M.Sc. / Tuberculosis, of which the causative agent is Mycobacterium tuberculosis, presents itself as a serious health problem globally, especially in Africa. Susceptibility to this infectious disease is influenced by the virulence of the strain of mycobacteria, environmental factors, and genetic variation within the host. The Vitamin D Receptor gene or VDR has been identified as a candidate gene for TB susceptibility. This gene codes for the VDR protein that mediates the biological actions of the active form of vitamin D. Vitamin D has been shown to impair growth of Mycobacterium tuberculosis in human monocytes and macrophages. Vitamin D also provides a link between Toll-like receptor activation and the antibacterial responses of innate immunity in its production of cathelicidin. The VDR protein is a transcription factor that mediates the effects of the active form of vitamin D. Vitamin D has an immunomodulatory role and variations in the VDR gene may result in variations in the functioning of the VDR protein, and hence variations in response to infection. The VDR gene includes the largely non-coding 5’ regulatory region exons 1a-1f and the coding exons 2-9. As a result of increased awareness of the heritability of gene expression and reports of disease associations with VDR promoter region variants, the focus of the research described in this dissertation was the regulatory region of the VDR gene. Polymorphisms that occur within the regulatory region were viii investigated, as were the effects these polymorphisms may have on gene expression, influencing host susceptibility to tuberculosis, with an emphasis on African populations. VDR polymorphisms have been shown to be involved in susceptibility to tuberculosis, particularly the FokI SNP in exon 2, BsmI and ApaI in intron 8 and the synonomous TaqI in exon 9. However, results have been inconsistent. SNPs shown to be associated with TB may serve as markers of truly functional SNP with which they are in linkage disequilibrium (LD). The majority of these association studies involve single nucleotide polymorphisms (SNPs) found in the introns or are silent mutations in the coding exons. Variations in the 5’ regulatory region have been shown to affect gene expression, in particular if they influence the binding sites of transcription factors.

Chitin Microparticles (CMPs, 1-10um), a special form of the ubiquitous and nontoxic polysaccharide Chitin (GlcNAc), is capable of inducing a switch in macrophages from the wound-healing M2 phenotype to the classically activated pro-inflammatory M1 phenotype; which has therapeutic implications in allergy and cancer. We hypothesized that TLR2 forms a complex with CMPs and Chitin-Binding Proteins (CBPs) at the surface of peritoneal macrophages and remains with that complex after internalization to initiate downstream signaling events, leading to the production of the M1 cytokine, TNFalpha. Our results from experiments performed in RAW 264.7 cells show that TLR2 and TLR1, but not TLR6, are associated with the CMP binding fraction, and that both TLR1 and TLR2 might be important for M1 activation as a result of CMP phagocytosis. This project sheds light on CMP as a potential therapeutic agent and provides more evidence for a phagocytosis-dependent TLR2 signaling pathway. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

