Dissertations / Theses on the topic 'Tumor Necrosis Factor Alpha α-SMA'

Die Haut ist das größte Organ des Menschen und bildet die Barriere gegenüber Einwirkungen aus der Umwelt. Die Störung der Hautbarriere durch exogene und endogene Reize führt zu einer Entzündungsreaktion in der Haut. In der Folge können Hauterkrankungen wie die irritative oder Atopische Dermatitis entstehen. Der Tumor Nekrose Faktor-α (TNF-α) ist ein pleiotrop wirksames Zytokin, das eine zentrale Rolle bei entzündlichen Prozessen spielt. Ziel der vorgelegten Promotionsarbeit war zu untersuchen, ob und wie TNF-α zu Entzündungsgeschehen, ausgelöst durch exogene und endogene Faktoren, beiträgt. Die Bedeutung von TNF-α wurde in TNF-ko Mäusen in verschiedenen Hautmodellen untersucht. Für das Irritationsmodell wurden chemische und physikalische Reize verwendet. TSLP (Thymic stromal lymphopoietin) wurde durch die verschiedenen Stimuli signifikant induziert. Diese Induktion war unabhängig von der endogenen TNF-α Produktion, gezeigt durch den Einsatz von TNF- ko Mäusen . Da endogenes TNF-α für die Hautirritation keine notwendige Bedingung darstellte, wurde die Bedeutung von TNF-α bei der atopischen Dermatitis (AD) untersucht. TNF-α defiziente Mäuse zeigen verstärkt Ekzeme im Vergleich zu Wildtyp Mäusen. Die Behandlung von TNF-ko Mäusen mit einem TSLP Antikörper führte zu einer Verminderung des Ekzems. Mastzellen wurden vermehrt in läsionaler Haut gefunden und korrelierten mit dem Schweregrad des atopischen Ekzems sowie der TSLP-Expression.
The skin is the largest organ of an individuum and builds the barrier for a host against the environment. Skin barrier disruption by exogenous or endogenous stimuli can lead to skin inflammation. As a consequence, irritant or atopic eczema, frequent skin diseases, may evolve. Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine which plays a central role in inflammatory processes. The main aim of this thesis was to clarify whether and how endogenous TNF-α is contributing to skin inflammation driven by exogenous and endogenous triggers. The role of endogenous TNF-α was studied using TNF knockout (-/-) mice. In an irritation model, chemical and physical stimuli were applied on to mouse skin. Thymic stromal lymphopoietin (TSLP) was significantly induced by the used irritants. This TSLP induction was independent from endogenous TNF-α proven by using TNF-/- mice. Next the role of TNF-α in atopic dermatitis (AD) promoting an allergic skin inflammation was investigated. TNF-/- mice developed more severe AD compared to the wildtype mice and TSLP was significantly increased and correlated with the severity of the eczema. To prove the pathophysiological role of TSLP for AD progression, TNF-/- mice were pretreated with an TSLP antibody. Indeed, these mice developed less AD symptoms compared to the control mice. Mast cells (MCs) were also significantly increased in lesional skin in the AD model and moreover, correlated with AD severity, but also with TSLP expression.

L'interleukine-17A (IL-17A) et le facteur de nécrose tumorale alpha (TNF-α) sont des cytokines pro-inflammatoires impliquées dans la pathogénèse de plusieurs maladies articulaires. Au cours de la polyarthrite rhumatoïde (PR), une augmentation de la destruction osseuse ainsi qu'un defaut de réparation sont responsables des dommages articulaires. Cependant au cours de la spondylarthrite ankylosante (AS), une importante ossification ectopique est observée, conduisant à la formation de syndesmophytes, associé à une perte de la masse osseuse systémique. Récemment, l'étude de ces cytokines a conduit à la publication de résultats contradictoires. Notre objectif a donc été d'étudier l'effet de ces deux cytokines sur la différenciation ostéogénique de cellules souches mésenchymateuses humaines isolées (hMSCs) et de fibroblastes de la membrane synoviale (FLS). Tous les modèles de cellules utilisés, ont démontré que l'IL-17A et le TNF-α augmentent de manière synergique l'ostéogénèse. Ceci semble se rapprocher du modèle de l'AS où une formation d'os ectopique est observée dans laquelle l'IL-17A et le TNF-α jouent un rôle majeur. En parallèle, ces deux cytokines stimulent localement les ostéoclastes, entraînant une perte de masse osseuse observée à la fois dans la PR et dans l'ostéoporose. Cibler simultanément l'IL-17A et le TNF-α pourrait conduire à une diminution de l'infiltration de cellules et de la destruction articulaire observée dans la PR et pourrait ainsi réduire les effets des FLS PR sur l'activation de l'ostéoclastogénèse
Interleukin-17A (IL-17A) and tumor necrosis factor alpha (TNF-α) are pro-inflammatory cytokines involved in the pathogenesis of several arthritic diseases. In rheumatoid arthritis (RA), joint damage is a result of an increase in bone destruction and a decrease in bone repair. In contrast, in ankylosing spondylitis (AS), a bone mass loss accompanied by a significant ectopic ossification is observed leading to the formation of syndesmophytes. Recent studies led to contradictory findings regarding the role of IL-17A and TNF-α in arthritic disease. Therefore, our objective was to study the effect of these two cytokines on the osteogenic differentiation of isolated human mesenchymal stem cells (hMSCs) and fibroblasts of the synovial membrane (FLS). In all the cell models used, we demonstrated that Il-17A and TNF α synergistically increase osteogenesis. This seems to approach the model of AS where ectopic bone formation is observed and in which IL-17A and TNF-α both are involved. These cytokines stimulate osteoclasts locally resulting in loss of bone mass observed in both RA and osteoporosis. Thus, targeting IL-17A and TNF-α could lead to a decrease in cell infiltration and joint destruction which is observed in RA and may reduce the effects of RA FLS on the activation of osteoclastogenesis

