Wong, Miu Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 140-152). / Abstracts in English and Chinese. / DECLARATION --- p.i / ACKNOWLEDGEMENT --- p.ii / ABBREVIATIONS --- p.iii / ABSTRACT IN ENGLISH --- p.iv / ABSTRACT IN CHINESE --- p.vii / TABLE OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xviii / Chapter CHAPTER I - --- INTRODUCTION / Chapter 1.1 --- Regulation of human airway surface liquid --- p.1 / Chapter 1.2 --- Sodium reabsorption and chloride secretion in airway epithelium --- p.3 / Chapter 1.3 --- Purinergic receptors --- p.7 / Chapter 1.4 --- P2Y receptors in epithelial cells --- p.11 / Chapter 1.5 --- Autocrine or paracrine regulation of ion transport in epithelial cells --- p.13 / Chapter 1.6 --- Signaling pathways underlying the regulation of ion transport by P2Y receptors stimulation --- p.16 / Chapter 1.7 --- The therapeutic potential of P2Y receptors in treating cystic fibrosis --- p.18 / Chapter 1.8 --- Particular interest on P2Y6 receptor as potential target for treatment of cystic fibrosis --- p.21 / Chapter 1.9 --- Properties of 16HBE14o- cell line --- p.23 / Chapter 1.10 --- Objectives of the present experiments --- p.25 / Chapter CHAPTER II - --- MATERIALS AND METHODS / Chapter 2.1 --- Solutions and Chemicals --- p.26 / Chapter 2.2 --- Cell culture --- p.28 / Chapter 2.3 --- Simultaneous measurement of short-circuit current (Isc) and intracellular calcium concentration ([Ca2+ ])i --- p.29 / Chapter 2.3.1 --- Preparation of 16HBE14o- cells for simultaneous measurement of Isc and [Ca2+]i --- p.29 / Chapter 2.3.2 --- Measurement of Isc and transepithelial resistance with Ussing chamber --- p.32 / Chapter 2.3.3 --- Simultaneous measurement of Isc and [Ca2+]i --- p.35 / Chapter 2.4 --- Measurement of protein kinase A activity --- p.38 / Chapter 2.5 --- Data analysis --- p.39 / Chapter CHAPTER III - --- RESULTS / Chapter 3.1 --- Apical and basolateral application of P2Y agonists induced Isc and [Ca2+］i responses in 16HBE14o- cells --- p.40 / Chapter 3.1.1 --- Effect of apical and basolateral application of ATP on Isc and [Ca2+̐]ư --- p.40 / Chapter 3.1.2 --- Effect of apical and basolateral application of UTP on Isc and [Ca2+̐]ư --- p.45 / Chapter 3.1.3 --- Effect of apical and basolateral application of UDP on Isc and [Ca2+̐]ư --- p.50 / Chapter 3.1.4 --- "Summary of the effects of apical and basolateral application of ATP, UTP and UDP on Isc and [Ca2+̐]ư" --- p.55 / Chapter 3.2 --- Ionic mechanisms underlying the effect of apical and basolateral UDP on 16HBE14o- cells --- p.56 / Chapter 3.2.1 --- Differential effect of apical and basolateral UDP on Isc --- p.56 / Chapter 3.2.2 --- Effect of various apical CI－ channel blockers on Isc response induced by apical and basolateral application of UDP --- p.59 / Chapter 3.2.3 --- Effect of various basolateral K+ channel blockers on Isc response induced by apical and basolateral application of UDP --- p.83 / Chapter 3.3 --- Involvement of other signaling molecules or pathways in regulation of the chloride secreting response evoked by apical and basolateral UDP --- p.108 / Chapter 3.3.1 --- Effect of apical and basolateral UDP on PKA activity --- p.109 / Chapter 3.3.2 --- Effect of PKC inhibitors on Isc response induced by apical and basolateral application of UDP --- p.111 / Chapter CHAPTER IV - --- DISCUSSION / Chapter 4.1 --- Simultaneous measurement of Isc and [Ca2+ ̐]ư upon apical and basolateral application of P2Y agonists in 16HBE14o- cells --- p.125 / Chapter 4.2 --- Ionic mechanism underlying the effect of apical and basolateral UDP on 16HBE14o- cells --- p.128 / Chapter 4.2.1 --- Possible ionic mechanism for chloride secretion mediated by apical P2Y6 receptors --- p.131 / Chapter 4.2.2 --- Possible ionic mechanism for chloride secretion mediated by basolateral P2Y6 receptors --- p.133 / Chapter 4.3 --- Involvement of other possible signaling molecules or pathway underlying the action of apical and basolateral UDP --- p.135 / Chapter 4.4 --- Summary --- p.138 / Chapter CHAPTER V - --- REFERENCES --- p.140 / Publications --- p.153

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2005" Includes bibliographical references.

