Introdução e objetivo: Existem poucos estudos a respeito da imunidade inata em dermatofitoses (tinhas), este trabalho estudou a expressão dos receptores toll-like 2 e 4 em dermatofitoses por T. rubrum. Casuística e método: Estudaram-se sete pacientes com dermatofitose extensa, definida como pelo menos três segmentos corporais acometidos, e oito com a forma localizada. Inexistia qualquer imunodepressão primária ou secundária nos pacientes. Realizou-se em cada doente biópsia de área da pele lesada e sã, esta última distando pelo menos 4 cm da lesão. Outros 20 fragmentos de pele foram obtidos a partir de cirurgias estéticas. Utilizou-se a imuno-histoquímica com anticorpos anti-TLR2 e TLR4. As imagens foram analisadas pelo programa Image Pro Plus. A epiderme foi dividida em superficial e profunda, para análise da imunomarcação, considerando-se o ponto de divisão como 50% da sua espessura. Resultados: A análise da expressão do TLR4 em pacientes com tinha na epiderme superficial apresentou menores índices de densidade óptica (IDO), média 108,8±7,73 e 110,86±15,57 em pacientes com tinha localizada e extensa, respectivamente, em relação aos controles (145,26 ±21,88) com significância estatística. Também houve redução da expressão do TLR4 na epiderme profunda com IDO de 111,19±13,45 em dermatofitoses extensas e 144,65±17,20 nos controles (p=0,001). A análise da expressão do TLR2 na epiderme profunda em indivíduos com dermatofitose localizada evidenciou menor expressão na área lesada do que na sã do mesmo indivíduo, média 109,28±30,9 e 118,75±36,84, respectivamente (p 0.018). Embora não tenha havido redução da expressão do TLR2 na epiderme superficial comparativamente aos controles, na epiderme profunda encontrou-se, na área lesada das dermatofitoses extensas, menor expressão em relação aos controles, sendo 6,29±9,73 e 27,8±18,6, respectivamente. Conclusões: Encontrou-se menor expressão de TLR4 na epiderme superficial e profunda em indivíduos com dermatofitose comparativamente aos controles sadios. Em dermatofitoses extensas também a expressão de TLR2 está diminuída na epiderme profunda. Não existe diminuição da expressão do TLR2 na epiderme superficial provavelmente para manter a função de barreira da epiderme, onde este receptor é importante para coesão dos queratinócitos. Observou-se ainda menor expressão de TLR2 na pele lesada comparada à sã de indivíduos com dermatofitose localizada o que justificaria o fato destas lesões permanecerem limitadas a uma única área. / Introduction & Objectives: There are few studies to concern the role of innate immune response in dermatophytosis, so we conducted an investigation to define the involvement of TLRs in the course of tinea due T. rubrum infection. Patients & Methods: We allocated 8 patients with localised dermatophytosis and 7 with widespread one, defined as at least on three body segments. The skin was biopsied in two points: from lesion (active lesion) and healthy skin distant at least 4 cm. Twenty controls were obtained from cosmetic surgery. We use immunohistochemical staining with antibodies for antigens TLR 2 and 4. Images were analyzed. Results: (i) analysis of the expression of TLR4 of patients with tinea, found on the upper epidermis, average optical density index of 108,8±7,73 and 110,86±15,57 in localised and widespread tinea, respectively, and 145,26 ±21,88 in control skin; similar reduction maintain at lower one with average optical density index 111,19±13,45 in widespread tinea and 144,65±17,20 in controls p=0,001; (ii) analysis of TLR2 expression in the lower epidermis of patients with tinea met reduced optical density index in skin with localised tinea than in healthy skin, average 109,28±30,9 and 118,75±36,84, respectively, p 0.018. There were no reduction in TLR2 expression in upper epidermis compared to controls, although it was significant reduced in lower epidermis in widespread tinea, average 27,8±18,6 and 6,29±9,73. Conclusions: We found reduced expression of TLR4 in the lower and upper epidermis skin with tinea compared to controls in widespread and localised dermatophytosis. There was no reduction of TLR2 at upper epidermis probably in order to mantain the epidermal barrier function. We found yet a reduced expression of TLR2 in the infected skin compared to healthy one of the same patient with localised dermatophytosis which could explain that in these cases the tinea was not spread in extension.

