Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 60,000 annual diagnoses in the U.S. and approximately 500,000 worldwide. About 90% of these individuals receive radiation therapy, and salivary hypofunction and xerostomia occur in 60-85% of these patients due to irreversible damage to the salivary glands. Current preventative and palliative care fail to improve quality of life, accentuating the need for regenerative therapies. Stem/progenitor-cell based therapies have been proposed to regenerate the irradiated glands; however, the identity of stem and progenitor cells in the adult salivary glands has remained somewhat elusive. Moreover, it is unclear how salivary progenitors respond to radiation and whether they can be stimulated to effectively reinstate salivary function. The second chapter of the present study describes the development of a label-retaining assay in salivary glands using EdU. The label-retaining cells (LRCs) identified in murine salivary glands have proliferative potential in vitro and expressed markers of putative salivary progenitors, such as Keratin 5, Keratin 14, and c-Kit. Interestingly, LRCs were still present 30 days following radiation, when chronic loss of saliva is evident. The significance of these findings lies in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining salivary gland homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. In the following chapter, we show that a unique population of murine salivary gland LRCs undergo compensatory proliferation in response to radiation. The initiation of compensatory proliferation is tightly associated with inactivation of the kinase aPKCζ and increased nuclear localization of YAP. This part of the study provides novel insights into the regulation of function of salivary gland progenitors, which can be utilized for the development of therapeutic agents to treat salivary hypofunction. Finally, the last chapter describes how the mechanisms found to initiate compensatory proliferation in acinar LRCs as a response to radiation are involved in the regeneration of salivary glands with IGF-1. Administration of IGF-1 post-radiation restores salivary function in mice, but the mechanisms of regeneration are still unknown. Here, we show that IGF-1 requires aPKCζ to restore saliva production. Further, IGF-1 inhibits nuclear translocation of Yap in an aPKCζ-dependent fashion. We propose that a tightly regulated balance in the levels of aPKCζ and Yap in acinar LRCs has to be maintained in order to restore function following radiation. In conclusion, the findings from this study provide new knowledge in regards to the regulation of function of salivary progenitors during a state of injury (by radiation) and during regeneration (with IGF), and offer potential targets of study for the development of new therapeutics for salivary gland dysfunction. Future studies will determine whether aPKCζ and Yap can be effectively targeted in salivary progenitors to restore salivary function in head and neck cancer patients who receive radiation therapy.

Doctor of Philosophy / Entomology / Yoonseong Park / Tick salivary secretion is crucial for survival and for successful feeding. Tick saliva includes excretory water/ions and bioactive components for compromising the hosts' immune responses, and provides a direct route for pathogen transmission. Control of the tick salivation involves autocrine/paracrine dopamine, the most potent stimulator of tick salivation. Our research group reported the presence of two dopamine receptors in the salivary glands of the blacklegged tick (Ixodes scapularis): dopamine receptor (D1) and invertebrate specific D1-like dopamine receptor (InvD1L). Dopamine-induced salivary secretion was orchestrated by two distinct physiological roles via activation of the two dopamine receptors (Chapter 2). Low concentration of dopamine activated D1 receptor on epithelial cells of salivary gland acini leading inward fluid transport. High concentration of dopamine activated InvD1L receptors on axonal projections innervating myoepithelial cells modulating pumping/gating actions for emptying luminal saliva into the main duct. Thus, ticks coordinated salivary secretion with duo dopamine receptors. Dopamine-mediated saliva production involves an important downstream component, Na/K-ATPase (Chapter 3). Na/K-ATPase was found in the epithelial cells of all types of acini. However, Na/K-ATPase had two different functions in salivary secretion in different acini: 1) dopamine-mediated production of primary saliva in distally located salivary gland acini type-2/- 3, and 2) dopamine-independent resorption in proximally located salivary gland acini type-1. Type-1 acini were also found to function in direct water absorption of off-host ticks, which could be a potential route for delivery of acaricides. Chapter 4 investigated the comparative transcriptomics of the lone star tick underlying the processes of pathogen acquisition. Differential expression analyses in pathogen-exposed ticks revealed a number of transcripts that are important in the tick-pathogen interaction. These included genes for tick immunity against pathogen and for modulation of tick physiology facilitating a pathogen’s invasion and proliferation. My study expanded the understanding of physiological mechanisms controlling tick salivation. In addition, transcriptomics of ticks in interaction with pathogen identified several genes that are relevant in vector/pathogen interactions. The knowledge obtained in my study will facilitate to the development of novel methods for the disruption of tick feeding and pathogen transmission.