Tumor necrosis factor-alpha (TNF-α) is a cytokine produced primarily by macrophages during acute inflammation. In this study we examined the differential effect of retinoic acid (RA) and phorbol 12-myristate 13-acetate (PMA) on the induction of TNF-α secretion from U937 monocytic cell populations by using the reverse hemolytic plaque assay (RHPA). The RHPA will allow us to investigate both changes in TNF-α secreting populations as well as monitor the relative amount of TNF-α released from individual cells. Our results indicate that treatment of U937 cells with RA (10-6M) moderately increases the secreting cell populations, and dramatically enhances the amount of TNF-α secreted from cells already committed to secretion. In contrast, treatment with PMA (250ng/ml) drastically increased the secreting population, but only slightly increasing the amount of TNF-α released. These results suggest that induction of TNF-α secretion from U937 cells occurs by different pathways.

Background: Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration dependently. Results: Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24–48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8–12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12–24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release. Conclusions: Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse.

Ginsenosides (GF) are a major bioactive constituent of ginseng and have been shown to elicit a multitude of actions ranging from the improvement of synaptic plasticity to the improved uptake of glucose into a cell. Furthermore, ginsenosides and their metabolites have been shown to be potent anti-cancer agents in multiple experimental cancer models. The aim of this study was to investigate the potential influence of GF derived from American ginseng root (Panax quinquefolius), and a ginsenoside metabolite Rh2, on tumor necrosis factor-α (TNF-α) cytotoxicity in MDA-MB 231 and MCF-7 human breast cancer cells. In combination, these agents significantly increased cell death in both cell lines. Together, ginsenosides and TNF-α induced a robust increase of the pre-G0/G1 and accompanying decrease in S phase cell populations in breast cancer cells. This cell death was the result of the induction of apoptosis, as determined by annexin-V/7-AAD and Hoechst staining. Furthermore, the mechanism of ginsenoside and TNF-α induced apoptosis is caspase-dependent, as determined by the pan-caspase inhibitor Z-VAD-FMK, with caspase-8, but not caspase-9, serving as initiator caspase in both cell lines. Additionally, ginsenoside treatment significantly XIAP expression in both MDA-MB 231 and MCF-7 cells, in the absence of TNF-α. In addition to enhancing apoptosis, it was also hypothesized that ginsenosides would abrogate pro-survival pathways induced by TNF-α. However, ginsenosides failed to block TNF-α effects on NFκB expression in either cell line. JNK which, when activated by TNF-α in MDA-MB 231 cells has a pro-survival function, was reduced by ginsenosides. However, JNK inhibition had no effect on cell death, suggesting that it does not play an integral role in the mechanism of action. In MCF-7 cells, JNK has been shown to have a pro-apoptotic function. Treatment with ginsenosides had no effect on TNF-α activation of JNK, but inhibition of JNK significantly reduced cell death in combined ginsenoside and TNF-α treated cells. To conclude, combined treatment with ginsenosides and TNF-α can enhance cell death in the sensitive MCF-7 cell line, and induce cell death in the insensitive MDA-MB 231 cell line in a caspase-dependent manner that is aided by the reduction of XIAP by ginsenosides.

Deux facteurs de croissance jouent un rôle prépondérant dans le processus lésionnel rénal : l’EGF et l’angiotensine II (AngII). Le but de mon travail de thèse a été d’évaluer si la transactivation du récepteur de l’EGF (R-EGF) était responsable de l’effet délétère de l’AngII lors du développement des lésions rénales, et, si c’était le cas, d’identifier les mécanismes moléculaires à l’origine de ce phénomène. Nous avons montré par des modèles expérimentaux de néphropathie chronique, des lignées de souris génétiquement modifiées et des inhibiteurs pharmacologiques, que les lésions rénales induites par l’AngII passent par l’activation du R-EGF, via la surexpression d’un de ces ligands, le TGF-, elle-même provoquée par l’activation de sa métalloprotéase de clivage, TACE. Puis nous avons débuté l’étude du rôle du TGF- et de sa modulation par la voie de l’AngII dans la pathologie rénale humaine et plus particulièrement dans la polykystose. Nous avons observé une surexpression de TGF- et de TACE dans l’épithélium kystique. L’excrétion urinaire de TGF- est associée à la sévérité de l’insuffisance rénale dans la polykystose et dans différentes néphropathies. Cette excrétion semble dépendre de la prise d’inhibiteur de la voie de l’AngII et être corrélée à une détérioration plus rapide de la fonction rénale. Ceci suggère que le TGF- est impliqué dans le processus lésionnel de diverses néphropathies chroniques humaines et que sa production pourrait être régulée par l’AngII. Ce travail ouvre de nouvelles perspectives dans la recherche de molécules susceptibles de modifier l’évolution des maladies rénales et de traitements capables de ralentir la progression des lésions rénales.