Trichomonas vaginalis é o agente etiológico da doença sexualmente transmissível não viral mais comum no mundo, sendo registrados aproximadamente 276 milhões de novos casos de tricomonose a cada ano. O estabelecimento da infecção se deve principalmente à capacidade de adesão do parasito às células epiteliais vaginais, cervicais ou de próstata, seguida pela intensa reação inflamatória, resultado da infiltração de neutrófilos no sítio da infecção. Nucleotídeos e nucleosídeos, especialmente ATP e adenosina, são liberados para o espaço extracelular por células em situações de estresse ou injúria tecidual e desenvolvem seus efeitos sinalizadores através da ativação de purinoceptores. Ainda, as ectonucleotidases, NTPDase e ecto-5’-nucleotidase, são capazes de hidrolisar os nucleotídeos gerando adenosina e finalmente, a enzima adenosina deaminase (ADA) é responsável pela conversão de adenosina em inosina. A expressão gênica de cinco NTPDases putativas presentes no genoma de T. vaginalis foram investigadas, assim como o envolvimento da sinalização purinérgica na relação parasito-hospedeiro. Nossos resultados mostraram que diferentes isolados de T. vaginalis expressam os genes TvNTPDase1, 2, 3, 4 e 5, sendo observado o maior número de transcritos para TvNTPDase1, 2 e 4. A sequência preditiva de aminoácidos revelou a presença das cinco regiões conservadas da apirase, domínios transmembrana, sítios de fosforilação, peptídeos sinais e os prováveis sítios ativos da enzima. A análise filogenética demonstrou maior similaridade das TvNTPDases com as formas intracelulares da enzima, como as NTPDases 4 e 7 humanas e a de Saccharomyces cerevisiae. Além disso, a restrição de soro promoveu aumento significativo da atividade da NTPDase de T. vaginalis, no entanto sem corresponder com o aumento de expressão gênica de determinada(s) sequência(s). Quanto à participação da sinalização purinérgica na resposta inflamatória de células do hospedeiro frente ao parasito, foram utilizados como modelos celulares as células epiteliais vaginais (HMVII), cervicais (HeLa) e neutrófilos humanos. As linhagens HMVII e HeLa mostraram expressar todos os subtipos de receptores P1, P2X e P2Y e XI os diferentes isolados de T. vaginalis, que foram cocultivados com as células, mostraram hidrolisar eficientemente os nucleotídeos ATP, ADP e AMP. Ainda, o isolado clínico fresco TV-LACM6 foi o único a apresentar elevada citotoxicidade frente às células epiteliais vaginais e cervicais, no entanto não foi detectado aumento da liberação de ATP pelas células após o cocultivo, provavelmente devido à alta atividade da enzima NTPDase observada nesse isolado. Os trofozoítos de T. vaginalis não foram capazes de aumentar a produção de IL-8 e IL-6 pelas linhagens HMVII e HeLa, e apenas os isolados ATCC30236 e TV-LACM6 causaram aumento na secreção da citocina MIP-3α pelas células epiteliais cervicais. Finalmente, o nucleotídeo ATP e o nucleosídeo adenosina não modularam a produção dos mediadores inflamatórios investigados. Em relação aos neutrófilos, estes mostraram aumentar a produção de espécies reativas de oxigênio (ERO) e IL-8 após incubação com os trofozoítos de T. vaginalis. Os nucleotídeos e nucleosídeos da adenina e guanina não produziram efeito na produção de ERO e IL-8; no entanto, quando o nucleosídeo adenosina foi incubado junto com o inibidor da enzima ADA (EHNA) observou-se uma redução significativa da produção de ERO e IL-8 pelos neutrófilos, devido à inibição da ADA e consequentemente, ao aumento da concentração de adenosina disponível no meio extracelular. Os nossos resultados indicaram a ativação do receptor A1 dos neutrófilos nessa condição. O conjunto de dados aqui obtidos contribuiu