As glândulas salivares são estruturas essenciais para a manutenção da homeostase da cavidade oral pela síntese e secreção do fluido salivar. A disfunção ou perda permanente das glândulas salivares causadas por radioterapia, doenças inflamatórias ou desordens congênitas elevam principalmente o risco de infecções da mucosa oral e de estruturas dentárias, além de potencialmente prejudicar funções fisiológicas como fala, mastigação e paladar, diretamente interferindo na qualidade de vida dos indivíduos afetados. Os tratamentos atualmente disponíveis são apenas paliativos, ressaltando a necessidade de se compreender melhor os processos embriogênicos a fim de desenvolver novas estratégias terapêuticas capazes de regenerar as glândulas salivares. O princípio da formação das glândulas salivares baseia-se na coordenação de diversos processos morfogenéticos, e este trabalho foca particularmente em investigar a formação do espaço luminal do sistema de ductos, uma vez que a adequada abertura dos lumens é um processo essencial para a secreção salivar. Relata-se que a remoção das células centrais dos cordões sólidos epiteliais por morte celular apoptótica é o principal mecanismo de abertura do espaço luminal dos futuros ductos glandulares em camundongos. Porém, pouco se sabe sobre o controle temporal da apoptose durante o desenvolvimento glandular e sobre seu comportamento em glândulas salivares humanas. Neste trabalho, o perfil de expressão de diversas proteínas envolvidas na cascata apoptótica em glândulas salivares fetais humanas foi analisado de acordo com cada estágio morfogenético por imunoistoquímica (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x, Bcl-xL, caspase-3 clivada, caspases-6, -7 e -9, apaf-1, survivina e citocromo c). As análises semi-qualitativas resultaram em negatividade apenas para as proteínas Bcl-2, Bad, Bid e caspase-3 clivada em todas as fases de desenvolvimento. A expressão nuclear de Bax e Bak foi identificada em presumidos espaços luminais em estágios precoces, enquanto Bcl-xL foi o fator antiapoptótico da família Bcl-2 que exibiu expressão nuclear mais importante. Caspases-6, -7 e -9 foram positivas em todas as fases, e a ausência de caspase-3 clivada sugere caspase-7 como principal caspase efetora da apoptose em desenvolvimento de glândulas salivares humanas. Ambos os componentes do complexo apoptossomo foram positivos durante o desenvolvimento glandular, e o inibidor survivina demonstrou mais positividade nuclear em estágios mais avançados. Ao observar a expressão de reguladores apoptóticos durante o desenvolvimento glandular humano, foram realizados experimentos funcionais com culturas de tecido glandular de camundongos para avaliar o papel das caspases durante a formação desta estrutura. Inicialmente detectou-se a atividade apoptótica em glândulas salivares de camundongos albinos no centro dos cordões epiteliais primários a partir de estágios precoces de desenvolvimento através de TUNEL e caspase-3 clivada. A partir disso, foi realizada a inibição apoptótica funcional in vitro durante o mesmo período, que resultou em ductos significativamente mais amplos e em defeitos morfológicos importantes nas estruturas luminal e acinar. Este trabalho evidenciou portanto atividade apoptótica durante a formação de glândulas salivares humanas e de camundongo, expressando-se em fases mais precoces do que reportadas anteriormente. Além disso, a ausência de Bad e Bid indica que a via intrínseca está mais ativa que a extrínseca, e distintos perfis de expressão da maioria das moléculas sugere adicionais funções não-apoptóticas durante a morfogênese glandular. / Salivary glands are essential structures for the maintenance of homeostasis of the oral cavity by synthesizing and secreting saliva. Permanent dysfunction or loss of salivary glands caused by radiotherapies, inflammatory diseases or congenital disorders increase mainly the risk of infections of the oral mucosa and tooth surface, also impairing physiological functions as speech, mastication and taste, directly interfering in quality of life. Current treatments are only palliative-based, which highlights the need of having a better understanding of embryonic processes to develop therefore new therapeutic strategies able to regenerate salivary glands. The development of glandular secretory units and ductal system involves the coordination of several morphogenetic processes, and this study particularly focuses in investigating the formation of the lumenal space of the ductal system, as the proper lumen opening is an essential step for the salivary secretion. The clearance of the central cells of developing solid epithelial stalks by apoptotic cell death is the main mechanism of lumen space opening within presumptive ducts in mouse salivary glands. However little is known about its temporal regulation and its function in human salivary glands. Here we analysed the profile expression of several apoptosis-related proteins during human salivary gland development in correlation to each morphogenetic stage by immunohistochemistry (Bax, Bak, Bad, Bid, Bcl-2, Bclx, Bcl-xL, cleaved caspase-3, caspases-6, -7 e -9, apaf-1, survivin e citocromo c). Immunohistochemical results were analysed semi-qualitatively, and proteins Bcl-2, Bad, Bid and cleaved caspase-3 were considered completely negative at all stages of development. The nuclear expression of Bax and Bak were observed within the presumptive luminal spaces at early stages, while Bcl-xL was the antiapoptotic factor of Bcl-2 family that showed more prominent nuclear expression. Caspases-6, -7 and -9 were positive at all stages, and the absence of cleaved caspase-3 suggests caspase-7 as the main effector caspase during human salivary gland development. Both components of the apoptosome complex were also positive through all development, and the inhibitor of apoptosis survivin has shown more nuclear positivity at later stages. As the expression of apoptotic regulators was observed during human salivary gland development, functional experiments were then performed in mouse salivary gland cultures to determine the apoptotic activity of during the glandular formation. Initially, the apoptotic activity was detected in mouse salivary glands within the centre of primary epithelial stalks from early stages of development by TUNEL and cleaved caspase-3. Thus the in vitro apoptotic inhibition was performed at the same stages, which resulted in significant wider ducts and important morphological defects within luminal and acinar structures. This work has therefore evidenced the existence of apoptotic role in salivary gland lumen formation of both human and mouse models, having an earlier start point as reported before. Moreover, the absence of Bad and Bid indicates that the intrinsic pathway is more active than the extrinsic during human development, and the distinct subcellular expression of most molecules suggests additional non-apoptotic functions.