The primary objective was to ascertain whether implementation of the e-tolling system will influence the spending on recreational activities by people staying in the Vaal Region. Thus the aim was to assess whether an incremental rise in expenses, leading to a decrease in available disposable income will impact people’s decisions to travel outside their residences to visit and engage in leisure destinations and activities respectively. A questionnaire was developed and distributed with the primary objective of determining whether people within the Vaal Region are aware of the costs associated with travelling using the e-toll Gauteng freeways, and whether it will have an impact on their decisions to travel from their respective residences to leisure properties elsewhere. It also probed the respondents for the type of leisure activities they engage in, frequency of visits and the reasons for participating in the respective activities. The study shows that the e-toll project will inevitably, from a monetary perspective, affect all road users travelling from the Vaal Region to the greater Johannesburg areas. This is significant and confirms that the e-toll project will be perceived to have an impact on people’s available and disposable income. With the implementation of the e-tolling project seemingly imminent, businesses and consumers will feel the belt tighten in the leisure and recreational (and in particular the casino) industry, which is dependent on the availability of disposable income, to be ultimately affected. Trends with regards to leisure activities were identified along with recommendations for future research. / MBA, North-West University, Potchefstroom Campus, 2014

The primary objective was to ascertain whether implementation of the e-tolling system will influence the spending on recreational activities by people staying in the Vaal Region. Thus the aim was to assess whether an incremental rise in expenses, leading to a decrease in available disposable income will impact people’s decisions to travel outside their residences to visit and engage in leisure destinations and activities respectively. A questionnaire was developed and distributed with the primary objective of determining whether people within the Vaal Region are aware of the costs associated with travelling using the e-toll Gauteng freeways, and whether it will have an impact on their decisions to travel from their respective residences to leisure properties elsewhere. It also probed the respondents for the type of leisure activities they engage in, frequency of visits and the reasons for participating in the respective activities. The study shows that the e-toll project will inevitably, from a monetary perspective, affect all road users travelling from the Vaal Region to the greater Johannesburg areas. This is significant and confirms that the e-toll project will be perceived to have an impact on people’s available and disposable income. With the implementation of the e-tolling project seemingly imminent, businesses and consumers will feel the belt tighten in the leisure and recreational (and in particular the casino) industry, which is dependent on the availability of disposable income, to be ultimately affected. Trends with regards to leisure activities were identified along with recommendations for future research. / MBA, North-West University, Potchefstroom Campus, 2014

Toll-like receptors (TLRs), named after toll proteins identified in Drosophila melanogaster, are the pattern recognition receptors in the innate immune system that detect microbes. TLRs are mono, membrane-spanning, as well as non-catalytic receptors, which are mainly expressed in sentinel cells, such as the dendritic cells, neutrophils and macrophages. While humans have ten TLRs (TLR 1 to 10), the mouse has another three (TLRs 11, 12, 13). TLRs are made up of glycoproteins, which have luminal ligand-binding sites consisting of leucine-rich repeat (LRR) for detection of pathogens leading to activation of immune cells. TLR1, 2, 4, and 6 are responsible for recognition of lipids (such as triacetylated lipopeptide), peptidoglycan, and lipopolysaccharide (LPS). However, the TLR3, 7, 8, and 9 mainly recognize nucleic acids, such as double-stranded RNA (dsRNA) and CpG DNA, while the TLR13 detects ribosomal RNA sequences. So far, there are no data on the localization and immunological functions of TLR10. I studied the expression, localization and role of TLR10 in S. pneumoniae infection. First, I examined the expression of TLR10 in lungs of pig, cattle, dog, rat, and chickens. The light and electron microscopic data show TLR10 expression in vascular endothelium and smooth muscles in lungs of control and inflamed animals. Further, we found altered basal level of expression and localization of TLR10 in bovine neutrophils treated with E. coli lipopolysaccharide. These data show the expression of TLR10 in the lungs of tested animal species, and its alteration by LPS in bovine neutrophils. The next study was designed to investigate the regulation of TLR10 expression and to address its role in neutrophil chemotaxis. E. coli LPS activated human neutrophils showed temporal and spatial change in TLR10 expression. Confocal microscopy showed cytosolic and nuclear distribution of TLR10 in normal and activated neutrophils. TLR10 in E. coli LPS-activated neutrophils colocalized with flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, suggested its endocytosis. Live cell imaging of LPS activated neutrophils showed TLR10 translocation to the leading edge. Neutrophils upon TLR10 knockdown were unable for fMLP-induced migration. TLR10 knockdown reduced the number of membrane pseudopods in activated neutrophils without altering the expression of key proteins of actin nucleation process, ARP-3 and Diap1. These data show TLR4-mediated pathway for regulation of TLR10 expression, and that TLR10 may have a role in neutrophil chemotaxis. Next, I examined the role of TLR10 in innate immune response to S. pneumoniae infection in U937 human macrophage cell line. S. pneumoniae are major causative agents of pneumonia, meningitis and bacteremia. A significant increase in TLR10 mRNA expression was found in S. pneumoniae (107 cfu for 6hr) challenged macrophages. TLR10 knockdown significantly reduced production of IL-1β, IL-8, IL-17 and TNF-α and no significant change in IL-10 expression, and also significantly diminished nuclear translocation of NF-κB but without affecting the phagocytosis of S. pneumoniae. Altogether, I report the that TLR10 is expressed in the normal and inflamed lungs in cattle, pigs, dogs, rats, chickens and humans. The expression of TLR10 is altered in activated neutrophils, and it plays a role in neutrophils chemotaxis and production of pro-inflammatory cytokines in macrophages infected with S. pneumoniae.

