Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 139-155).

Orientador: Fabio Papes / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T08:04:04Z (GMT). No. of bitstreams: 1 Nakahara_ThiagoSeike_M.pdf: 27685441 bytes, checksum: c755af1be54c3ba9b204bed05559dd88 (MD5) Previous issue date: 2014 / Resumo: O Sistema Olfativo é um Sistema Sensorial complexo, composto por diversos subsistemas cuja integração no cérebro resulta na interação entre os animais e seus respectivos ambientes de maneira adequada. Essa adequação pode significar respostas comportamentais e fisiológicas distintas para situações diversas a que esses animais tenham sido expostos. Esse Sistema exibe compartimentos especializados na detecção de estímulos de uma mesma natureza e nesse contexto, o Sistema Olfativo Principal é responsável pela detecção de odorantes voláteis em geral e o Sistema Olfativo Acessório é responsável pela detecção de feromônios. Apesar dessa divisão formal, estudos recentes questionam essa divisão e propõem sobreposição entre a função desses subsistemas. Nesse estudo investigamos a expressão de receptores OR sendo expressos no Órgão Vomerosasal em níveis comparáveis aos receptores V2R ("endógenos"). Desses receptores, isolamos o receptor Olfr692 que possui o nível de expressão mais alto entre os OR estudados ou relatados anteriormente na literatura. As células que expressam o receptor Olfr692 foram caracterizadas molecularmente e foram feitos estudos preliminares a fim de investigar a função do receptor Olfr692 frente a possíveis funções biológicas que fossem capazes de explicar a expressão robusta de um receptor de classe OR no Órgão Vomeronasal / Abstract: The Olfactory System is a complex Sensorial System, comprised of some subsystems whose integration in the brain results in the appropriated interaction between animals and their environment, that is, proper behavioral or physiological answers to diverse situations to which these animals are exposed. This System exhibits specialized features for detection of a given kind of stimuli. The Main Olfactory System detects volatile odorants in general while the Accessory Olfactory System detects pheromones. Apart from this formal distinction, recent studies have questioned this division and propose some overlap between them. In the present study, we have investigated the expression of OR receptors in the Vomeronasal Organ whose expression level is compared to V2R Receptors (endogenous). We have isolated from these genes the Olfr692, which has the higher levels among the VNO-OR here studied and those discussed in the literature. These cells have been molecularly characterized and preliminary functional studies were also performed, searching for the possible biological functions of this Receptor, which could explain its expression in the Vomeronasal Organ / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

The airway epithelium plays a central role in respiratory physiology through its transport and immunological functions. Our previous study suggested that P2Y receptors are expressed in airway epithelia and play a significant role in regulating transepithelial ion transport. P2Y receptors belong to the family of purinergic receptors, which can be stimulated by nucleotides such as UTP and UDP. P2Y receptors are G protein-coupled receptors and classically signal through G[subscript q], resulting in an increase in intracellular Ca²⁺ concentration ([Ca²⁺]ᵢ) and thereby in the activation of Ca²⁺-dependent ion channels and downstream signalling pathway(s). Furthermore, P2Y receptors are involved in asthmatic inflammation. / Estrogen (or E₂) is an important hormone in human physiology. In addition to the classical nuclear hormone receptors ERα and ERβ, a novel estrogen receptor, G protein-coupled estrogen receptor (GPER), was recently identified and found to be involved in both rapid