para uma melhor caracterização da família de enzimas NTPDases de T. vaginalis assim como para um maior conhecimento acerca da influência da sinalização purinérgica na relação parasito-hospedeiro. / Trichomonas vaginalis is the agent of the most common non-viral sexually transmitted disease worldwide, causing 276.4 million new cases a year. The establishment of the infection is closely related to the parasite ability to adhere to vaginal, cervical and prostate epithelial cells, followed by an intense inflammatory response as result of neutrophil infiltration. Nucleotides and nucleosides, mainly ATP and adenosine, are released into the extracellular space by cells under stress or injury and they exert their signaling effects through activation of the purinoceptors. Moreover, the ectonucleotidases, NTPDase and ecto-5'-nucleotidase, are capable of hydrolyzing the nucleotides producing adenosine and finally, the adenosine deaminase (ADA) is responsible for the conversion of adenosine to inosine. We investigated the gene expression of five putative NTPDases found in T. vaginalis genome as well as the involvement of purinergic signaling on the host-parasite relationship. Our results showed that different T. vaginalis isolates are able to express TvNTPDase1, 2, 3, 4 and 5 and that TvNTPDase1, 2 and 4 are the most expressed genes. Predictive amino acid sequence revealed the presence of the five apyrase conserved regions, transmembrane domains, phosphorylation sites, signal peptides and the active sites. Phylogenetic analysis showed that TvNTPDases share more similarity with the intracellular enzymes, such as human NTPDase 4 and 7 and Saccharomyces cerevisiae NTPDase. In addition, the serum limitation caused a significant increase in NTPDase activity, but without association with the gene expression of a specific TvNTPDase sequence. Regarding the participation of purinergic signaling on the inflammatory responses against the parasite, the vaginal (HMVII) and cervical (HeLa) epithelial cells and the human neutrophils were used as cellular models. HMVII and HeLa cell lines showed to express all subtypes of P1, P2X and P2Y receptors and the different T. vaginalis isolates, which were co-cultured with the cells, showed to hydrolyze efficiently ATP, ADP and AMP. Furthermore, only the fresh clinical isolate, TV-LACM6, caused a profound cytotoxicity against the vaginal and cervical epithelial cells. Interestingly, it was not detected an increase in ATP release by the cells after cocultivation, probably due to the high NTPDase activity dislplayed by TV-LACM6 isolate. The T. vaginalis trophozoites were not able to increase the production of IL-8 and IL-6 by HMVII and HeLa cells and only ATCC30236 and TV-LACM6 isolates enhanced MIP-3α secretion by the cervical epithelial cells. Finally, neither ATP nor adenosine has modulated the production of the inflammatory mediators here investigated. Considering the neutrophils, T. vaginalis stimulated the production of reactive oxygen species (ROS) and IL-8 by these immune cells and both adenine as guanine nucleotides and nucleosides did not cause any effect on ROS and IL-8 levels. However, when adenosine was incubated with an ADA inhibitor (EHNA) we observed a significant reduction of ROS and IL-8 production by neutrophils, due to inhibition of ADA with a subsequent increase of adenosine concentration in the extracellular milieu. . Our results suggested the participation of A1 receptor in this condition. The data set obtained in this study contributed to the characterization of T. vaginalis NTPDases family as well as to a better understanding of the influence of purinergic signaling on host-parasite relationship.