Histonas de glândulas salivares de larvas de Rhynchosciara americana foram extraídas de preparações de núcleos e de cromatina purificada e analisadas por eletroforese em géis de acrilamida. As mobilidades eletroforéticas das histonas ARE e GRR são idênticas e as das histonas KAP, LAR e KAS são levemente diferentes das histonas correspondentes encontradas em vertebrados. É particularmente interessante o comportamento da histona KAP de R. americana, que possui em géis de poliacrilamida mobilidade menor que a histona correspondente do timo de bezerro. Verificou-se, por análise em géis de poliacrilamida-SDS que o peso molecular desta histona é igual a 33.000 daltons, valor maior que o da histona correspondente de timo de bezerro e de outros vertebrados (21.000 a 25.000 daltons). Este resultado coincide com dados obtidos por outros investigadores em Drosophila e Acheta. Isto sugere que o maior peso molecular da histona KAP possa ser uma característica geral dos insetos. Verificou-se, por experimentos da dupla marcação com lísina- 14 C e triptofano-3 H, que a síntese de histonas em glândulas salivares de larvas de R. americana, ocorre no citoplasma em pequenos polirríbossomos contendo 3-4 ribossomos. Estes polirribissomos são particularmente ativos na síntese de histonas por ocasião da abertura máxima do puff B-2 na extremidade proximal da glândula,quando é máxima a síntese de DNA. O estudo da síntese das várias frações de histonas durante os 6-7 dias finais do período larval mostra um estreito acoplamento entre a síntese dessas frações e a do DNA. Não foram detectadas variações relativas na síntese de nenhuma das frações de histonas durante este período. Os resultados deste estudo sugerem também que algumas das frações de histonas são modificadas durante o desenvolvimento. Estas modificações podem ser correlacionadas com o aparecimento dos \"puffs de DNA\" nos cromossomos das células glandulares. Foi também estudada a síntese de histonas em larvas jovens (fim do segundo período do 4o estádio) injetadas com ecdisterona. Observou-se, conforme esperado de resultados anteriores, que a injeção do hormônio causou grande estímulo na síntese de DNA. Contudo, não houve estímulo apreciável na síntese de histonas. Estes resultados sugerem que a cromatina formada nestas condições é deficiente em histonas. Nestas experiências notou-se também, que após tratamentos com hormônio durante 44 horas ocorre uma profunda modificação nos perfis de radioatividade de histonas recém sintetizadas analisadas em géis de poliacrilamida. Sugere-se que a alteração desses perfis se deva à modificação química de alguma histona induzida pelo hormônio. / The histones of the salivary gland of Rhynchosciara americana larvae were extracted from both nuclearand purified chromatin preparations and then analyzed by polyacrylamide gel electrophoresis.The electrophoretic mobilities of the ARE and GRK histones are identical and those from the KAP, LAK and KAS histones are slightly different from the corresponding calf thrymus histones The behavior of the Rhynchosciara KAP histone is particularly interesting. In normal polyacrylamide gels it has a smaller mobility than that observed for the same protein from calf thymus.It was found by analysis in SDS-polyacrylamide gels that the molecular weight for this histone is approximately 33000 dalton, a value which is higher than that found for the same histone obtained from calf thymus and other vertebrates (21-250 00 dalton). This result is in agreement with those observed in Drosophila and Acheta, and suggests that the molecular weight of the KAP histone could be a characteristic feature for insects. Double label experiments with 14C-lysine and 3H-tryptophan show that histone synthesis in Rhynchoschiara salivary glands occurs in the cytoplasm in small polyribosomes containing 3-4 ribosomes. These polysomes are particularly active in the synthesis of these proteins at the time when the degree of opening of the 2B puff is maximal in the proximal cells of the gland, i.e when the DNA synthesis in the gland reach a maximum. The study of the synthesis of the different histone fractions during the last 6-7 days of the larval period shows a tight coupling between the synthesis of these fractions and that of DNA. However, variations in the relative proportions of those proteins during this period of larval development was not detected. The results of this study also suggest that a histone or histones are chemically modified during the development. These modifications can be correlated with the appearance of DNA puffs in the salivary gland. The study of histone synthesis was also carried out with younger