L'effet de l'Interleukine 1 (IL-1) et du Tumor Necrosis Factor α (TNF-α) sur la production de collagène a été étudiée in vitro chez les fibroblastes dermiques et les cellules synoviales. L'IL-1 exerce un effet inhibiteur sur la synthèse collagénique, effet accompagné par une forte accumulation de prostaglandine E2 (PGE2) dans les milieux de culture. Le blocage de la voie de la cyclooxygénase du métabolisme de l'acide arachidonique empêche l'action de l'IL-1 chez les synoviocytes, ce qui suggère l'implication de la PGE2 dans l'effet exercé par cette cytokine, mais pas chez les fibroplastes. Les taux d'ARNm des procollagènes I et III sont augmentés par l'IL-1 chez les deux types cellulaires. Des mécanismes post-transcriptionnels interviennent donc pour s'opposer à l'augmentation de l'expression des gènes des procollagènes. Le TNF-α réduit la production de collagène des fibroplastes dermiques. Le blocage de la production de PGE2 par l'indométacine n'empêche pas l'action du TNF-α. Celui-ci induit une forte diminution des taux d'ARNm des procollagènes I et III. La réduction de la synthèse collagénique par le TNF-α s'exerce essentiellement au niveau transcriptionnel. La mesure des taux d'ARNm codant pour l'IL-1ß chez les fibroblastes dermiques indique une forte stimulation sous l'effet de l'IL-1 ainsi que du TNF-α. Ceci laisse présager une régulation locale de la synthèse de matrice conjonctive par les fibroblastes eux-mêmes.

Oxidative stress and inflammation are important factors involved in the progression of heart failure. An important cytokine produced during myocardial infarction (MI) is tumor necrosis factor alpha (TNF-α). TNF-α may induce oxidative stress, cell damage, apoptosis and cardiac dysfunction. Considering the anti-inflammatory and anti-oxidant properties of extra-virgin olive oil and its major component (80%) oleic acid (OA), and their benefits to the cardiovascular system, we hypothesized that the negative effects of TNF-α in the pathogenesis of heart failure will be mitigated by olive oil consumption. This hypothesis was tested by examining the effects of a special diet supplemented with 10% olive oil, in coronary artery ligated animal model of MI. Corn oil (10%) supplementation was used as a control for matching caloric intake. Animals in the sham and ligated groups fed regular chow, olive oil, and corn oil were studied at 4 and 16 weeks post myocardial infarction (PMI). Mortality, diet consumption, weight gain and conduction system abnormalities were comparable among all ligated groups. Echocardiography showed that MI deteriorated cardiac function, and olive oil restored the function. At 16 weeks PMI, only corn oil fed groups showed significant increase in both total cholesterol and HDL. Corn oil was not able to offer protection to the heart, suggesting that the beneficial effects of olive oil are not due to increased caloric intake or increased HDL. MI increased myocardial TNF-α, oxidative stress, lipid peroxidation, pro-apoptotic protein expression (Bax, cleaved Caspase 3, cleaved PARP, TGFβ, Bnip3), cytochrome C release, MAP kinase activation (p38, JNK) and decreased anti-apoptotic protein Bcl-xL expression at both 4 and 16 weeks PMI, and these changes were modulated by olive oil. In order to further test the central role of TNF-α PMI, we examined the possible miti-gation of TNF-α induced changes by OA in isolated adult rat cardiomyocytes. TNF-α in-creased oxidative stress, cell damage, cell death, and apoptosis, while OA treatment miti-gated these TNF-α induced effects. We concluded that TNF-α is implicated in the progression of heart failure subsequent to MI and that OA in olive oil may prevent this progression, through its anti-oxidant, anti-inflammatory, anti-hypertensive, and inotropic effects.

BACKGROUND AND AIMS: Congenital Hepatic Fibrosis (CHF) is caused by mutations in PKHD1, a gene encoding for fibrocystin, a protein of unknown function, expressed in cholangiocyte cilia and centromers. In CHF, biliary dysgenesis is accompanied by severe progressive portal fibrosis and portal hypertension. The mechanisms responsible for portal fibrosis in CHF are unclear. The αvβ6 integrin mediates local activation of TGFβ1 and is expressed by reactive cholangiocytes during cholestasis. To understand the mechanisms of fibrosis in CHF we studied the expression of αvβ6 integrin and its regulation in Pkhd1del4/del4 mice. METHODS: In Pkhd1del4/del4 mice we studied, at different ages (1-12 months): a) portal fibrosis (Sirius Red) and portal hypertension (spleen weight/body weight); b) αvβ6 mRNA and protein expression (RT-PCR, IHC); c) α-SMA and TGFβ1 mRNA expression (RT-PCR); d) portal inflammatory infiltrate (IHC for CD45 and FACS analysis of whole liver infiltrate); f) cytokines secretion from cultured monolayers of primary cholangiocytes (Luminex assay); g) cytokine effects on monocyte/macrophage proliferation (MTS assay) and migration (Boyden chamber); h) TGFβ1 and TNFα effects on β6 integrin mRNA expression by cultured cholangiocytes before and after inhibition of the TGFβ receptor type II (TGFβRII); i) TGFβ1 effects on collagen type I (COLL1) mRNA expression by cultured cholangiocytes. RESULTS: Pkhd1del4/del4 mice showed a progressive increase in αvβ6 integrin expression on biliary cyst epithelia. Expression of αvβ6 correlated with portal fibrosis (r=0.94, p<0.02) and with enrichment of a CD45+ve cell infiltrate in the portal space (r=0.97, p<0.01). Gene expression of TGFβ1 showed a similar age-dependent increase. FACS analysis showed that 50-75% of the CD45+ve cells were macrophages (CD45/CD11b/F4/80+ve). Cultured polarized Pkhd1del4/del4 cholangiocytes secreted from the basolateral side significantly increased amounts of CXCL1 and CXCL10 (p<0.05). Both cytokines were able to stimulate macrophage migration (p<0.05). Basal expression of β6 mRNA by cultured Pkhd1del4/del4 cholangiocytes (0.015±0.002 2^-dCt) was potently stimulated by the macrophage-derived cytokines TGFβ1 (0.017±0.002 2^-dCt, p<0.05) and TNFα (0.018±0.003 2^-dCt, p<0.05). Inhibition of TGFβRII completely blunted TGFβ1 (0.014±0.003 2^-dCt, p<0.05) but not TNFα effects (0.017±0.001 2^-dCt, p=ns) on β6 mRNA. COLL1 mRNA expression by cultured Pkhd1del4/del4 cholangiocytes (0.0009±0.0003 2^-dCt) was further and significantly increased after TGFβ1 stimulation (0.002±0.0005 2^-dCt, p<0.05). CONCLUSIONS: Pkhd1del4/del4 cholangiocytes possess increased basolateral secretory functions of chemokines (CXCL1, CXCL10) able to orchestrate macrophage homing to the peribiliary microenvironment. In turn, by releasing TGFβ1 and TNFα, macrophages up-regulate αvβ6 integrin in Pkhd1del4/del4 cholangiocytes. αvβ6 integrin activates latent TGFβ1, further increasing the fibrogenic properties of cholangiocytes.