Trichomonas vaginalis é o agente etiológico da doença sexualmente transmissível não viral mais comum no mundo, sendo registrados aproximadamente 276 milhões de novos casos de tricomonose a cada ano. O estabelecimento da infecção se deve principalmente à capacidade de adesão do parasito às células epiteliais vaginais, cervicais ou de próstata, seguida pela intensa reação inflamatória, resultado da infiltração de neutrófilos no sítio da infecção. Nucleotídeos e nucleosídeos, especialmente ATP e adenosina, são liberados para o espaço extracelular por células em situações de estresse ou injúria tecidual e desenvolvem seus efeitos sinalizadores através da ativação de purinoceptores. Ainda, as ectonucleotidases, NTPDase e ecto-5’-nucleotidase, são capazes de hidrolisar os nucleotídeos gerando adenosina e finalmente, a enzima adenosina deaminase (ADA) é responsável pela conversão de adenosina em inosina. A expressão gênica de cinco NTPDases putativas presentes no genoma de T. vaginalis foram investigadas, assim como o envolvimento da sinalização purinérgica na relação parasito-hospedeiro. Nossos resultados mostraram que diferentes isolados de T. vaginalis expressam os genes TvNTPDase1, 2, 3, 4 e 5, sendo observado o maior número de transcritos para TvNTPDase1, 2 e 4. A sequência preditiva de aminoácidos revelou a presença das cinco regiões conservadas da apirase, domínios transmembrana, sítios de fosforilação, peptídeos sinais e os prováveis sítios ativos da enzima. A análise filogenética demonstrou maior similaridade das TvNTPDases com as formas intracelulares da enzima, como as NTPDases 4 e 7 humanas e a de Saccharomyces cerevisiae. Além disso, a restrição de soro promoveu aumento significativo da atividade da NTPDase de T. vaginalis, no entanto sem corresponder com o aumento de expressão gênica de determinada(s) sequência(s). Quanto à participação da sinalização purinérgica na resposta inflamatória de células do hospedeiro frente ao parasito, foram utilizados como modelos celulares as células epiteliais vaginais (HMVII), cervicais (HeLa) e neutrófilos humanos. As linhagens HMVII e HeLa mostraram expressar todos os subtipos de receptores P1, P2X e P2Y e XI os diferentes isolados de T. vaginalis, que foram cocultivados com as células, mostraram hidrolisar eficientemente os nucleotídeos ATP, ADP e AMP. Ainda, o isolado clínico fresco TV-LACM6 foi o único a apresentar elevada citotoxicidade frente às células epiteliais vaginais e cervicais, no entanto não foi detectado aumento da liberação de ATP pelas células após o cocultivo, provavelmente devido à alta atividade da enzima NTPDase observada nesse isolado. Os trofozoítos de T. vaginalis não foram capazes de aumentar a produção de IL-8 e IL-6 pelas linhagens HMVII e HeLa, e apenas os isolados ATCC30236 e TV-LACM6 causaram aumento na secreção da citocina MIP-3α pelas células epiteliais cervicais. Finalmente, o nucleotídeo ATP e o nucleosídeo adenosina não modularam a produção dos mediadores inflamatórios investigados. Em relação aos neutrófilos, estes mostraram aumentar a produção de espécies reativas de oxigênio (ERO) e IL-8 após incubação com os trofozoítos de T. vaginalis. Os nucleotídeos e nucleosídeos da adenina e guanina não produziram efeito na produção de ERO e IL-8; no entanto, quando o nucleosídeo adenosina foi incubado junto com o inibidor da enzima ADA (EHNA) observou-se uma redução significativa da produção de ERO e IL-8 pelos neutrófilos, devido à inibição da ADA e consequentemente, ao aumento da concentração de adenosina disponível no meio extracelular. Os nossos resultados indicaram a ativação do receptor A1 dos neutrófilos nessa condição. O conjunto de dados aqui obtidos contribuiu para uma melhor caracterização da família de enzimas NTPDases de T. vaginalis assim como para um maior conhecimento acerca da influência da sinalização purinérgica na relação parasito-hospedeiro. / Trichomonas vaginalis is the agent of the most common non-viral sexually transmitted disease worldwide, causing 276.4 million new cases a year. The establishment of the infection is closely related to the parasite ability to adhere to vaginal, cervical and prostate epithelial cells, followed by an intense inflammatory response as result of neutrophil infiltration. Nucleotides and nucleosides, mainly ATP and adenosine, are released into the extracellular space by cells under stress or injury and they exert their signaling effects through activation of the purinoceptors. Moreover, the