larvae (end of the 2nd period of the 4th instar) treated with ecdysterone. It was found, as expected from previous results, that the hormone caused a great increase in the rate of DNA synthesis in the gland. Despite this, there was not an appreciable increase in the amount of newly synthesized histones which can be extracted from gland chromatin. These results indicate that the chromatin formed in the gland under these conditions might be deficient in histones In these experiments it was observed a great modification in the radioactivity profile of newly synthesided histones as analysed by polyacrylamide gel electrophoresis. This change occurs 44 hours after ecdysterone treatment. Tentatively it is suggested that this alteration in the radioactivity profiles would be a result of a chemical modification of some histone fraction which was induced by the hormone

A família Malpighiaceae é típica no Cerrado brasileiro; a maioria de seus gêneros possuem elaióforos na base de suas sepálas que secretam óleo como recompensa aos seus polinizadores. Objetivou-se caracterizar os elaióforos e folhas de Byrsonima coccolobifolia Kunth e Byrsonima verbascifolia (L) DC, em três áreas de cerrado stricto sensu, presentes nas fazendas Canaã, Providência e São Judas Tadeu. Neste estudo morfoanatômico, foram realizadas micromorfologia nos elaióforos, anatomia nos elaióforos e folhas, e micromorfometria nas folhas. As análises micromorfológicas dos elaióforos, nas diferentes áreas de Cerrado, demonstraram que esta estrutura mantém seu padrão oval e séssil, como um conservadorismo floral, o que garante o sucesso reprodutivo dessas espécies, o que foi observado nas três áreas estudadas Canaã, Providência e São Judas Tadeu. As folhas apresentaram diferenças anatômicas frente às diferentes áreas estudadas, para B. coccolobifolia a presença de bainha fenólica nas populações de Providência e São Judas Tadeu, e em B. verbascifolia a bainha esclerificada nas plantas de Canaã, Providência e São Judas Tadeu, ambas agem contra a perda de água em ambientes onde o solo possui pouca retenção de água, característica encontrada no solo de Providência. A análise micromorfométrica revelou diferenças no parênquima esponjoso de B. coccolobifolia, nas plantas de São Judas Tadeu, demonstrando que a maior espessura desse tecido facilita a difusão do CO2, e a epiderme abaxial mais espessa em B. verbascifolia, típica de plantas que estão expostas à alta luminosidade. Nas plantas de B. verbascifolia, da população Providência, o parênquima paliçádico foi menor, devido sua distribuição em áreas mais sombreadas. Características xeromórficas como: cutícula espessa, tricomas e muitos estômatos, demonstram que estas espécies estão bem adaptadas ao Cerrado. / The Malpighiaceae family is typical in the Brazilian Cerrado; most of their genera have elaiophores in the basis of its sepals that secretion oil like reward to its pollinators. The objective was to characterize the elaiophores and leaves of Byrsonima coccolobifolia Kunth and Byrsonima verbascifolia (L) DC, in three areas of cerrado stricto sensu, present in the farms Canaã, Providência and São Judas Tadeu. In this morpho-anatomical study, micromorphology was performed on the elaiophores, anatomy on the elaiophores and leaves, and micromorphometry on the leaves. The micromorphological analysis of elaiophores in the different areas of Cerrado showed that this structure maintains its oval and sessile pattern, as a floral conservatism, which guarantees the reproductive success of these species, which was observed in the three studied areas Canaã, Providência and São Judas Tadeu. The leaves presented anatomical differences to the different areas studied, for B. coccolobifolia the presence of phenolic sheath in the populations of Providência and São Judas Tadeu, and in B. verbascifolia the sclerified sheath in the plants of Canaã, Providência and São Judas Tadeu, both act against loss of water in environments where the soil has little water retention, a characteristic found in the soil of Providência. The micromorphometric analysis revealed differences in the spongy parenchyma of B. coccolobifolia in the plants of São Judas Tadeu, demonstrating that the greater thickness of this tissue facilitates the diffusion of CO2, and the thicker abaxial epidermis in B. verbascifolia, typical of plants that are exposed to high luminosity. In the B. verbascifolia plants of the Providência population, the palisade parenchyma was smaller, due to its distribution in more shaded areas. Xeromorphic features such as: thick cuticle, trichomes and many stomata, show that these species are well adapted to the Cerrado.