Cytomegaloviruses (CMV), the prototypical β-herpesviruses, have co-evolved with their hosts and thus acquired multiple strategies for modulation of the immune response. Viral engagement of pattern recognition receptors (PRR), such as toll-like receptors (TLRs) and cytosolic nucleic acids sensors, initiates the host immune response through activation of elaborate signalling programs. The ensuing inflammatory response is further sustained and amplified through cytokines, such as IL-1β, activating signalling pathways greatly overlapping those utilized by TLRs. The central hypothesis of this thesis is that a viral counter-measure by murine CMV (MCMV) involves specific targeting of TLR- and IL-1β-induced signalling along the MyD88 to NF-κB pathway. To test this hypothesis MCMV inhibition of IL-1β signalling was initially investigated in a fibroblast cell line. It was demonstrated that in MCMV infected cells IL-1β-induced IκBα degradation is largely inhibited. Comparison of productive and non-productive infection showed this modulation requires de-novo viral gene expression beyond the immediate early region. Further investigations utilising a ORF M45 deletion mutant identified viral gene M45 as necessary for mediating the observed modulation of IL-1β- induced IκBα degradation. To further test the hypothesis, studies were extended to include TLR stimulation in the context of bone marrow-derived macrophages (BMDM) infection. It was found that TLR7/9-induced NF-κB activation is inhibited in MCMV infected BMDM. Overall, data presented in this study demonstrate a previously unrecognised MCMV inhibition of IL-1β- and TLR7/9-induced NF-κB activation, and indicate a role for viral gene M45 in mediating this effect.

Morphine has been and continues to be one of the most potent and widely used drugs for the treatment of pain. Clinical and animal models investigating sex differences in pain and analgesia demonstrate that morphine is a more potent analgesic in males than in females; indeed, we report the effective dose of morphine for female rats is twice that of male rats. In addition to binding to the neuronal mu opioid receptor, morphine binds to the innate immune receptor toll-like receptor 4 (TLR4) on microglia. Morphine action at TLR4 initiates a neuroinflammatory response and directly opposes morphine analgesia. Our recent studies demonstrate that administration of chronic morphine activates microglia within the ventrolateral periaqueductal gray (vlPAG), a critical brain region for the antinociceptive effects of morphine, while blockade of vlPAG microglia increases morphine analgesia and suppresses the development of tolerance in male rats. Despite increasing evidence of the involvement of microglia in altering morphine efficacy, no studies have examined sex differences in microglia within the PAG. The present experiments seek to characterize the distribution and activity of vlPAG microglia in males and females using behavioral, immunohistochemical and molecular techniques, while demonstrating the sufficiency and necessity of vlPAG microglia to produce sex differences in morphine analgesia using site-specific pharmacological manipulation of TLR4. We also investigate a novel pharmacokinetic mechanism underlying the sexually dimorphic effects of morphine administration on microglial activity. Here, we address a fundamental gap in our current understanding of sex differences in morphine analgesia and establish a mechanistic understanding of how the activation of vlPAG microglia sex-specifically influences morphine analgesia.