HIV/AIDS is raging and causing millions of deaths around the world. The major challenge in treating HIV/AIDS is the establishment of HIV reservoirs where the viruse escapes both drug and immune system attempts at eradication. Throughout the course of HIV/AIDS, productive HIV infection occurs primarily in the lymphoid follicles or germinal centers (GC) surrounding follicular dendritic cells (FDC). In the GCs, FDCs trap and maintain infectious HIV for years and provide these infectious viruses to the host cells. FDCs also attract B and T cells into the GCs and increase the ability of CD4+ T cells to be infected. Additionally, FDCs also mediate the increase of HIV replication in HIV-infected CD4+ T cells. Recently, several clinical cases and in vitro studies suggest that alpha-1-antitrypsin (AAT) might inhibit HIV infection and replication. Therefore, I hypothesized that AAT inhibited both the infection and replication of HIV in primary CD4+ T cells. I also postulated that AAT inhibited the FDC-mediated contributions that potentiate HIV infection and replication. To test whether AAT inhibited HIV infection in lymphocytes, CD4+ T cells were pretreated with AAT and then incubated with HIV to detect HIV infection. To exam whether AAT inhibited HIV replication, infected CD4+ T cells were cultured with AAT to detect the replication of HIV. To determine whether AAT blocked the FDC-mediated contributions to HIV pathogenesis, activated or resting FDCs were treated with AAT to detect the trapping and maintenance of HIV. The results suggested that AAT inhibited HIV entry into CD4+ T cells by directly interacting with gp41 and thereby inhibiting the interaction between HIV and CD4+ T cells. AAT also inhibited HIV replication in infected CD4+ T cells. Further study revealed that AAT interacted with low-density lipoprotein-receptor related protein to mediate the internalization of AAT through a clathrin-dependent endocytic process in CD4+ T cells. Subsequently, internalized AAT was transported from the endosome to the lysosome and then released into the cytosol. In the cytosol, AAT directly interacted with IκBα to block its polyubiquitinylation at lysine residue 48, which resulted in the accumulation of phosphorylated/ubiqutinylated IκBα in the cytosol. In turn, the dissociation of IκBα from NF-κB was blocked, which thereby inhibited the nuclear translocation and activation of NF-κB. Additionally, AAT also down-regulated FDC-CD32 and FDC-CD21 expression, which are regulated by NF-kB, thereby inhibiting the trapping and maintenance of HIV on FDCs. Hence, AAT not only suppresses HIV replication, but also blocks HIV replication in CD4+ T cells. Moreover, AAT also inhibits the activation of FDCs thereby affecting the trapping and maintenance of HIV.