Capítulo 1: Dimorfismo sexual em aberturas glandulares das pernas de três subfamílias de Gonyleptidae (Arachnida: Opiliones: Laniatores) Glândulas sexualmente dimórficas podem não apenas ser informativas taxonomicamente como podem ainda trazer informações importantes sobre a comunicação entre os sexos. Nesse trabalho estudamos a evolução de aberturas glandulares sexualmente dimórficas nas pernas I (basitarso) e pernas IV (presença/ausência de intumescimento e quantidade de poros no metatarso) em uma linhagem monofilética pertencente ao clado K92 composta pelas subfamílias Progonyleptoidellinae, Sodreaninae e parte de Gonyleptinae. Utilizamos Microscopia Eletrônica de Varredura para descrever as aberturas glandulares e compará-las entre exemplares das três subfamílias analisadas. Encontramos aberturas glandulares sexualmente dimórficas na perna I de uma espécie em Progonyleptoidellinae (de seis analisadas), de uma espécie de Gonyleptinae (de quatro analisadas) e em três espécies de Sodreaninae (de seis analisadas). Com relação às aberturas da perna IV, todos os machos estudados possuem maiores quantidades de poros na perna IV do que as fêmeas exceto uma espécie de Gonyleptinae e uma de Sodreaninae. Os machos de todas as espécies estudadas possuem o astrágalo das pernas IV intumescidos, exceto uma espécie em Progonyleptoidellinae. Portanto as aberturas glandulares sexualmente dimórficas na perna I nos machos mostram-se variáveis na linhagem interna ao clado K92 estudada nesse trabalho. Por outro lado, o intumescimento e quantidade de poros na perna IV dos machos parecem ser conservados na linhagem estudada. Capítulo 2: Morfologia interna das glândulas sexualmente dimórficas de Progonyleptoidellus striatus Roewer 1913 (Opiliones: Laniatores: Gonyleptidae) Apesar de diversos trabalhos sugerirem a presença de glândulas sexualmente dimórficas nas quatro subordens de Opiliones, há poucos trabalhos que investigam essas glândulas histologicamente. Morfologia interna fornece importantes informações sobre o funcionamento e composição das secreções. Para isso, utilizamos técnicas histológicas e ultraestruturais para estudar a morfologia interna de duas glândulas de Progonyleptoidellus striatus (Progonyleptoidellinae): uma no basitarso das pernas I e a outra no metatarso das pernas IV. Adicionalmente, investigamos a composição histológica e ultraestrutural da área glandular no metatarso IV. Encontramos diferenças entre os sexos, tanto na morfologia interna como externa: machos possuem intumescimentos das áreas glandulares e internamente possuem células glandulares ausentes nas fêmeas. Encontramos abundantes retículos endoplasmáticos rugosos, lisos e mitocôndrias, além de Complexo de Golgi bem desenvolvido na perna I dos machos. Diferentemente da perna I, não encontramos retículos endoplasmáticos lisos na perna IV, o que pode implicar em composições de produtos secretórios diferentes para as pernas. Nas duas pernas encontramos abundância de vesículas secretoras / Sexually dimorphic glands may not only be taxonomically informative but can also bring important information about communication between the sexes. In this work we studied the evolution of sexually dimorphic glandular openings on legs I (basitarsus) and legs IV (presence/absence of swollen area and quantity of pores on metatarsus) in a monophyletic lineage belonging to clade K92 that include the subfamilies Progonyleptoidellinae, Sodreaninae and part of Gonyleptinae. We used Scanning Electron Microscopy to describe the glandular openings and compare them among specimens of the three subfamilies analyzed. We found sexually dimorphic glandular openings on legs I of one species in Progonyleptoidellinae (out of six analyzed), one specie of Gonyleptinae (out of four analyzed) and three species of Sodreaninae (out of six analyzed). Concerning glandular openings on legs IV, all males studied have more pores than females except for one species in Gonyleptinae and another in Sodreaninae. Males of all species studied have a swollen astragalus IV, except for one species in Progonyleptoidellinae. Therefore, the glandular openings in leg I in males are shown to be variable for the internal lineage to the Clade K92 studied in this work. On the other hand, the swollen area and the greater quantity of pores on legs IV of males seem to be conserved in the lineage studied. Although several studies suggest the presence of sexually dimorphic glands in the four suborders of Opiliones, there are few studies that investigate these glands histologically. Internal morphology provides important information about the functioning of the glands and the composition of secretions. Here we used histological and ultrastructural techniques to study the internal morphology of two glands of Progonyleptoidellus striatus (Progonyleptoidellinae): one in the basitarsus of legs I and another in the metatarsus of legs IV. We found differences between the sexes, both in internal and external morphology, in which males have swollen of the glandular areas and internally the males have glandular cells, absent in females. We found abundant rough, smooth endoplasmic reticulum and mitochondria, as well as Golgi Complex well developed in legs I of males. In contrast, we did not find smooth endoplasmic reticulum in legs IV, which may result in different compositions of glandular products in each leg. In both legs we found abundance great number of secretory vesicles