Mutation, whether spontaneous or induced, is the premier tool for understanding gene function. One approach is to create mutations in a specific gene, and then use the resulting cell or organism to search for a phenotype. An alternative is to create mutations at random, and focus first on the identification of phenotypes. The mutation that underlies a phenotype can then be tracked down, forming the foundation of testable hypotheses. Using random chemical mutagenesis in mice, I have identified 20 heritable phenotypes affecting either the innate or adaptive branches of immunity. The genetic basis of 18 of these phenotypes was solved, caused by mutations in at least 16 unique genes. Five of these genes were not previously known to be involved in immunity, and a detailed analysis of four of them is provided in this thesis. These include genes encoding the following proteins: the inactive rhomboid protease iRhom2, which is specifically required for the secretion of tumour necrosis factor alpha; the hypothetical phospholipid flippase ATP11C, required for B cell development in the adult bone marrow; the folliculin-interacting protein FNIP1, also required for B cell development; and the zinc finger transcription factor ZBTB1, essential for the development of all lymphocyte lineages. These findings uncover new entry points for the understanding of mammalian immunity, and highlight the value of mouse forward genetics in the understanding of mammalian phenomena in general.

Memoria para optar al título de Químico Farmacéutico / El daño ocasionado por la ocurrencia de un infarto cardiaco es complejo, generando la pérdida de viabilidad de las células cardiacas entre otros efectos deletéreos ocurridos tanto en el periodo isquémico de falta de oxígeno y nutrientes, así como también en la posterior reperfusión sanguínea. Frente a esta condición patológica los fibroblastos cardiacos (FC) son capaces de reaccionar, secretando y renovando la matriz extracelular; lo que los convierte en elementos celulares claves en la cicatrización y remodelamiento del tejido cardiaco dañado post-infarto al miocardio. Esto hace necesario el intentar regular la viabilidad de estas células para una correcta cicatrización y mantención de la función cardiaca. En relación a esto se ha reportado el efecto cardioprotector ejercido por el empleo de LPS en condiciones de daño celular causado por isquemia/reperfusión (I/R). Sin embargo, dicho efecto ha sido descrito principalmente en cardiomiocitos, por lo que su efecto en FC es aún desconocido. Nuestro trabajo estudió la capacidad cito-protectora ejercida por LPS sobre FC de ratas neonatas sometidos a un modelo de I/R in vitro e indagó las vías transduccionales implicadas en este efecto. El tratamiento de los FC con LPS (1 μg/mL) durante la isquemia y reperfusión previno la pérdida de viabilidad inducida por I/R. Sin embargo, en el pre-condicionamiento con LPS durante 24 o 16 h, y en el tratamiento con LPS durante la isquemia o reperfusión, no se observó el efecto cito-protector. En esta misma línea, demostramos que el efecto cito-protector ejercido por LPS es mediado a través del receptor TLR4 vía PI3K/Akt y ERK1/2, ya que el empleo de TAK-242, Ly29002 y PD98059, inhibidores del receptor y de las vías transduccionales respectivamente, bloquearon completamente el efecto cito-protector. La activación de TLR4 por LPS previno, además, el procesamiento de la procaspasa 8 y 3 inducido por I/R. Conjuntamente, demostramos que las vías PI3K/Akt y ERK1/2 participaban en la prevención del procesamiento de la procaspasa 8 ejercido por LPS, pero no tuvieron efecto en la activación de la caspasa 3. Nuestros resultados dan cuenta del efecto cito-protector y antiapoptótico ejercido por LPS a través de TLR4 vía PI3K/Akt y ERK1/2 frente a la muerte de FC inducida por I/R / The damage caused by a myocardial infarct is complex, causing cardiac cell viability loss, among other deleterious effects that occur during the ischemia period, such as lack of oxygen and nutrients; as well as the posterior reperfusion with blood. In this situation, cardiac fibroblasts react by secreting proteins and renewing the extracellular matrix. These properties make them a key element in the scar and remodeling process of the injured cardiac tissue. Thus, the viability of these cells are required to preserve the correct functioning of the heart. In this regard, the use of LPS as a cytoprotector agent in cardiac Ischemia/reperfusion (I/R) has been reported. However such effect has been described mostly in cardiomyocytes cells, whereas its role in cardiac fibroblasts remains unknown. Our work studied the LPS cytoprotector effect in neonate cardiac fibroblast exposed to an in vitro model of I/R, and transductional pathways involved in this process were explored. Incubation with LPS (1μg/mL) during both ischemia and reperfusion periods prevented the I/R-induced cell loss. However, LPS used only during preconditioning, ischemia or reperfusion did not induced cytoprotection. Furthermore we demonstrated that the LPS-dependent protective effects were completely abolished when TLR4 receptor (TAK-242), and PI3K/Akt and ERK1/2 (Ly29002 and PD98059, respectively) inhibitors were used. These data suggest that LPS-dependent cytoprotector effects are mediated through TLR4 receptor activation and PI3K/Akt and ERK1/2 signalling pathways. Additionally the activation of TLR4 by LPS prevented the cleave of procaspases 3 and 8 induced by I/R. Both PI3K/Akt and ERK1/2 were necessary for the prevention of the caspase 8 activation, but not of caspase 3. Our results conclude that LPS protects cardiac fibroblasts from I/R apoptosis by the activation of TLR4 and both PI3K/Akt and ERK1/2 signalling pathways / FONDECYT