signalling and transcriptional regulations. The action of GPER is unclear, but it has been implicated in mediating anti-inflammatory responses. / In our experiments, both human bronchial epithelial cell line, 16HBE14o-, and primary normal human bronchial epithelial cells expressed GPER at mRNA and protein levels, as demonstrated by RT-PCR and western blotting, respectively. ERα and ERβ expression were also detected at mRNA and protein level. Expression of GPER receptors was localized in the human bronchial epithelial cells by immunofluorescence staining and western blotting of fractionated cell lysates. / [Ca²⁺]ᵢ induced by nucleotides were monitored by calcium imaging technique using MetaFluor fluorescence ratio imaging system. Stimulation of epithelial cells with E₂ or with the specific agonist of GPER, G1, rapidly attenuated a UDP-, UTP- or ATPyS- evoked increase in [Ca²⁺]ᵢ in both 16HBE14o- cell line and primary cells. This inhibitory effect of E₂ and G1 were concentration dependent, while this effect was reversed by GPER specific antagonist, G15. To examine the effect of E₂ and G1 on UDP-activated intracellular Ca²⁺ release and influx, the epithelia were exposed to nominally Ca²⁺ -free solution in the presence or absence of G1 or E₂, and then stimulated with UDP. Subsequently, Ca²⁺ was added to the perfusate. Both E₂ and G1 could inhibit UDP-induced Ca²⁺ release. However, only E₂ but not G1 could inhibit UDP-induced Ca²⁺ influx. / E₂ or G1 inhibited the secretion of two pro-inflammatory cytokines, interleukin (IL)-6 or IL-8, in cells stimulated by different nucleotides or the cationic protein, poly-L-arginie, as quantified by ELISA. CFP-Epac-YPF, an Epac-based polypeptide FRET reporter was used to monitor the real-time cAMP changes in 16HBE14o- cells. Both G1 and E₂ induced an increase in cAMP production. The transepithelial chloride (Cl⁻) secretion was measured using short circuit current technique in cells grown on permeable support. Cl⁻ secretion induced by apical UDP was partially inhibited by G1 in a concentration dependent manner. / Our results provide the first evidence that human bronchial epithelia express GPER, which interact with the P2Y receptor-mediated calcium signalling pathway and cytokine secretion. Moreover, the anti-inflammatory role of GPER may be due to its opposing effect on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia. / 气道上皮具有调节运输以及参与免疫反应等功能，在呼吸生理学研究中有着十分关键的意义。我们曾经的研究发现P2Y受体在气道上皮中表达并调节上皮细胞离子运输过程。P2Y受体属于嘌呤受体，可被三磷酸尿苷（UTP），二磷酸尿苷（UDP）等核苷酸激活。同时，P2Y受体也是一类G蛋白偶联受体，可通过活化G[subscript q]蛋白调控细胞内钙离子浓度而激活钙依赖性离子通道及其他下游信号通路。此外P2Y受体还参与哮喘炎症的调控。 / 雌激素（或雌二醇，E₂）是人体一类十分重要的激素。除传统的核受体ERα与ERβ外，一类新型雌激素受体GPER已被发现和鉴定。GPER属于G蛋白偶联受体，可同时参与转录调控和非基因依赖的快速信号调节。尽管具体机理尚不明确，但研究发现GPER可介导抗炎症反应。 / 实验结果显示，在支气管上皮细胞株16HBE14o-和原代人支气管上皮细胞中GPER都被检测到基因和蛋白水平的表达。GPER在人支气管上皮细胞中的定位也通过免疫荧光染色（immunofluorescence）和亚细胞组分蛋白质印迹（western blot of fractionated cells）得到鉴定。 / 本研究中，荧光显微技术（fluorescence microscopy）被用于测定核苷酸介导的细胞内钙离子浓度（[Ca²⁺]ᵢ）。在16HBE14o- 和原代培养人支气管上皮细胞中，E₂和GPER特异性激动剂G1都可抑制核苷酸介导的 [Ca²⁺]ᵢ增加，且这种抑制作用呈浓度依赖。GPER特异性拮抗剂G15可抵消G1的抑制作用。进一步研究表明，E₂和G1都可抑制UTP诱导的胞内钙库释放，然而只有E₂抑制UTP诱导的胞外钙离子内流。 / 除钙离子调节外，E₂和G1还可抑制支气管上皮细胞中核苷酸或聚精氨酸（poly-L-arginine）刺激介导的两种促炎症细胞因子，白介素6（IL-6）和白介素8（IL-8）的分泌。酶联免疫法（ELISA）被用于细胞因子的定量。同时，CFP-Epac-YPF作为一类多肽荧光共振能量转移（FRET）探针被转染入16HBE14o- ，探测细胞内腺苷-3',5'-环化一磷酸（cAMP）的实时变化。结果显示在人支气管上皮细胞中E₂和G1都可引导cAMP生成。此外，我们使用短路电流（short-circuit current, Isc）技术测定单层上皮细胞的氯离子（Cl⁻）分泌，并发现人支气管上皮顶膜面UDP诱导的Cl⁻ 分泌可被G1部分抑制，且抑制效果呈浓度依赖。 / 本研究首次证明GPER表达于人支气管上皮, 且激活GPER对P2Y受体介导的钙离子信号通路以及细胞因子生成起到抑制作用。这些结果表明在气道炎症反应中，GPER可通过反向调节P2Y受体激活的促炎症作用，达到抗炎症的效果。 / Hao, Yuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 187-211). / Abstracts also in Chinese. / Title from PDF title page (viewed on 03, November, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Introdução: A apneia do sono é condição prevalente e apresenta forte correlação com as principais causas de morbidade e mortalidade na sociedade ocidental. A perda do controle neuromotor proveniente de estágios mais profundos do sono está associada ao colapso faríngeo e a patogênese da apneia obstrutiva do sono (SAOS). A língua é implicada como principal protagonista na patogênese da obstrução das vias aéreas superiores (VAS) durante o sono. Não há farmacoterapia para SAOS. Novas tecnologias moleculares para controle neuronal através da inserção de um receptor de membrana geneticamente modificado, denominado Designer Receptor Exclusively Activated by Designer Drugs [DREADDs], o qual pode ser ativado por uma droga inicialmente inerte, de alta especificdade, clozapina-n-oxide (CNO). Objetivos: 1) Modificar geneticamente os neurônios motores do núcleo do hipoglosso utilizando-se de DREADDs, o qual permite regular sua atividade; 2) Analisar a atividade do músculo genioglosso sob administração de CNO; 3) Desenvolver abordagens para rastreamento dos músculos protrusores e retratores da língua por marcadores retrógrados, injecção de subunidade B de toxina colérica (CTB-AF) e do vírus de pseudoraiva (PRV) 267 transportando um gene repórter, nos músculos efetores. Métodos: Receptores muscarínicos mutados em um vetor adenoviral associado (AAV) foram infundidos no núcleo do hipoglosso de camundongos via injeção esterotáxica bilateral. Após quatro semanas, período para expressão fenotípica, foram comparadas a atividade eletromiográfica do músculo genioglosso (EMGGG) em resposta a administração de ligante (CNO) versus solução salina. Em um segundo grupo foram realizadas infusões com vírus controle e comparação da EMGGG pré e pós infusão de CNO. Para rastreamento neural a CTB-AF foi injetado nos músculos protusores e retratores da língua e para expressão de Cre o vírus PRV-267 foi injetado no músculo genioglosso. A expressão dessas substâncias no núcleo do hipoglosso foi avaliada através de microscopia de fluorescência. Resultados: Dos dezoito camundongos injetados com DREADDs, dezesseis foram transfectados pelo vetor de AAV. Em camundongos onde o núcleo motor do hipoglosso foi corretamente atingido a EMGGG apresentou importante aumento após a administração de CNO. Em contraste, a atividade do genioglosso não apresentou alteração após a administração de soro fisiológico. Em três camundongos onde a transfecção ultrapassou os limites do núcleo foi observado arritmia respiratória após infusão do ligante. Todos animais infundidos com vírus controle foram adequadamente transfectados mas não apresentaram alteração eletromiográfica após a infusão de CNO. Foram diferenciados no núcleo do hipoglosso os neurônios motores da musculatura retratora e protusora da língua. A expressão intranuclear da enzima Cre-recombinase foi identificada no núcleo hipoglosso. Conclusão: A utilização de métodos quimiogenéticos para ativar grupos selecionados de neurônios motores em áreas cerebrais específicas representa técnica promissora para o estudo do controle neuromotor das VAS. Estes resultados sugerem que a terapia transgênica pode ser eficaz no tratamento da SAOS além de uma vasta gama de patologias que resultam de perturbações no controle neural das VAS. Através da manipulação das fibras musculares efetoras na língua foi possível a identificação do seu respectivo neurônio motor no núcleo do hipoglosso e induzi-lo a sintetizar uma enzima específica (Cre-recombinase) / Introduction: Sleep Apnea is a prevalent condition and strongly correlates with the major causes of morbidity and mortality in Western Society. The loss of motor input from deeper sleep stages is associated with pharyngeal collapsibility and the pathogenesis of obstructive sleep apnea (OSA). The tongue plays a major role in the pathogenesis of upper airway (UA) obstruction during sleep. There is no pharmacotherapy for OSA. New molecular techniques allow to control neuronal function by inserting a genetically modified