ectonucleotidases, NTPDase and ecto-5'-nucleotidase, are capable of hydrolyzing the nucleotides producing adenosine and finally, the adenosine deaminase (ADA) is responsible for the conversion of adenosine to inosine. We investigated the gene expression of five putative NTPDases found in T. vaginalis genome as well as the involvement of purinergic signaling on the host-parasite relationship. Our results showed that different T. vaginalis isolates are able to express TvNTPDase1, 2, 3, 4 and 5 and that TvNTPDase1, 2 and 4 are the most expressed genes. Predictive amino acid sequence revealed the presence of the five apyrase conserved regions, transmembrane domains, phosphorylation sites, signal peptides and the active sites. Phylogenetic analysis showed that TvNTPDases share more similarity with the intracellular enzymes, such as human NTPDase 4 and 7 and Saccharomyces cerevisiae NTPDase. In addition, the serum limitation caused a significant increase in NTPDase activity, but without association with the gene expression of a specific TvNTPDase sequence. Regarding the participation of purinergic signaling on the inflammatory responses against the parasite, the vaginal (HMVII) and cervical (HeLa) epithelial cells and the human neutrophils were used as cellular models. HMVII and HeLa cell lines showed to express all subtypes of P1, P2X and P2Y receptors and the different T. vaginalis isolates, which were co-cultured with the cells, showed to hydrolyze efficiently ATP, ADP and AMP. Furthermore, only the fresh clinical isolate, TV-LACM6, caused a profound cytotoxicity against the vaginal and cervical epithelial cells. Interestingly, it was not detected an increase in ATP release by the cells after cocultivation, probably due to the high NTPDase activity dislplayed by TV-LACM6 isolate. The T. vaginalis trophozoites were not able to increase the production of IL-8 and IL-6 by HMVII and HeLa cells and only ATCC30236 and TV-LACM6 isolates enhanced MIP-3α secretion by the cervical epithelial cells. Finally, neither ATP nor adenosine has modulated the production of the inflammatory mediators here investigated. Considering the neutrophils, T. vaginalis stimulated the production of reactive oxygen species (ROS) and IL-8 by these immune cells and both adenine as guanine nucleotides and nucleosides did not cause any effect on ROS and IL-8 levels. However, when adenosine was incubated with an ADA inhibitor (EHNA) we observed a significant reduction of ROS and IL-8 production by neutrophils, due to inhibition of ADA with a subsequent increase of adenosine concentration in the extracellular milieu. . Our results suggested the participation of A1 receptor in this condition. The data set obtained in this study contributed to the characterization of T. vaginalis NTPDases family as well as to a better understanding of the influence of purinergic signaling on host-parasite relationship.

This study demonstrates the presence of protein kinase C activity in both cytosolic and membrane fractions of bovine lens epithelial cells in culture. Protein kinase C activity is similar in normal and hyperglycemic cells. Furthermore, the ability of the enzyme to translocate from the cytosol to the membrane following phorbol ester treatment is unimpeded by hyperglycemic conditions. Moreover, protein kinase C activation had no effect on myoinositol uptake either in normal cells or in cells exposed to hyperglycemic conditions.

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.

皮膚是人體最大的器官，具有多種功能，其中最重要的功能之一就是作為身體內部和外界環境之間的的保護屏障。完整地修復這一保護屏障是創傷癒合和組織再生領域的一個重要內容。本論文探討了人類臍帶被覆上皮細胞 (cord lining epithelial cells, CLECs)作為一種幹細胞來源，可用于表皮重建的潛能. / 本論文的第二章對CLECs的體外分離和增殖進行了詳細地描述。這一類細胞具有較長的染色體端粒，較高的增殖潛能和傳代能力。同時，它們表達上皮幹細胞和多能性幹細胞的標誌性表面抗原。它們還具有多種分化潛能，包括成脂、成骨和成軟骨。然而當皮下異種移植後，它們並不會形成畸胎瘤。 / 本論文的第三章對CLECs的免疫特性進行了評估。結果顯示CLECs不但具有低免疫原性，還具有免疫調節功能。它們表達典型性的一型主要組織相容性複合體(MHC class I)，即人白細胞ABC抗原(HLA-ABC)，但不表達典型性的二型主要組織相容性複合體(MHC class II)，即人白細胞DR抗原(HLA-DR)。它們同時還表達非典型性的MHC class I, 包括人白細胞G抗原和人白細胞E 抗原(HLA-G和HLA-E), 但不表達共激分子(CD40, CD80和CD86)。此外，體外檢測還發現它們表達適度的促炎/抗炎細胞因子和大量的生長因子. / 本論文的第四章對CLECs在表皮重建應用中的潛能進行了考察。結果顯示無論在體外器官培養還是異種移植動物模型中，CLECs都能形成分層的上皮結構，與用表皮細胞構建的分層上皮結構相類似。而且在CLECs構建的皮膚替代物中證實了有表皮分化標誌性抗原的表達。 / 結論：本論文證明了CLECs具有幹細胞樣特性但無致瘤性，具有低免疫原性和表皮分化的可塑性。