by Shan Sze Wan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (leaves 145-166).
Abstracts in English and Chinese.
Title --- p.i
Abstract --- p.ii
摘要 --- p.v
Acknowledgements --- p.vii
Table of Contents --- p.viii
List of Abbreviations --- p.xiv
List of Figures --- p.xvii
List of Tables --- p.xx
Chapter Chapter 1 --- Introduction
Chapter 1.1 --- What are the general functions of cytokines? --- p.2
Chapter 1.2 --- What is TNP-α? --- p.4
Chapter 1.3 --- Actions of TNF-α --- p.5
Chapter 1.4 --- General functions of TNF-α in astrocytes --- p.6
Chapter 1.5 --- TNF-α receptors (TNF-Rs) --- p.8
Chapter 1.6 --- Second messengers induced by TNP-α --- p.10
Chapter 1.7 --- Glial Cells --- p.11
Chapter 1.7.1 --- Oligodendroglia --- p.12
Chapter 1.7.2 --- Brain Macrophages (Microglia) --- p.12
Chapter 1.7.3 --- Astrocytes --- p.14
Chapter 1.7.3.1 --- Functions of astrocytes --- p.15
Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.20
Chapter 1.9 --- β-Adrenergic receptors (β-ARs) --- p.21
Chapter 1.9.1 --- The active functional unit: the receptor complex --- p.22
Chapter 1.9.2 --- General functions and distribution of β-ARs --- p.22
Chapter 1.10 --- Functions of β-ARs in astrocytes --- p.24
Chapter 1.10.1 --- Regulations of astrogliosis by β-ARs --- p.24
Chapter 1.10.1.1 --- β-ARs are expressed in normal optic nerves and up-regulated after nerve crush --- p.24
Chapter 1.10.1.2 --- Injury-induced alterations in endogenous catecholamine leads to enhanced β-AR activation --- p.25
Chapter 1.10.1.3 --- β-AR blockade suppresses glial scar formation --- p.25
Chapter 1.10.1.4 --- β-AR agonists affect the proliferation of astrocytes in normal brain --- p.26
Chapter 1.11 --- Manganese Superoxide Dismutase (MnSOD) --- p.27
Chapter 1.11.1 --- MnSOD is the target gene of NF-kB --- p.29
Chapter 1.11.2 --- Induction of MnSOD by proinflammatory cytokines in rat primary astrocytes --- p.29
Chapter 1.11.3 --- SMase and ceramides induce MnSOD in various cell types --- p.30
Chapter 1.12 --- Why do we use C6 glioma cells? --- p.31
Chapter 1.13 --- Aims and Scopes of this project --- p.32
Chapter Chapter 2 --- MATERIALS AND METHODS
Chapter 2.1 --- Materials --- p.36
Chapter 2.1.1 --- Cell Line --- p.36
Chapter 2.1.2 --- Cell Culture Reagents --- p.36
Chapter 2.1.2.1 --- Complete Dulbecco´ةs modified Eagle medium (CDMEM) --- p.36
Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.37
Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.37
Chapter 2.1.3 --- Recombinant cytokines --- p.38
Chapter 2.1.4 --- Chemicals for signal transduction study --- p.38
Chapter 2.1.4.1 --- Modulators of protein kinase C (PKC) --- p.38
Chapter 2.1.4.2 --- Modulator of protein kinase A (PKA) --- p.39
Chapter 2.1.4.3 --- β-Adrenergic agonist and antagonist --- p.39
Chapter 2.1.5 --- Antibodies --- p.40
Chapter 2.1.5.1 --- Anti-TNF-receptor type 1 (TNF-R1) antibody --- p.40
Chapter 2.1.5.2 --- Anti-TNF-receptor type 2 (TNF-R2) antibody --- p.41
Chapter 2.1.5.3 --- Anti-βi-adrenergic receptor (βl-AR) antibody --- p.42
Chapter 2.1.5.4 --- Anti-β2-adrenergic receptor (β2-AR) antibody --- p.42
Chapter 2.1.5.5 --- Antibody conjugates --- p.43
Chapter 2.1.6 --- Reagents for RNA isolation --- p.43
Chapter 2.1.7 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.43
Chapter 2.1.8 --- Reagents for electrophoresis --- p.45
Chapter 2.1.9 --- Reagents and buffers for Western blot --- p.45
Chapter 2.1.10 --- Other chemicals and reagents --- p.47
Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.47
Chapter 2.3 --- RNA isolation --- p.48
Chapter 2.3.1 --- Measurement of RNA yield --- p.49
Chapter 2.4 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.50
Chapter 2.5 --- Western blot analysis --- p.52
Chapter Chapter 3 --- RESULTS
Chapter 3.1 --- Effect of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.55
Chapter 3.1.1 --- Effect of TNF-α on TNF-R1 and -R2 mRNA expression in C6 cells --- p.56
Chapter 3.1.2 --- The signaling systems mediating TNP-α-induced TNF-R2 expression in C6 cells --- p.57
Chapter 3.1.2.1 --- The involvement of PKC in TNF-α-induced TNF-R2 expression in C6 cells --- p.57
Chapter 3.1.2.2 --- Effect of PMA on the TNF-R protein levels in C6 cells --- p.63
Chapter 3.1.2.3 --- Effect of Ro31 on the TNF-α-induced TNF-R protein level in C6 cells --- p.65
Chapter 3.1.2.4 --- Effect of PKA activator on the level of TNF-R2 mRNA in C6 cells --- p.67
Chapter 3.2 --- Effect of TNP-α on the expression of β1- and β2-adrenergic receptors (β1- and β2-ARs) in C6 glioma cells --- p.69
Chapter 3.2.1 --- Effect of TNF-α on β1- and β2-ARs mRNA expression in C6 cells --- p.70
Chapter 3.2.2 --- The signaling systems mediating TNF-α-induced β1- and β2-AR expression in C6 cells --- p.70
Chapter 3.2.2.1 --- The involvement of PKC mechanism between TNF-α and β-ARs in C6 cells --- p.71
Chapter 3.2.2.2 --- Effect of PMA on the β1- and β2-ARs protein level in C6 cells --- p.76
Chapter 3.2.2.3 --- Effect of Ro31 on the TNF-α-induced β1- and β2-AR protein levels in C6 cells --- p.78
Chapter 3.2.2.4 --- Effect of dbcAMP on the levels of βl- and β2-ARs mRNA in C6 cells --- p.80
Chapter 3.3 --- Relationship between TN1F-R2 and β-adrenergic mechanism in C6 cells --- p.82
Chapter 3.3.1 --- Effects of isproterenol and propranolol on endogenous TNF-α mRNA levels in C6 cells --- p.82
Chapter 3.3.2 --- Effects of isoproterenol and propranolol on TNF-R2 mRNA levels in C6 cells --- p.83
Chapter 3.3.3 --- Effects of β1-agonist and antagonist on endogenous TNF-α mRNA expression in C6 cells --- p.87
Chapter 3.3.4 --- Effects of β1-agonist and antagonist on TNF-R2 mRNA expression in C6 cells --- p.91
Chapter 3.3.5 --- Effects of β2-agonist and antagonist on endogenous TNF-α mRNA in C6 cells --- p.93
Chapter 3.3.6 --- Effects of β2-agonist and antagonist on TNF-R2 mRNA in C6 cells --- p.100
Chapter 3.4 --- Effect ofTNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.102
Chapter 3.4.1 --- Effect ofTNF-α on NF-kB (p50) mRNA expression in C6 cells --- p.106
Chapter 3.4.2 --- Effect of β-agonist and antagonist on NF-kB (p50) mRNA expression in C6 cells --- p.108
Chapter 3.4.3 --- Effect of PMA and Ro31 on the levels of NF-kB mRNA in C6 cells --- p.109
Chapter 3.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.111
Chapter 3.5.1 --- Effects of TNF-α on MnSOD and Cu-ZnSOD mRNAs expression in C6 cells --- p.114
Chapter 3.5.2 --- Effects of β-agonist and β-antagonist on MnSOD mRNA expression in C6 cells --- p.115
Chapter 3.5.3 --- Effects of PKC activator and inhibitor on the levels of MnSOD mRNA in C6 cells --- p.117
Chapter Chapter 4 --- DISCUSSION AND CONCLUSION
Chapter 4.1 --- Effects of TNF-α on the expression of TNF-receptors (TNFRs) in C6 glioma cells --- p.122
Chapter 4.2 --- Effects of TNF-a on the expression of β1- and β2-adrenergic receptors (β1 and β2-ARs) in C6 glioma cells --- p.126
Chapter 4.3 --- Relationship between TNF-α and β-adrenergic mechanism in C6 cells --- p.128
Chapter 4.4 --- Effects of TNF-α on the expression of a transcriptional factor nuclear factor kappa B (NF-kB) in C6 glioma cells --- p.131
Chapter 4.5 --- Effects of TNF-α on the expression of manganese superoxide dismutase (MnSOD) in C6 glioma cells --- p.133
Chapter 4.6 --- Possible sources of β-agonists --- p.136
Chapter 4.7 --- Conclusions --- p.137
Appendix A --- p.143
References --- p.145