A gengivite e a periodontite constituem doenças periodontais infecciosas comuns do homem, nas quais as bactérias periodontais e seus produtos participam ativamente para indução da inflamação local e efeitos sistêmicos (ex.: coração). Sabendo que a saliva representa a primeira e grande barreira às infecções orais, este trabalho se propôs a: i) avaliar a perda óssea induzida pela doença periodontal em ratos submetidos à indução de doença, via implante de ligadura, após os intervalos de 3, 7 e 14 dias; ii) Investigar possíveis alterações de fluxo (estimulado ou não com pilocarpina) e composição salivar nesses animais; iii) avaliar a concentração / expressão de marcadores de estresse oxidativo e inflamatórios em glândulas salivares e em amostras de saliva; iv) avaliar o papel funcional da função das glândulas salivares ex-vivo na produção da amilase. Para isto, ratos Wistar machos (180 -200g) foram submetidos à indução da periodontite através do implante da ligadura, e os parâmetros inflamatórios e bioquímicos foram avaliados nesses animais. Ratos com periodontite no dia 3, quando comparado ao grupo sham, exibiram aumento significativo do fluxo salivar (estimulada com pilocarpina), produção de Ca2+, secreção de proteínas e produção de amilase na saliva, bem como aumento do conteúdo de TBARs em glândulas parótida e da amilase liberada das glândulas submandibulares (GSM). Observou-se, ainda, aumento da expressão de mRNA para iNOS e nNOS em GSM. Em contrapartida, ratos com periodontite após 7 dias exibiram redução da taxa de salivação estimulada (e não estimulada), da produção de proteínas totais e da concentração e secreção de amilase na saliva, muito embora o conteúdo sérico e salivar de TBARs e da atividade de MPO na saliva desses animais mostrou-se elevado em relação ao grupo sham. Não foram observadas diferenças significativas quanto ao conteúdo de TBARs em glândulas salivares, secreção e concentração de Ca2+ na saliva e tampouco sobre o conteúdo de proteínas nitradas em amostras de GSM desses animais. Já no 14<font face=\"Symbol\">&#176; dia visualizou-se um aumento da atividade de NOS Ca2+ dependente e da expressão mRNA das iNOS e nNOS em GSM. Ratos com periodontite, após 14 dias de indução, não exibiram aumento significativo na taxa de salivação, concentração e secreção de Ca2+ salivar, produção e concentração de proteínas totais, amilase na saliva e conteúdo de TBARs em amostras de saliva e glândulas salivares. Ainda, observou-se aumento das atividades da peroxidase/MPO, concentração de nitrato em saliva e proteínas nitradas em GSM e maior concentração de citocinas Th1 / Th2 (IL-4, IL-13 e IL-10) em amostras de GSM. Conclui-se que a indução experimental da doença periodontal em ratos , influencia o funcionamento de glândulas salivares de acordo com os dias de indução, inicialmente estimulando, em um segundo momento inibindo e posteriormente retornando aos níveis basais. Após 7 dias, caracteriza-se como o tempo ideal para a manifestação dos efeitos inibitórios na glândula. / Gingivitis and periodontitis are common infectious periodontal diseases in man, in which periodontal bacteria and their products participate actively to induce local inflammation and systemic effects (eg heart). Knowing that saliva represents the first major barrier to oral infections, this study aimed to: i) to assess bone loss due to induced periodontitis in rats, after intervals of 3, 7 and 14 days, ii) to investigate possible changes in flow (or not stimulated with pilocarpine) and salivary composition in these animals, and iii) evaluate the concentration and expression of markers of oxidative stress and inflammation in the salivary glands and saliva samples, and iv) assess the functional role of salivary gland function in ex vivo production of amylase. For this purpose, male Wistar rats (180-200g) underwent induction of periodontitis by implanting the ligature, and biochemical and inflammatory parameters were assessed. Rats with periodontitis on day 3 when compared to sham group, exhibited a significant increase in salivary flow (stimulated with pilocarpine), production of Ca2+, protein secretion and production of amylase in saliva, as well as increased contents of TBARS in the parotid and amylase released from submandibular glands (GSM). There was also increased expression of mRNA for iNOS and nNOS in GSM. In contrast, rats with periodontitis after 7 days exhibited a reduction in stimulated saliva (not stimulated), production and total protein concentration and secretion of salivary amylase, although the content of serum and salivary TBARS and the activity of MPO in saliva of these animals was high compared to the sham group. There were no significant differences in TBARS content in salivary gland secretion and Ca2+ concentration in saliva, nor on the content of nitrated proteins in samples from these animals GSM. By the 14th day envisioned an increase of NOS activity and Ca2+ dependent mRNA expression of iNOS and nNOS in GSM. Rats with periodontitis, 14 days after induction, exhibited a significant increase in the rate of salivation, concentration and salivary secretion of Ca2+, production and concentration of total protein, salivary amylase and content of TBARS in samples of saliva and salivary glands. Still, there was increased activity of peroxidase / MPO, nitrate concentration in saliva and proteins nitrated in GSM and higher concentration of Th1 / Th2 (IL-4, IL-13 and IL-10) in samples of GSM. We conclude that the experimental induction of periodontal disease in rats, influence the functioning of the salivary glands according to the days of induction, initially stimulating, in a second time and subsequently inhibiting return to baseline levels. After 7 days, is characterized as the ideal time for the expression of an inhibitory effect on the gland function.