研究結果支持CLECs在創傷癒合和皮膚再生領域的臨床應用可行性. / The skin is the largest organ in the body and has multiple functions. One of the most important functions is to serve as a protective barrier between the internal and external environments of the body. Restoration of the integrity of this protective barrier is an essential aspect of wound healing and tissue regeneration. In this thesis, the potential of human umbilical cord lining epithelial cells (CLECs) as a source of stem cells with appropriate differentiation capacity for epidermal reconstitution has been explored. / The isolation and propagation of CLECs from human umbilical cord lining epithelium were described in Chapter II. The cells presented a long telomere length and had high proliferative potential and passaging capability. They were also shown to display both epithelial and pluripotent stem cell markers. They were capable of multipotent differentiation, including adipogenesis, osteogenesis and chondrogenesis. However, they didn’t form teratoma after subcutaneous xenotransplantation until 12 weeks. / The immune properties of CLECs in vitro were assessed in Chapter III. The cells were shown to have low immunogenicity but high immunosuppressive function. They expressed classical major histocompatibility complex (MHC) class I antigens (HLA-ABC), but not MHC class II antigen (HLA-DR). They also expressed non-classical MHC class I antigens (HLA-G and HLA-E), but lacked the expression of the co-stimulatory molecules (CD40, CD80 and CD86). Moreover, they expressed moderate pro/anti-inflammatory cytokines and multiple growth factors both in cell supernatants and cell lysates. / The potential of CLECs for epidermal reconstitution was investigated in Chapter IV. In both organotypic culture and xenotransplantation model, CLECs were capable of generating a stratified epithelial structure, which is similar to that constructed by using keratinocytes. Furthermore, the expression of epidermal differentiation markers was verified in CLEC-constructed skin substitutes. / In conclusion, the stem cell-like properties of CLECs have been demonstrated in the present study. In addition to the lack of tumorigenicity, CLECs also have low immunogenicity and significant plasticity in epidermal differentiation. The findings support the potential clinical application of CLECs in wound healing and skin regeneration. / 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. / Cai, Yijun. / "October 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-129). / Abstract also in Chinese. / Abstrac --- p.i / Table of Contents --- p.v / Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Chapter Chapter I --- Introduction --- p.1 / Skin --- p.3 / Wound healing --- p.6 / Wound regeneration and repair --- p.6 / Recent history of wound treatment --- p.9 / Skin substitutes --- p.11 / Stem cells for wound treatment --- p.14 / Stem cells overview --- p.15 / Adult stem cells --- p.16 / Fetal stem cells --- p.18 / Amniotic membrane derived stem cells --- p.19 / Umbilical cord stem cells --- p.22 / Hypothesis and Specific aims --- p.24 / Chapter Chapter II --- The Isolation and Characterization of the Stem Cell-like Properties of Human Umbilical Cord Lining Epithelial Cells --- p.28 / Introduction --- p.28 / Materials and methods --- p.30 / Results --- p.47 / Discussion --- p.62 / Conclusion --- p.67 / Chapter Chapter III --- The assessment of the Immune Properties of Human Umbilical Cord Lining Epithelial Cells --- p.69 / Introduction --- p.69 / Materials and methods --- p.72 / Results --- p.75 / Discussion --- p.83 / Conclusion --- p.88 / Chapter Chapter IV --- The Investigation of the Potential of Human Umbilical Cord Lining Epithelial Cells for the Epidermal Reconstitution --- p.89 / Introduction --- p.89 / Materials and methods --- p.91 / Results --- p.94 / Discussion --- p.101 / Conclusion --- p.104 / Chapter Chapter V --- Summary and Future Plan --- p.105 / Summary --- p.105 / Future plan --- p.108 / Acknowledgements --- p.113 / References --- p.114 / Appendix --- p.130

Acute colitis was induced by exposing male SD rats to 4% DSS in drinking water for 8 days. Rats were divided into four groups as follows: DSS group---DSS-induced colitis; DSS + SRE group---SRE, 100 mg/kg/day in addition to DSS; Ctr + SRE group---SRE alone; and Ctr group---sham control group. The colon damage was elucidated by macroscopic, histological, electrophysiological and biochemical assessment. Orally administered SRE significantly reduced the colonic damage in all four aspects. However, baicalein did not show similar effect in the same experiment. / In summary, our finding indicated that both SRE and its major flavonoid, baicalein, could stimulate chloride secretion in human colonic