Ngan, Kit Shan.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 176-187).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.

by Yuen Wai Fan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 211-232).
Abstracts in English and Chinese.
Acknowledgements --- p.i
List of Publications and Abstracts --- p.ii
Abbreviations --- p.iv
Abstract --- p.xi
Abstract in Chinese --- p.xiv
List of Figures --- p.xvii
List of Tables --- p.xxiii
Contents --- p.xxiv
Chapter Chapter 1. --- General Introduction --- p.1
Chapter 1.1 --- Hyperthermia --- p.2
Chapter 1.1.1 --- History of Hyperthermia --- p.2
Chapter 1.1.2 --- Biological Functions of Hyperthermia --- p.3
Chapter 1.1.3 --- Clinical Application of Hyperthermia --- p.4
Chapter 1.1.3.1 --- Whole-body Hyperthermia --- p.4
Chapter 1.1.3.2 --- Regional Hyperthermia --- p.4
Chapter 1.1.3.3 --- Local Hyperthermia --- p.5
Chapter 1.1.4 --- Combination Therapy --- p.5
Chapter 1.1.4.1 --- Combined treatment with Hyperthermia and Radiotherapy --- p.6
Chapter 1.1.4.2 --- Combined treatment with Hyperthermia and Chemotherapy --- p.6
Chapter 1.2 --- Tumour Necrosis Factor --- p.9
Chapter 1.2.1 --- History of Tumour Necrosis Factor --- p.9
Chapter 1.2.2 --- Sources of TNF-α and TNF-β --- p.9
Chapter 1.2.3 --- Biological Roles of TNF --- p.10
Chapter 1.2.3.1 --- Receptors of TNF-α --- p.11
Chapter 1.2.4 --- Signaling Pathway of TNF --- p.12
Chapter 1.2.4.1 --- Activation of Death Domain --- p.12
Chapter 1.2.4.2 --- Activation of Sphingomyelin Pathway --- p.13
Chapter 1.2.4.3 --- Activation of NF-kB pathway --- p.13
Chapter 1.3 --- Types of Cell Death: Necrosis and Apoptosis --- p.16
Chapter 1.3.1 --- Necrosis --- p.16
Chapter 1.3.2 --- Apoptosis --- p.16
Chapter 1.4 --- Signaling Pathway in Apoptosis --- p.19
Chapter 1.4.1 --- Factors Involved in Apoptotic Pathway --- p.19
Chapter 1.4.1.1 --- Caspases --- p.19
Chapter 1.4.1.2 --- Death Substrates --- p.20
Chapter 1.4.1.3 --- Bcl-2 Protein Family --- p.21
Chapter 1.4.1.4 --- Role of Mitochondria --- p.23
Chapter 1.5 --- Objectives of the Project --- p.26
Chapter Chapter 2. --- Materials and Methods --- p.28
Chapter 2.1 --- Materials --- p.29
Chapter 2.1.1 --- Culture of Cells --- p.34
Chapter 2.1.1.1 --- "TNF-α Sensitive Cell Line, L929" --- p.34
Chapter 2.1.1.2 --- "TNF-α Resistance Cell Line, L929-11E" --- p.34
Chapter 2.1.1.3 --- Preservation of Cells --- p.35
Chapter 2.1.2 --- Culture Media --- p.36
Chapter 2.1.2.1 --- RPMI 1640 (Phenol Red Medium) --- p.36
Chapter 2.1.2.2 --- RPMI 1640 (Phenol Red-Free Medium) --- p.36
Chapter 2.1.3 --- Buffers and Reagents --- p.37
Chapter 2.1.3.1 --- Preparation of Buffers --- p.37
Chapter 2.1.3.2 --- Buffer for Common Use --- p.37
Chapter 2.1.3.3 --- Reagents for Annexin-V-FITC/PI assay --- p.37
Chapter 2.1.3.4 --- Reagents for Cytotoxicity Assay --- p.37
Chapter 2.1.3.5 --- Reagents for Molecular Biology Work --- p.38
Chapter 2.1.3.6 --- Reagents for Western Blotting Analysis --- p.38
Chapter 2.1.4 --- Chemicals --- p.40
Chapter 2.1.4.1 --- Recombinant Murine TNF-α --- p.40
Chapter 2.1.4.2 --- Dye for Cytotoxicity Assay --- p.41
Chapter 2.1.4.3 --- Fluorescence Dyes --- p.41
Chapter 2.1.4.4 --- Chemicals Related to Mitochondrial Studies --- p.41
Chapter 2.1.4.5 --- Inhibitors of Caspases --- p.42
Chapter 2.1.4.6 --- Antibodies for Western Blotting --- p.42
Chapter 2.1.4.7 --- Other Chemicals --- p.43
Chapter 2.2 --- Methods --- p.44
Chapter 2.2.1 --- Treatment with TNF-α --- p.44
Chapter 2.2.2 --- Treatment with Hyperthermia --- p.44
Chapter 2.2.3 --- In vitro Cell Cytotoxicity Assay --- p.45
Chapter 2.2.4 --- Flow Cytometry --- p.46
Chapter 2.2.4.1 --- Introduction --- p.46
Chapter 2.2.4.2 --- Analysis by FCM --- p.48
Chapter 2.2.4.3 --- Determination of Apoptotic and Late Apoptotic/Necrotic Cells with Annexin-V-FITC/PI Cytometric Analysis --- p.50
Chapter 2.2.4.4 --- Determination of Mitochondrial Membrane Potential (ΔΨm) --- p.51
Chapter 2.2.4.5 --- Determination of Hydrogen Peroxide (H202) Release --- p.52
Chapter 2.2.4.6 --- Determination of Intracellular Free Calcium ([Ca2+]i) Level --- p.52
Chapter 2.2.4.7 --- Determination of the Relationship of ΔΨm and [Ca2+]i Level --- p.53
Chapter 2.2.5 --- Western Blotting Analysis --- p.53
Chapter 2.2.5.1 --- Preparation of Proteins from Cells --- p.53
Chapter 2.2.5.2 --- SDS Polyacrylamide Gel Electophoresis (SDS- PAGE) --- p.56
Chapter 2.2.5.3 --- Electroblotting of Proteins --- p.57
Chapter 2.2.5.4 --- Probing Antibodies for Proteins --- p.57
Chapter 2.2.5.5 --- Enhanced Chemiluminescence (ECL) assay --- p.58
Chapter 2.2.6 --- Reverse Transcriptase Polymerase Chain Reaction --- p.58