Os tecidos periodontais estão em confronto contínuo com microrganismos capazes de disparar mecanismos da resposta imune inata, dando origem ao infiltrado inflamatório neutrofílico. A participação dos receptores tipo Toll (TLRs) na resposta de neutrófilos frente à periodontopatógenos associados à doença periodontal precisam ser determinados. Nesse estudo procuramos caracterizar o infiltrado inflamatório presente no peritônio de animais deficientes de TLR2-/-, avaliar a atividade fagocítica, bem como a produção de óxido nítrico (NO) e a atividade de mieloperoxidase (MPO) no curso da infecção por Aggregatibacter actinomycetemcomitans (Aa). Os resultados revelaram um menor recrutamento de leucócitos para o peritônio de animais deficientes de TLR2 (TLR2-/-), com predomínio de macrófagos no exsudato peritoneal tanto de animais selvagens (WTTLR2-/-) como nos animais nocautes. A análise da atividade fagocítica revelou uma menor taxa de fagocitose pelas células do exsudato de animais TLR2-/- e, ainda, a deficiência do receptor TLR2 inibiu a produção de NO (óxido nítrico) e aatividade de MPO (mieloperoxidase). Adicionalmente, são apresentados os resultados referentes ao protocolo de doença periodontal experimentalmente induzida (DPEI) com Aggregatibacter actinomycetemcomitans (Aa) em camundongos deficientes de TLR2. Os resultados mostraram que 100% dos animais deficientes de TLR2 sobreviveram à infecção durante o período de observação. Em relação à análise de perda óssea os dados revelaram uma maior perda progressiva de osso alveolar na região dos molares de animais deficientes de TLR2. A ausência do receptor interferiu com a disseminação da bactéria, uma vez que se observou um grande número de bacilos no linfonodo e baço dos animais que não expressaram TLR2, diferente do observado para os animais selvagens (WTTLR2-/-). Os resultados indicam a importância da sinalização via TLR2 durante a resposta imune contra Aggregatibacter actinomycetemcomitans. / Polymorphonuclear leukocytes (PMNs) are the first line of defense against bacterial infections. PMNs express a numerous pattern-recognition receptors (PRR) that facilitate identification of invading microorganisms. Toll-like receptors (TLRs) represent the main class of PRR involved to a recognize pathogenic microorganism. However, the role played by TLR-2 in the recognition and killing of Aggregatibacter actinomycentemcomitans by PMNs is unknown. Thus, we investigated the ability of TLR-2 to mediate neutrophil phagocytosis and bactericidal activity. To determine the role of A. actinomycentemcomitans in triggering neutrophil infiltration, TLR2-/- mice were infected intraperitoneally (2x109 bacteria) and sacrificed after 24 hours. Peritoneal inflammatory cells were isolated and analyzed by optical microscopy. Examination of local inflammatory infiltrates revealed that neutrophil influx into peritoneal cavity of TLR2-/- mice was similar than that observed into their littermate controls wild type C57BL/6 mice (WT). A. actinomycentemcomitans was detected in the spleen of the TLR2-/- mice but not in WT. In the phagocytic assays, TLR2-/- presented minor number of ingested inflammatory cells than WT. Peritoneal cells were stimulated with A. actinomycetemcomitans antigens, and NO and myeloperoxidase was measured after 48 hours; results showed that TLR2-/- cells produce less NO and MPO than WT. In addition, TLR2-/- deficient mice presented higher bone loss following oral infection with A. actinomycetemcomitans when compared with WT and higher tissue destruction.

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