T84 cells and mucosa freshly isolated from human colon. Although SRE was effective in treating acute DSS-induced ulcerative colitis, baicalein is unlikely the active anti-inflammatory component of SRE. Nevertheless, the results demonstrated that this TCM has a scientific basis for its effectiveness. Our data support further evaluation of the therapeutic potential of SRE for the treatment of IBD. / In TCM, Scutellariae radix and Coptidis Rhizoma (CR) derived compounds have been frequently used for gastroenteritis and secretory diarrhea. Our laboratory findings suggested that the major flavonoid component of SR, baicalein, stimulates chloride secretion in rat distal colon, probably via CFTR activation (Ko et al., 2002). In contrast, limited information about the cellular mechanism of chloride secretion induced by SR in human colonic epithelia is available. Therefore, the effect of Scutellariae radix extract (SRE) on electrolyte transport in a human colonic epithelial cell line, T84, was examined using the short-circuit current (ISC) technique. Results demonstrated that SRE stimulated a Cl--dependent secretion across T84 cells, probably via both Ca2+- and cAMP-mediated pathway. / On the other hand, the cellular mechanism of baicalein-induced Cl - secretion in T84 cells was further investigated. It was found that the secretory mechanisms involve protein kinase A (PKA)-, adenylate cyclase (AC)- and luminal cAMP-dependent Cl- channels, most likely cystic fibrosis transmembrane conductance regulator (CFTR) and serosal 293B-sensitive K + channels. However, the action of baicalein cannot be solely explained by its cAMP-elevating effect. In addition, the effect of baicalein could be potentiated by the inhibition of mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K). Furthermore, it was found that inhibition of protein kinase C (PKC) delta limited the baicalein-induced chloride secretion. / Our laboratory has found that baicalein (Ko et al., 2002 and Yue et al., 2003) stimulates chloride secretion in rat distal colon and human colonic T84 cells. As it is known that responses in the animal model or the cell line may not completely reflect the in vivo physiology, it is important to study the above responses in human colon. With scarce supply of freshly isolated human colonic mucosa, the results showed that the effect of SRE and baicalein on ion transport in human samples is similar to that obtained in T84 cell line and rat model. / Scutellariae radix (SR) is the dry root of Scutellariae baicalensis Georgi (Huangqin). SR has been employed for centuries as a traditional Chinese medicine (TCM) for various purposes. It contains a large amount of flavonoids such as baicalein, baicalin, and wogonin, which possess a number of beneficial bioactivities including anti-oxidant, anti-bacterial and anti-inflammatory, etc. / Ulcerative colitis (UC), an inflammatory bowel disease (IBD), has been known for more than half a century. Recent studies have shown that two flavonoids derived from SR, baicalein and wogonin, might alleviate the symptoms of IBD. Moreover, SR is the major component of Hange-shasshin-to (HST), one of the Chinese herbal formulas, which has been reported to suppress the pathogenesis of IBD. The above scientific background led us to examine the effect of SRE administration on DSS-induced colitis in rats in a way to evaluate new treatments potentially applicable to UC in humans. / Chung, Ho Lam. / "August 2007." / Adviser: W. H. Ko. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0925. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Airway bronchial epithelial cells play important roles in host defense, inflammation and regulation of immune responses. Activated bronchial epithelial cells are potent sources of a wide variety of soluble and cell-surface molecules that can alter the biological functions of inflammatory cells in the airways. Molecular mechanisms regulating the production of inflammatory mediators from bronchial epithelial cells remain to be fully elucidated. / All of the above findings suggest that human bronchial epithelial cells could be activated by a variety of stimuli in airway inflammatory reactions. Besides, different intracellular signaling pathways could regulate the activation of human bronchial epithelial cells in response to different stimuli. Our results therefore provide new insight into the molecular mechanisms involved in airway inflammatory diseases and may have important therapeutic