Chapter 2.2.6.1 --- Extraction of RNA by Trizol Reagent --- p.59
Chapter 2.2.6.2 --- Determination of the Amount of RNA --- p.60
Chapter 2.2.6.3 --- Agarose Gel Electrophoresis --- p.60
Chapter 2.2.6.4 --- Reverse Transcription --- p.63
Chapter 2.2.6.5 --- Polymerase Chain Reaction (PCR) --- p.63
Chapter 2.2.6.6 --- Design of Primers for Different Genes --- p.64
Chapter 2.2.6.7 --- Determination of the Number of Cycles in PCR for Different Genes --- p.67
Chapter 2.2.7 --- Caspase Fluorescent Assay --- p.67
Chapter 2.2.7.1 --- Caspase-3 or ´ؤ8 Assay --- p.67
Chapter Chapter 3. --- Results --- p.59
Chapter 3.1 --- Studies of the Characteristics of L929 and L929-11E cells --- p.70
Chapter 3.1.1 --- Determination of the Growth Curve of L929 and L929-11E Cells --- p.70
Chapter 3.2 --- Studies on the Effect of TNF-α on L929 and L929-11E Cells --- p.73
Chapter 3.2.1 --- TNF-α Induced Cell Death in L929 Cells but not in L929- 11E Cells --- p.73
Chapter 3.2.2 --- TNF-α Induced Apoptosis in a Time-dependent Manner in L929Cells but not in L929-11E Cells --- p.80
Chapter 3.2.3 --- TNF-α Induced Mitochondrial Membrane Depolarization in a Time-dependent Manner in L929 Cells but notin L929-11E Cells --- p.87
Chapter 3.2.4 --- TNF-α Induced Cytochrome c Release in a Time- dependent Manner in L929 Cells but not in L929-11E Cells --- p.92
Chapter 3.3 --- Effect of Hyperthermia on L929 and L929-11E Cells --- p.96
Chapter 3.3.1 --- Introduction --- p.95
Chapter 3.3.2 --- Hyperthermia Induced Apoptosis in L929 and L929-11E Cells --- p.96
Chapter 3.3.3 --- Effect of Hyperthermia on Mitochondrial Membrane Depolarization --- p.100
Chapter 3.3.4 --- Hyperthermia Induced Cyto c Release in a Time-dependent Manner in L929 and L929-11E Cells --- p.105
Chapter 3.4 --- Relationship of Hyperthermia and TNF-α with PTP in L929 Cells --- p.107
Chapter 3.5 --- Effect of TNF-α and Hyperthermia on the Level of Hydrogen Peroxide (H202) in L929 and L929-11E Cells --- p.114
Chapter 3.5.1 --- Introduction --- p.114
Chapter 3.5.2 --- TNF-α Enhanced the Level of H202 in L929 cells but not in L929-11E Cells --- p.115
Chapter 3.5.3 --- Hyperthermia Enhanced the Level of H202 in L929 and L929-11E cells --- p.117
Chapter 3.6 --- Effect of TNF-α and Hyperthermia on the Level of Intracellular Calcium in L929 and L929-11E Cells --- p.122
Chapter 3.6.1 --- Increase in the Intracellular Calcium Level Induced by TNF-α Was Related to the Mitochondrial Membrane Depolarization in L929 Cells but not in L929-11E Cells --- p.122
Chapter 3.6.2 --- Hyperthermia Increased the Level of [Ca2+]i in L929 and L929-11E Cells in a Time-dependent Manner --- p.124
Chapter 3.7 --- Effect of Combined Hyperthermia and TNF-α Treatment on the Induction of Apoptosis in L929 and L929-1 1E Cells --- p.129
Chapter 3.7.1 --- Combined Treatment with Hyperthermia and TNF- α Induced Apoptosis in Both L929 and L929-11E cells --- p.129
Chapter 3.7.2 --- Hyperthermia and Its Combined Treatment with TNF-α Induced Mitochondrial Membrane Depolarization in L929 and L929-11E Cells --- p.135
Chapter 3.8 --- Investigation of the Downstream Apoptotic Pathway in L929 and L929-11E Cells Upon Hyperthermia and TNF-a treatment --- p.142
Chapter 3.8.1 --- Introduction --- p.142
Chapter 3.8.2 --- Effect ofTNF-α and Hyperthermia on p53 Expression --- p.142
Chapter 3.8.3 --- Effect of Hyperthermia and TNF-α on PARP --- p.146
Chapter 3.8.4 --- Effect of Hyperthermia and TNF-α on Caspase-3 Activity --- p.149
Chapter 3.8.5 --- Effect of Hyperthermia and TNF-α on Bid protein --- p.158
Chapter 3.8.6 --- Effect of Hyperthermia and TNF-α on Caspase-8 Activity --- p.165
Chapter 3.8.7 --- Effect ofTNF-α on TNFR1 Expression --- p.169
Chapter Chapter 4. --- Discussion
Chapter 4.1 --- TNF-α Induced Apoptosis and Changed the Mitochondrial Activities in L929 Cells --- p.176
Chapter 4.2 --- L929-11E cells Possessed Resistance Towards TNF-α --- p.187
Chapter 4.3 --- Hyperthermia Triggered Apoptosis and Changed Mitochondrial Activities in L929 and L929-11E cells --- p.190
Chapter 4.4 --- Combined hyperthermia and TNF-α treatment induced cell death and changed mitochondria activities in L929 and L929-11E cells --- p.195
Chapter 4.5 --- Reversal of the TNF-α resistance and Enhancement of Sensitivity Towards Hyperthermia in L929-11E cells --- p.197
Chapter 4.6 --- Proposed Pathway in the TNF-α- and Hyperthermia-mediated Apoptosis --- p.200
Chapter 4.7 --- Application of TNF-α and Hyperthermia on Clinical Cancer Treatment --- p.203
Chapter Chapter 5. --- Future Perspective of the Project --- p.206
References --- p.210