membrane receptor termed the Designer Receptor Exclusively Activated by Designer Drugs [DREADDs] which can be activated by a highly specific and otherwise pharmacologically inert drug clozapine-N-oxide (CNO) Objectives: 1) To genetically modify the hypoglossal nucleus motor neurons using DREADDs, which allows to regulate their activity; 2) To analyze the genioglossal activity upon administration of CNO; 3) to develop novel approaches to targeting tongue protruders and retractors by retrograde tracers, cholera toxin subunit B (CTB-AF) and pseudorabies virus (PRV) 267 injection carrying a reporter gene into the effector muscles. Methods: Mutated muscarinic receptors in an adenoviral associated vector (AAV) were delivered to the hypoglossal nucleus via stereotactically bilateral injection. Four weeks after adenoviral delivery (expression period), responses in genioglossal electromyography (EMGGG) activity to intraperitoneal administration of CNO ligand vs. Saline (control) were compared in mice. In a second group, control-virus was infused and genioglossus muscle EMGGG was compared before and after CNO infusion. For neuronal tracing CTB-AF was injected into the protrusor and retractor muscles of the tongue and for Cre induction PRV-267 virus was injected in the genioglossus muscle. Expression of these substances in the hypoglossal nucleus were evaluated by fluorescence histology. Results: Of eighteen DREADDs injected mice, sixteen were transfected with AAV vector. After CNO administration EMGGG activity increased in mice where the hypoglossal motor nucleus was correctly targeted. In contrast, genioglossal activity was not augmented following saline administration. In three mice where transfection surpassed the nucleus limits, breathing arrhythmia was observed following ligand infusion. All animals infused with control virus were adequately transfected but did not present electromyographic change following CNO infusion. The motor neurons of the rectractor and protrusor musculature of the tongue were well differentiated in the hypoglossal nucleus. Intranuclear expression of Cre recombinase enzyme was identified in the hypoglossal nucleus. Conclusion: Utilizing chemogenetic methods to activate motor neuron groups in selected brain areas show promise to UA neuromotor control, and suggest that transgenic therapy may be effective in treating OSA and a wide range of pathologies that result in disturbances of UA neural control. By manipulating the effector muscle fibers of the tongue, it was possible to identify its respective motor neuron in the hypoglossal nucleus and to induce synthesis of a specific enzyme (Cre recombinase)

Introdução: A apneia do sono é condição prevalente e apresenta forte correlação com as principais causas de morbidade e mortalidade na sociedade ocidental. A perda do controle neuromotor proveniente de estágios mais profundos do sono está associada ao colapso faríngeo e a patogênese da apneia obstrutiva do sono (SAOS). A língua é implicada como principal protagonista na patogênese da obstrução das vias aéreas superiores (VAS) durante o sono. Não há farmacoterapia para SAOS. Novas tecnologias moleculares para controle neuronal através da inserção de um receptor de membrana geneticamente modificado, denominado Designer Receptor Exclusively Activated by Designer Drugs [DREADDs], o qual pode ser ativado por uma droga inicialmente inerte, de alta especificdade, clozapina-n-oxide (CNO). Objetivos: 1) Modificar geneticamente os neurônios motores do núcleo do hipoglosso utilizando-se de DREADDs, o qual permite regular sua atividade; 2) Analisar a atividade do músculo genioglosso sob administração de CNO; 3) Desenvolver abordagens para rastreamento dos músculos protrusores e retratores da língua por marcadores retrógrados, injecção de subunidade B de toxina colérica (CTB-AF) e do vírus de pseudoraiva (PRV) 267 transportando