly aid to an improved future management and the study of salivary gland tumors angiogenic potential represents an interesting target of investigation. It has been shown that semaphorins induce tumor cell apoptosis, modulate tumor cell migration and inhibit angiogenesis in different human neoplasms, competing with vascular endothelial growth factor (VEGF) for biding to their main receptors, the neuropilins-1 and -2, thereby inhibiting mitogenic and pro-angiogenic effects of VEGF. Hence, the objective of this study is to investigate the expression of class 3 Semaphorins A and B, and their receptors neuropilins-1 and -2 in salivary gland tumors, determining their clinical significance. Two hundred and forty eight benign and malignant salivary gland tumors selected from four Brazilian institutions were organized in tissue microarray paraffin blocks and submitted to immunohistochemical reactions against CD34, Sema3A, Sema3B, Np-1 and Np-2. The immunoreactions were quantified using digital algorithms and the results were correlated with clinicopathological parameters and survival rates. Malignant tumors presented an increased vascular density but a lower vascular area than their benign counterparts. In normal salivary glands Np-1 and Np-2 expression was restricted to ductal cells, whereas Sema3A and Sema3B were positive mainly in serous acinar compartment. Benign and malignant tumors revealed a similar expression of all markers and the co-expression of Np-1/Np-2 significantly correlated with the occurrence of paresthesia and higher stages of the tumors. In addition, although not statistically significant, simultaneous overexpression of both receptors also indicated an inferior survival rate. Hence, these results suggest that Sema3A, Sema3B, Np-1 and Np-2 may be involved in the development of normal salivary glands and in the pathogenesis of benign and malignant neoplasms derived from these structures; however, the expression of these proteins did not present a statistically significant prognostic potential in the current study / Doutorado / Patologia / Doutor em Estomatopatologia

The author of this work deals with the problems of the sialolithiasis. He summarizes current knowledge of the etiology of the disease, diagnostic and therapeutic procedures. There are described conventional therapeutic approaches, but the main part is dedicated to current practices which are used after the introduction of new technology-sialoendoscopy. The endoscopic technique is described in this work, the evaluation of its benefits, but also its limits. The author's efforts for conservative approach to the issue sialolithiasis, the exact localization of the sialith and extraction with preservation of the salivary gland is based on publications in the scientific literature. He documents this procedure with the resultes of his own research. There is evaluated using sialoendoscopy in treatment of the sialolithiasis in cases where the sialolith is located in hilum of the submandibular gland. This localization was in the past often an indication for extirpation of the submandibular gland. The author statistically rates files of extirpated submandibular glands in the author's workplace in two five-years periods. The first period is in the time when the sialoendoscopy is not used for examination and treatment of the sialolithiasis yet. In the second period the sialoendoscopy is available, there is also...