implications. / Basophils are the accessory cell type required for T helper (Th)2 induction and initiators in IgE-mediated chronic allergic inflammation in response to allergens. Number of basophils and Th17 cells increases at the sites of allergic inflammation in the airways of allergic asthmatic patients. To elucidate the interaction among the activation of human bronchial epithelial cells, Th17 cells, and basophils, we investigated the activation effects of Th17 hallmark cytokine IL-17A on the human primary bronchial epithelial cells/BEAS-2B bronchial epithelial cells and human primary basophils/ KU812 basophilic cells. Human bronchial epithelial cells and basophils were cultured either together or separately in the presence or absence of IL-17A stimulation. Co-culture of human bronchial epithelial cells and basophils could significantly increase the release of inflammatory cytokine IL-6 and mononuclear chemoattractant protein-1 (MCP-1/CCL2), a chemokine for basophils, eosinophils and monocytes, while human bronchial epithelial cells were the main source for releasing IL-6 and CCL2. Such induction was synergistically enhanced upon the activation of IL-17A. The use of transwell inserts in the co-culture system demonstrated that the direct interaction between these two cell types was necessary for IL-6 and CCL2 release induced by IL-17A. Surface expression of intercellular adhesion molecule-1 (ICAM-1) on the human bronchial epithelial cells was also up-regulated upon their interaction. The interaction of human bronchial epithelial cells and basophils under IL-17A stimulation was differentially regulated by extracellular signal-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK), p38 mitogen activated protein kinase (MAPK) and nuclear factor (NF)-kappaB pathways. Our findings therefore suggest a novel immunopathological role of human Th17 cells and basophils in allergic asthma through the activation of granulocytes-mediated inflammation initiated by the direct interaction between human basophils and bronchial epithelial cells. / IL-27 is a novel member of the IL-6/IL-12 family cytokines that are produced early by antigen-presenting cells (APCs) during immune responses. IL-27 can drive the commitment of naive T cells to a Th1 phenotype and inhibit inflammation in later phases of infection. Recent evidence has suggested that human bronchial epithelial cells with the expression of IL-27 receptor complex are potential target cells of IL-27. Here we investigated the in vitro effects of IL-27, alone or in combination with inflammatory cytokine TNF-alpha on the pro-inflammatory activation of human bronchial epithelial cells, and the underlying intracellular signaling molecules were also studied. IL-27 was found to up-regulate ICAM-1 expression on the surface of human bronchial epithelial cells, and a synergistic effect was observed in the combined treatment of IL-27 and TNF-alpha on the surface expression of ICAM-1. Although IL-27 did not alter the basal IL-6 secretion from human bronchial epithelial cells, it could significantly enhance TNF-alpha-induced IL-6 production. The synergistic effects on the induction of ICAM-1 and IL-6 were partially due to the up-regulated expression of TNF-alpha receptor (p55TNFR) on the surface of human bronchial epithelial cells induced by IL-27. Further investigations showed that the enhanced production of ICAM-1 and IL-6 in human bronchial epithelial cells activated by IL-27 and TNF-alpha was differentially regulated by phosphatidylinositol 3-OH kinase (PI3K)-Akt, p38 MAPK and NF-kappaB pathways. Our study therefore suggests a potential role of IL-27 and TNF-alpha in the pathogenesis of airway infection or inflammatory diseases. / In the present study, we investigated the mechanisms of the activation of human bronchial epithelial cells induced by various stimuli including interleukin (IL)-17A, IL-27, tumor necrosis factor (TNF)-alpha and human basophils. The activation of human bronchial epithelial cells was studied in terms of the expression of cytokines, chemokines and adhesion molecules. Using intracellular staining with flow cytometry and selective pharmacological inhibitors, we further investigated the underlying intracellular signaling mechanisms regulating the activation of human bronchial epithelial cells. / Cao, Ju. / Advisers: Chun K. Wong; Christopher W. K. Lam. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 175-202). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.