um gene repórter, nos músculos efetores. Métodos: Receptores muscarínicos mutados em um vetor adenoviral associado (AAV) foram infundidos no núcleo do hipoglosso de camundongos via injeção esterotáxica bilateral. Após quatro semanas, período para expressão fenotípica, foram comparadas a atividade eletromiográfica do músculo genioglosso (EMGGG) em resposta a administração de ligante (CNO) versus solução salina. Em um segundo grupo foram realizadas infusões com vírus controle e comparação da EMGGG pré e pós infusão de CNO. Para rastreamento neural a CTB-AF foi injetado nos músculos protusores e retratores da língua e para expressão de Cre o vírus PRV-267 foi injetado no músculo genioglosso. A expressão dessas substâncias no núcleo do hipoglosso foi avaliada através de microscopia de fluorescência. Resultados: Dos dezoito camundongos injetados com DREADDs, dezesseis foram transfectados pelo vetor de AAV. Em camundongos onde o núcleo motor do hipoglosso foi corretamente atingido a EMGGG apresentou importante aumento após a administração de CNO. Em contraste, a atividade do genioglosso não apresentou alteração após a administração de soro fisiológico. Em três camundongos onde a transfecção ultrapassou os limites do núcleo foi observado arritmia respiratória após infusão do ligante. Todos animais infundidos com vírus controle foram adequadamente transfectados mas não apresentaram alteração eletromiográfica após a infusão de CNO. Foram diferenciados no núcleo do hipoglosso os neurônios motores da musculatura retratora e protusora da língua. A expressão intranuclear da enzima Cre-recombinase foi identificada no núcleo hipoglosso. Conclusão: A utilização de métodos quimiogenéticos para ativar grupos selecionados de neurônios motores em áreas cerebrais específicas representa técnica promissora para o estudo do controle neuromotor das VAS. Estes resultados sugerem que a terapia transgênica pode ser eficaz no tratamento da SAOS além de uma vasta gama de patologias que resultam de perturbações no controle neural das VAS. Através da manipulação das fibras musculares efetoras na língua foi possível a identificação do seu respectivo neurônio motor no núcleo do hipoglosso e induzi-lo a sintetizar uma enzima específica (Cre-recombinase) / Introduction: Sleep Apnea is a prevalent condition and strongly correlates with the major causes of morbidity and mortality in Western Society. The loss of motor input from deeper sleep stages is associated with pharyngeal collapsibility and the pathogenesis of obstructive sleep apnea (OSA). The tongue plays a major role in the pathogenesis of upper airway (UA) obstruction during sleep. There is no pharmacotherapy for OSA. New molecular techniques allow to control neuronal function by inserting a genetically modified membrane receptor termed the Designer Receptor Exclusively Activated by Designer Drugs [DREADDs] which can be activated by a highly specific and otherwise pharmacologically inert drug clozapine-N-oxide (CNO) Objectives: 1) To genetically modify the hypoglossal nucleus motor neurons using DREADDs, which allows to regulate their activity; 2) To analyze the genioglossal activity upon administration of CNO; 3) to develop novel approaches to targeting tongue protruders and retractors by retrograde tracers, cholera toxin subunit B (CTB-AF) and pseudorabies virus (PRV) 267 injection carrying a reporter gene into the effector muscles. Methods: Mutated muscarinic receptors in an adenoviral associated vector (AAV) were delivered to the hypoglossal nucleus via stereotactically bilateral injection. Four weeks after adenoviral delivery (expression period), responses in genioglossal electromyography (EMGGG) activity to intraperitoneal administration of CNO ligand vs. Saline (control) were compared in mice. In a second group, control-virus was infused and genioglossus muscle EMGGG was compared before and