肥大細胞是過敏和炎症的主要效應細胞，其激活機制包括了IgE依賴性和非IgE依賴性的激活。IgE依賴性激活是指抗原與IgE的高親和力受體FcεRI上的IgE結合，促使FcεRI受體交聯而引起變態反應。其它的肥大細胞促分泌素如神經肽P物質，能夠激活百日咳毒素（PTX）敏感性的G蛋白而介導非IgE依賴性的細胞激活。最近的研究指出，肥大細胞表達Toll樣受體家族，提示肥大細胞也積極參與固有免疫反應。本研究主要探討Toll樣受體2激動劑肽聚糖（PGN）和合成激動劑Pam3CSK4對人類肥大細胞的影響，及其對抗原和P物質引起的肥大細胞激活的調控。 / Toll樣受體2激動劑本身不引起人類肥大細胞脫顆粒，但抑制抗原和P物質引起的肥大細胞脫顆粒。鈣動員是引起肥大細胞脫顆粒的關鍵因素。Pam3CSK4通過抑制抗原和P物質鈣動員來抑制肥大細胞脫顆粒。PGN只抑制抗原鈣動員，卻對P物質沒有影響。 / PGN和Pam3CSK4皆刺激人類肥大細胞釋放白細胞介素8(IL-8)和腫瘤壞死因子α（TNF-α）。Pam3CSK4通過激活G₀蛋白，Erk，Ca²⁺/calcineurin/NFAT和TAK信號通路引起肥大細胞釋放IL-8。其間，Go蛋白的激活介導Erk和Ca²⁺/calcineurin/NFAT信號通路的活化。與Pam3CSK4不同，PGN通過激活JNK, Erk, PI3K和TAK信號通路引起肥大細胞釋放IL-8。此外，雖然PTX敏感性G蛋白不影響PGN刺激引起的IL-8釋放，它卻抑制PGN刺激引起的Erk激活。 / Pam3CSK4與抗原協同作用刺激肥大細胞釋放IL-8和TNF-α，PGN與抗原卻並無協同作用。PGN與P物質協同作用刺激肥大細胞釋放IL-8和TNF-α，Pam3CSK4卻幹擾P物質的作用。在Pam3CSK4與抗原的協同作用中，Erk，Ca²⁺/calcineurin/NFAT和TAK信號通路起重要作用。PGN與P物質的協同作用則通過Erk, Ca²⁺/calcineurin/NFAT，NF-κB，PI3K和TAK這五條信號通路。 / 本研究表明，不同的Toll樣受體2激動劑能通過不同的作用機制介導和調控人類肥大細胞的反應。同時，我們首次發現G₀蛋白參與人類肥大細胞Toll樣受體2信號的激活。由於Toll樣受體2與感染和炎症息息相關，繼續研究Toll樣受體2激活對人類肥大細胞的調控機制，有助於促進開發抗感染和炎症藥物，意義深遠。 / Mast cells are activated by IgE-dependent and -independent mechanisms and play a pivotal role in both allergic and inflammatory responses. The classical IgE-dependent mechanism involves the binding of antigens to the receptor-bound IgE and crosslinking of the high-affinity receptor for IgE (FcεRI). For the poly-basic secretagogues, such as the neuropeptide substance P, they can directly stimulate pertussis toxin (PTX)-sensitive G proteins in mast cells in an IgE-independent manner. Recent studies also discover the expression of the Toll-like receptors on mast cells, indicating that mast cells are active players in innate immunity against a wide variety of pathogens. In this study, we investigated the effects of Toll-like receptor 2 (TLR2) ligands peptidoglycan (PGN) and Pam3CSK4 on human mast cell line LAD2 cells activation and the modulatory effects of these TLR2 ligands on LAD2 cells activities in response to anti-IgE and substance P. / TLR2 ligands did not cause significant degranulation on their own, but inhibited anti-IgE and substance P induced degranulation. Pam3CSK4 acted through TLR2, while the inhibitory effect of PGN involved other non-TLR2 related mechanisms. Pretreatment of Pam3CSK4 inhibited calcium mobilization induced by anti-IgE and substance P. However, pretreatment of PGN only inhibited calcium mobilization induced by anti-IgE, but failed to demonstrate similar effect on substance P. / Both TLR2 ligands triggered the release of IL-8 and TNF-α from LAD2 cells in TLR2-dependent manner. G protein, MAPKs, Ca²⁺/calcineurin/NFAT, PI3K/Akt and TAK pathways were differentially activated by PGN and Pam3CSK4. Release of IL-8 induced by Pam3CSK4 required the involvement of G₀ protein, Erk, Ca²⁺/calcineurin/ NFAT and TAK signaling pathways, but not PI3K/Akt and NF-κB. Meanwhile, G₀ protein was required for the upstream regulation of Erk and Ca²⁺/calcineurin/NFAT signaling cascades activated by Pam3CSK4. In contrast to Pam3CSK4, IL-8 release induced by PGN required the activation of JNK, Erk, PI3K and TAK signaling pathways, but not Ca²⁺ /calcineurin/NFAT and NF-κB. PTX-sensitive Gi/o protein was also involved in PGN induced Erk phosphorylation without influencing IL-8 release. / Pam3CSK4 acted in synergy with anti-IgE to augment the release of IL-8 and TNF-α, but PGN failed to demonstrate similar effect. In contrast, PGN acted in synergy with substance P, while co-stimulation of Pam3CSK4 with substance P failed to demonstrate similar synergism. Erk, Ca²⁺/calcineurin/NFAT and TAK signaling pathways were required for the synergistic action of Pam3CSK4 combined with anti-IgE, while synergistic release of IL-8 induced by PGN and substance P required the activation of Ca²⁺/calcineurin/NFAT, Erk, NF-κB, PI3K, and TAK signaling networks and was enhanced by Ca²⁺/calcineurin/NFAT and NF-κB signaling cascades in LAD2 cells, although NF-κB was not required for IL-8 release induced by PGN or substance P. / These ndings suggest that activation of human mast cells LAD2 can be differentially modified by different TLR2 ligands via distinct signaling pathways. We identify for the first time the involvement of G₀ protein in TLR2 signaling transduction in human mast cells. Further studies of the regulation of mast cells by Toll-like receptors will provide important opportunities for the therapeutic manipulation of infection and allergic diseases. / 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. / Yu, Yangyang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 205-233). / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / Publication --- p.vii / Abbreviations --- p.viii / Contents --- p.x / Chapter 1 --- Introduction --- p.1 / Origin of mast cells --- p.1 / Cytokines and growth factors required for mast cells development --- p.3 / Mediators release from mast cell --- p.7 / Mast cells activation by classical IgE-dependent pathway --- p.13 / Substance P and mast cells --- p.20 / Mast cells in host defense --- p.23 / Toll-like receptors and mast cells --- p.25 / Aims --- p.31 / Chapter 2 --- Materials and Methods --- p.33 / Materials --- p.33 / Methods --- p.42 / LAD2 mast cells culture --- p.42 / Degranulation assay --- p.43 / IL-8 and TNF-α measurement --- p.44 / Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.44 / Western Blotting --- p.46 / Calcium mobilization assay --- p.47 / Flow cytometry assay --- p.48 / siRNA Transfection --- p.48 / Statistical analysis --- p.49 / Chapter 3 --- Functional Studies of Toll-Like Receptor 2 on Human Mast Cells Activation --- p.51 / Experimental conditions --- p.56 / Results --- p.57 / Discussions --- p.62 / Chapter 4 --- Modulatory Effects of Toll-Like Receptor 2 on Human Mast Cells in Response to Anti-IgE and the Signaling Pathways Involved in the Events --- p.80 / Experimental conditions --- p.92 / Results --- p.93 / Discussions --- p.102 / Chapter 5 --- Modulatory Effects of Toll-Like Receptor 2 on Human Mast Cells Activation in Response to Substance P and Signaling Pathways Involved in the Event --- p.136 / Experimental conditions --- p.140 / Results --- p.141 / Discussions --- p.152 / Chapter 6 --- General Discussion --- p.188 / Chapter 7 --- References --- p.205