by Vincent, Wai-ip Law. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 128-134). / Abstract also in Chinese. / Chapter Chapter I. --- Literature Review / Chapter I.1. --- "Structure, functions and general physiology of equine sweat gland" / Chapter I.1.1. --- Ultrastructure of equine sweat gland --- p.1 / Chapter I.1.2. --- Functions and physiology of equine sweat gland --- p.3 / Chapter I.1.3. --- Experimental studies on equine sweat gland by functional approaches --- p.4 / Chapter I.1.4. --- Hormonal and neuronal regulation of sweat secretion in equidaes --- p.5 / Chapter I.1.5. --- Possible role(s) of extracellular ATP in equine sweat gland epithelia --- p.6 / Chapter I.1.6. --- Measurement of electrogenic anion secretion by short-circuit current (Isc) technique --- p.8 / Chapter I.2. --- Classification of purinergic receptors and its existence in biological systems / Chapter I.2.1. --- Functional classification of purinergic receptors --- p.13 / Chapter I.2.2. --- Basic structure of G-protein coupled P2Y receptors --- p.17 / Chapter I.2.3. --- Physiological function and significance of purinergic receptors --- p.20 / Chapter I.3. --- Objectives of study --- p.22 / Chapter Chapter II. --- Methods and Materials / Chapter II.l. --- Culture technique of the equine epithelial cells --- p.23 / Chapter II.2. --- Conventional short-circuit current (Isc) measurement technique / Chapter II.2.1. --- Introduction --- p.25 / Chapter II.2.2. --- Preparation of permeable supports and electrodes --- p.25 / Chapter II.2.3. --- Experimental set up and measurement of Isc --- p.28 / Chapter II.2.4. --- Measurement of Isc during experiment --- p.30 / Chapter II.3. --- Measurement of intracellular free calcium ( [Ca2+ ]ii) by microspectrofluorimetry / Chapter II.3.1. --- Preparation of cells --- p.31 / Chapter II.3.2. --- The set up and procedures for experiment --- p.31 / Chapter II.4. --- Simultaneous measurement of changes in [Ca2+ ]i and Isc / Chapter II.4.1. --- Experimental set up and manipulation --- p.35 / Chapter II.4.2. --- Other preparations before experiment --- p.37 / Chapter II.5. --- Material and solutions used for experiment / Chapter II.5.1. --- Culture media and enzyme --- p.40 / Chapter II.5.2. --- Chemicals and Drugs --- p.40 / Chapter II.5.3. --- Preparation of solution for experiments --- p.42 / Chapter II.6. --- Statistical analysis --- p.44 / Chapter Chapter III. --- Results / Chapter III.l. --- Effects of nucleotides on transepithelial ion transport / Chapter III.1.1. --- Basic electrophysiological properties of cultured equine sweat gland epithelia --- p.45 / Chapter III. 1.2. --- Short-circuit current (Isc) induced by nucleotides --- p.45 / Chapter III. 1.3. --- Identification of ion species responsible for the change in Isc --- p.50 / Chapter III. 1.4. --- Effects of chloride channels blockers on the UTP-induced Isc --- p.51 / Chapter III.2. --- Signal transduction mechanisms of P2Y-nucleotide receptors / Chapter III.2.1. --- The involvement of Gi-proteins --- p.56 / Chapter III.2.2. --- Effect of BAPTA on the increases in Isc induced by nucleotides --- p.58 / Chapter III.2.3. --- Study of P2Y-receptor mediated increase in [Ca2+]i --- p.62 / Chapter III.3. --- Characterization of the P2Y subtype(s) by cross desensitization experiments / Chapter III.3.1. --- Autologous desensitization experiments --- p.70 / Chapter III.3.2. --- Classical cross desensitization experiments --- p.70 / Chapter III.3.3. --- Characterization of the ATP-insensitive P2Y-receptor --- p.80 / Chapter III.3.4. --- Interaction between ATP and bradykinin --- p.87 / Chapter III.4. --- Simultaneous measurement of [Ca2+]i and Issc / Chapter III.4.1. --- Effect ofUDP and ADP --- p.89 / Chapter III.4.2. --- Correlation of Isc and [Ca2+]i --- p.92 / Chapter III.4.3. --- Cross desensitization experiments --- p.97 / Chapter III.5. --- Evidence of a [Ca2+]i-independent Isc-component induced by nucleotides / Chapter III.5.1. --- The time course of the ΔRf and ΔISC --- p.102 / Chapter III.5.2. --- Effect of ionomycin on the ΔISC and ΔRf induced by nucleotides --- p.110 / Chapter III.5.3. --- Effect of thapsigargin on the ΔISC and ΔRf induced by nucleotides --- p.110 / Chapter III.5.4. --- Effect of thapsigargin in nominal Ca2+-free solution --- p.115 / Chapter Chapter IV. --- Discussion / Chapter IV. 1. --- Role of extracellular nucleotides in epithelial tissues --- p.119 / Chapter IV.2. --- Characterization of an ATP-insensitive P2Y-nucleotide receptor --- p.120 / Chapter IV.3. --- Expression of the novel ATP-insensitive receptor on a functionally polarized epithelia --- p.122 / Chapter IV.4. --- Involvement of a [Ca2+]i -independent Isc induced by nucleotides --- p.124 / Chapter Chapter V. --- References --- p.128