after CNO infusion. For neuronal tracing CTB-AF was injected into the protrusor and retractor muscles of the tongue and for Cre induction PRV-267 virus was injected in the genioglossus muscle. Expression of these substances in the hypoglossal nucleus were evaluated by fluorescence histology. Results: Of eighteen DREADDs injected mice, sixteen were transfected with AAV vector. After CNO administration EMGGG activity increased in mice where the hypoglossal motor nucleus was correctly targeted. In contrast, genioglossal activity was not augmented following saline administration. In three mice where transfection surpassed the nucleus limits, breathing arrhythmia was observed following ligand infusion. All animals infused with control virus were adequately transfected but did not present electromyographic change following CNO infusion. The motor neurons of the rectractor and protrusor musculature of the tongue were well differentiated in the hypoglossal nucleus. Intranuclear expression of Cre recombinase enzyme was identified in the hypoglossal nucleus. Conclusion: Utilizing chemogenetic methods to activate motor neuron groups in selected brain areas show promise to UA neuromotor control, and suggest that transgenic therapy may be effective in treating OSA and a wide range of pathologies that result in disturbances of UA neural control. By manipulating the effector muscle fibers of the tongue, it was possible to identify its respective motor neuron in the hypoglossal nucleus and to induce synthesis of a specific enzyme (Cre recombinase)

Cerebral ischemia is the principal cause of morbidity and mortality worldwide. In addition to neuronal loss associated with hypoxic-ischemic damage, cerebral ischemia is characterized by a neuromicrovascular injury. Nitrative stress and lipid peroxidation increase in hypoxic-ischemic damages and play an essential role in neuromicrovascular injury leading to cerebral ischemia. We hypothesized that newly described lipid peroxidation products, termed trans-arachidonic acids (TAA), could be implicated in the pathogenesis of hypoxia-ischemia by affecting the cerebral vasomotricity and microvascular integrity. / The effects of TAA on neuromicrovascular tone were tested ex vivo by monitoring the changes in vascular diameter of rat cerebral pial microvessels. Four isomers of TAA, namely 5 E-AA, 8E-AA, IIE-AA and 14 E-AA induced an endothelium-dependent vasorelaxation. Possible mechanisms involved in TAA-induced vasorelaxation were thoroughly investigated. Collectively, data enclosed revealed that TAA induce cerebral vasorelaxation through the interactive activation of BKCa channels with heme oxygenase-2. This interaction leads to generation of carbon monoxide which in turn activates soluble guanylate cyclase and triggers vasorelaxation. / Chronic effects of TAA on microvascular integrity were examined by generating a unilateral hypoxic-ischemic (HI) model of cerebral ischemia on newborn rat pups. Our HI model showed microvascular degeneration as early as 24h post-HI, preceded by an increase in cerebral TAA levels. HI-induced microvascular lesions were dependent on nitric oxide synthase activation and ensued TAA formation. Although the molecular mechanisms leading to TAA-induced microvascular degeneration were, in part uncovered for the retina, the primary site of action of TAA remains unknown. We demonstrated that TAA binds and activates GPR40 receptor, a newly described free fatty acid receptor. Importantly, GPR40 receptor knock-out prevents TAA-induced reduction in cerebral microvascular density and limits HI-induced brain infarct.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 137-167).

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 142 pages. Includes Vita. Includes bibliographical references.

No description available.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 6 uppsatser.