No description available.

Ischaemic stroke has become one of the leading causes of death and disability in the world. Protease activated receptors (PARs, PAR-1 to PAR-4) belong to G protein coupled receptors that can be self-activated by tethered ligands (TL) revealed through proteolytic cleavage. Based on these TL, many activating peptides (APs) and antagonists have been synthesized to investigate PARs actions. / In the present study, the roles of PARs were examined in two models of ischaemic stroke. For the in vivo model, transient middle cerebral artery occlusion (tMCAO) was performed to establish cerebral ischaemia in rats. For the in vitro model, oxygen and glucose deprivation (OGD) was used to mimic an ischaemia insult in primary cultured rat embryonic cortical neurones. / Western blot studies showed that expressions of PAR-1 and PAR-2 were increased in the rat ischaemic brain cortex, whereas PAR-1 was reduced in the rat cortical neurones subjected to OGD. Pretreatments of PAR-1 AP (SFLLRN-NH₂) and PAR-2 AP (SLIGRL-NH₂) produced significant protection against ischaemia-induced damage. Pretreatment of PAR-3 AP (SFNGGP-NH₂) only improved ischaemic symptoms in in vivo but not in in vitro model. When treated after ischaemia, only PAR-1 AP produced significant reductions on ischaemia-induced damage. Protective actions of PAR-1 and PAR-2 APs were inhibited by PAR-1 antagonist (BMS-200261) and PAR-2 antagonist (ENMD-1068) respectively, but PAR-1 antagonist did not affect posttreatment effects of PAR-1 AP in in vitro model. Pre- and posttreatments of thrombin, and pretreatment of trypsin also protected ischaemia-induced damage in the two models. / PAR-1 AP produced marked increase in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and ratio of bcl-2/bax, but reduced contents of reactive oxygen species (ROS), nitric oxide (NO) and malondialdehyde (MDA) in both ipsilateral ischaemic brain cortices and in rat cortical neurones subjected to OGD. In the in vitro model, PAR-1 AP greatly decreased caspase-3 activity and TUNEL positive cells, while markedly increased mitochondrial membrane potential (MMP). All these protective actions were inhibited by its antagonist, which suggests it was mediated via activation of PAR-1. / In MCA isolated from normal and ischameic rats, PAR-2 AP and trypsin produced vasodilatation while PAR-3 AP elicited vasoconstriction. However, another PAR-3 AP had no effect in the two types of MCA. A high concentration of PAR-1 AP relaxed MCA isolated form ischaemic rats, and it was not inhibited by a PAR-1 antagonist. The vasodilator action of PAR-2 AP was inhibited by one of two PAR-2 antagonists tested. The vasodilator actions induced by PAR-1 and PAR-2 APs involved NO production since L-NAME was effective in inhibiting their actions. / In conclusion, PAR-1 AP was found to be the most efficacious in protecting the brain from ischaemia-induced damage when administered either before or after ischaemia insults. The protective actions were likely to be attributed to its anti-oxidant properties in the ischaemic brain that reduced apoptosis of brain cells. Therefore, PAR-1 was identified as a promising target for development of novel prophylactic and therapeutic treatments of ischaemic brain disease. / 缺血性腦中風已經成為全世界導致死亡和殘疾的最主要的疾病之一。蛋白酶激活受體（PARs, PAR-1 to PAR-4）屬於G蛋白偶聯受體並且可以通過蛋白水解生成系鎖配體（TL）從而作用於受體本身而激活信號通路。根據TL的序列已經合成了很多激活肽和拮抗劑，它們可以作為有價值的工具藥進行PAR的作用研究。 / 當前，PAR的作用在兩個缺血性腦中風模型中進行研究。體內模型是通過大鼠大腦中動脈阻塞手術而建立；體外模型是通過對大鼠胚胎大腦皮層神經元進行氧糖剝奪模擬缺血性損傷。 / 蛋白質印跡法的實驗表明PAR-1和PAR-2的表達在缺血側大腦皮層中有所增多，而PAR-1在氧糖剝奪的大鼠皮層神經元中表達卻有所降低。預處理PAR-1（SFLLRN-NH₂）和PAR-2（SLIGRL-NH₂）的激活肽顯著改善了缺血導致的損傷。預處理PAR-3激活肽（SFNGGP-NH₂）僅僅改善了體內缺血症狀，卻對體外缺血模型沒有效果。然而，當這些激活肽在缺血后給予的時候，只有PAR-1的激活肽顯著改善了缺血損傷。PAR-1的拮抗劑（BMS-200261）和PAR-2的拮抗劑（ENMD-1068）抑制了PAR-1和PAR-2激活肽的保護作用，但是體外實驗後處理PAR-1激活肽的保護作用卻未收影響。預處理及後處理凝血酶，預處理胰酶都在這兩個模型中顯示出保護缺血性損傷的作用。 / PAR-1激活肽在缺血同側大腦皮層以及經受氧糖剝奪的大鼠皮層神經元中，顯著提高了超氧化物歧化酶（SOD）、過氧化氫酶（CAT）、谷胱甘肽過氧化物酶（GSH-Px）的活力以及bcl-2/bax的比例，同時顯著降低了活性氧自由基（ROS）、一氧化氮（NO）以及丙二醛（MDA）的含量。在體外模型中，PAR-1激活肽還顯著降低了caspase-3的活力以及TUNEL陽性細胞的比例，同時顯著提高了線粒體膜電位（MMP）。所有這些作用都可以被拮抗劑抑制，說明PAR-1激活肽的保護作用是通過激活PAR-1介導的。 / 不管是從正常還是缺血的大鼠中分離出來的大腦中動脈，PAR-2激活肽和胰酶都可以使之舒張，PAR-3激活肽卻對其有收縮作用。然而，另外一種PAR-3激活肽卻未顯現出對血管活性的影響。高劑量的PAR-1激活肽只可以在分離于缺血大鼠的大腦中動脈中引起舒張，但此作用不能被其拮抗劑所抑制。PAR-2激活肽導致的血管舒張只可以被檢測的兩個拮抗劑中的其中一個所抑制。PAR-1和PAR-2激活肽引起的血管舒張與NO的產生有關，因為L-NAME可以有效抑制它們的作用。 / 總之，不管是預處理還是後處理的給藥方式，PAR-1的激活肽在保護大腦的缺血性損傷中都是最有效果的。保護作用可能可以歸因于其抗氧化以及抗凋亡的特性。所以，PAR-1是研究防治缺血性腦疾病的發展中富有希望的一個靶點。 / Zhen, Xia. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 194-206). / Abstracts also in Chinese. / Title from PDF title page (viewed on 11, October, 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.

No description available.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisors: Dionisios G. Vlachos and Jeremy S. Edwards, Dept. of Chemical Engineering. Includes bibliographical references.

Thesis (M.S.)--University of Montana, 2006. / Title from title screen. Description based on contents viewed Mar. 9, 2007. Includes bibliographical references (p. 21-22).

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 134-172). Also available in electronic version. Access restricted to campus users.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 118-131). Also available in print.