Resumo: A espermateca dos insetos estoca os espermatozóides desde a cópula até a fecundação dos ovos, e consiste de uma bolsa que freqüentemente possui glândulas associadas. Considerações sobre a anatomia, histologia e ultra-estrutura de espermatecas tornam-se muito mais complexas quando são considerados os insetos sociais, que apresentam diferenças no tamanho e na forma entre rainhas e operárias, e ainda, entre as espécies. Apesar desta complexidade, os resultados morfológicos, histoquímicos e ultra-estruturais aqui obtidos mostraram as diferenças entre as espermatecas das espécies: Atta sexdens rubropilosa, Atta laevigata, Atta sexdens sexdens, Atta sexdens piriventris, Atta capiguara, Acromyrmex balzani, Acromyrmex landolti, Acromyrmex landolti balzani, Acromyrmex coronatus e Cyphomyrmex muelleri. As espécies Atta sexdens rubropilosa, Atta laevigata, Acromyrmex coronatus e Cyphomyrmex muelleri apresentaram apenas a glândula espermatecal (porção secretora externa à espermateca). Atta sexdens sexdens, Atta capiguara e Acromyrmex landolti balzani apresentaram tanto a glândula espermatecal como um epitélio secretor interno. Atta sexdens piriventris, Acromyrmex balzani e Acromyrmex landolti, no entanto, apresentaram apenas epitélio secretor interno, sugerindo que nestas espécies a espermateca seja mais independente na manutenção dos espermatozóides. As formigas cortadeiras vêm sendo cada vez mais estudadas do ponto de vista filogenético, tendo como principal marcador o DNA mitocondrial. Estabelecer a filogenia das formigas atines auxiliaria na resposta de algumas questões evolutivas sobre a relação destas com seus fungos. Realizou-se aqui a correlação das informações obtidas através das seqüências de DNA e do estudo morfológico das espermatecas de rainhas Atta sexdens rubropilosa, Atta laevigata, Atta sexdens sexdens, Atta sexdens piriventris... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The insects spermatheca stores the spermatozoa from the copulation until the egg fecundation, and consists of a sac frequently associated with glands. Considerations about anatomy, histology and ultrastructure of spermathecae become much more complex in social insects, because they have queens and workers with different sizes and shapes, as well as among the species. Besides this complexity, the morphological, histochemical and ultrastructural results show the differences between the spermathecae of the following species: Atta sexdens rubropilosa, Atta laevigata, Atta sexdens sexdens, Atta sexdens piriventris, Atta capiguara, Acromyrmex balzani, Acromyrmex landolti, Acromyrmex landolti balzani, Acromyrmex coronatus and Cyphomyrmex muelleri. The species Atta sexdens rubropilosa, Atta laevigata, Acromyrmex coronatus and Cyphomyrmex muelleri presented only the spermathecal gland (external secretory portion). Atta sexdens sexdens, Atta capiguara and Acromyrmex landolti balzani presented both the spermathecal gland and an internal secretory epithelium. Atta sexdens piriventris, Acromyrmex balzani and Acromyrmex landolti, however, presented only the internal secretory epithelium, suggesting that the spermatheca in these species is more independent in spermatozoa maintenance. The leaf-cutter ants are being studied more from a phylogenetic point of view, having as a principal marker mitochondrial DNA. The establishment of the phylogeny of the attines ants would help in the response of some evolutionary questions about their relation with their fungus. The present study correlated the information obtained from the DNA sequences and morphological studies of spermathecae of Atta sexdens rubropilosa, Atta laevigata, Atta sexdens sexdens, Atta sexdens piriventris, Atta capiguara, Acromyrmex balzani, Acromyrmex landolti, Cyphomyrmex muelleri, Apterostigma sp. and Mycocepurus goeldii queens... (Complete abstract click electronic access below) / Orientador: Maria Izabel Camargo Mathias / Coorientador: Mauricio Bacci Júnior / Banca: José Eduardo Serrão / Banca: Maria Santina de Castro Morini / Banca: Odair Correa Bueno / Banca: José Roberto Feitosa Silva / Doutor

Enteroendocrine cells (EEs) are critical components in our bodies' ability to maintain homeostasis after eating a meal. Hormones released by EEs mediate processes ranging from triglyceride processing to glucose balance to hydration maintenance. Despite their importance, they remain relatively poorly understood in terms of development as well as function. Drosophila melanogaster is a promising model in which to study EEs. I performed a gene expression assay in Drosophila, and found 19 transcription factors likely to be specific to EEs. I am in the process of analyzing their mutant phenotypes in the fly midgut. Additionally, by a limited screen of the homologs to the fly EE-specific transcription factors, I was able to identify two candidates for novel transcriptional regulators involved in EE specification or functionality. I will be analyzing the mutant phenotypes for these two genes, Lmx1a and Lmx1b, in addition to a third mutant Prox1, chosen because of the strong phenotype of its homologous gene's knockdown in the fly. I am hoping I will be able to add to the ever-growing body of knowledge in reference to enteroendocrine development. Additionally, several assays were performed on flies lacking EEs. I found that flies without EEs lay significantly fewer eggs, and have apparent defects in oviposition and defecation. I will outline several experiments to continue the phenotype analysis of flies lacking EEs.