碩士
國立陽明大學
生理學研究所
101
Abstrat Smoking caused physical stress and had been widely known for leading to chronic disease, such as cancer. According to studies, the relative risk for active smokers to develop adenocarcinoma is greater when comparing with non-smoking people. Acrolein (2-propenal), anα,ß-unsaturated aldehyde, exists in a wide range of sources. It is also present in a haze of cigarette smoke as well as the environment ubiquitously. Previous studies indicated that the immune system was correlated with physical stress, but the cell response by circulation in acrolein-pretreated rats was still unknown. It has been well known that the change of cytokine level appears to be an important initiated pre-immunity effect of the splenic monocyte (i.e. splenocytes) in rats. Although the toxicity of acrolein has been extensively studied, there is little information about its impact on stress induced immunity changes through cytokine release. This study aimed to explore the stimulatory effects of acrolein on the production of the tumor necrosis factor-alpha (TNF-α) in rat splenocytes. Splenocytes were isolated and incubated with or without Lipopolysaccharide (LPS, 0~ 40 μg/ml) from rats, which was used as a positive control. In the present study, rat splenocytes were administrated in vitro with acrolein for different doses (1x10-10 M~1x10-7 M). The results showed that the splenocytes exposure on acrolein resulted in a decrease of TNF-α release in vitro. Furthermore, the rats were administrated with acrolein (2 mg/ml/kg) for 1- or 3-day in vivo study. The results suggested that administration of acrolein decreased the release of TNF-α via an inhibition on monocytes in response to acute physical stress. The adrenocorticosteroid hormones with further speculation might be involved in changes of cytokine production in rats. In conclusion, these results suggested that the release of cytokines (e.g.TNF-α) from splenocytes can be inhibited by the administration of acrolein in rats.

Skeletal muscle injury results in functional deficits that can take several weeks to fully recover. Ultimate recovery of function is dependent on the muscle’s ability to regenerate, a highly coordinated process that involves transient muscle inflammation and the replacement of damaged myofibers. Instrumental in the inflammatory response, is the pro-inflammatory cytokine TNF-α. Expression of TNF-α is thought to be regulated, in part, by the stress sensing 70 kDa heat shock protein (Hsp70). However, it remains unclear how Hsp70 alters TNF-α following injury, and if so, how these changes affect skeletal muscle repair. Therefore, we up-regulated Hsp70 expression using 17-allylamino-17-demethoxygeldanamycin (17-AAG) prior to and following BaCl2-induced injury, and assessed TNF-α and myogenin content. Regenerating fiber cross-sectional area (CSA) and in vivo isometric torque were also analyzed in the weeks following the injury. Treatment of 17-AAG resulted in a ~5 fold increase in Hsp70 of the uninjured muscle, but did not affect any other biochemical, morphological or functional variables compared to controls. In the days following the injury, TNF-α and myogenin were elevated and directly correlated. At these earlier time points (≤7 days), treatment of 17-AAG increased TNF-α above that of the injured controls and resulted in a sustained increase in myogenin. However, no differences were observed in regenerating fiber CSA or in vivo torque production between the groups. Together, these data suggest that Hsp70 induction increases TNF-α and myogenin content following BaCl2-induced injury, but does not appear to alter skeletal muscle regeneration or attenuate functional deficits in otherwise healthy young mice.

Kar Wing To.
Thesis submitted in: December 1999.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (leaves 200-223).
Abstracts in English and Chinese.
Acknowledgements --- p.i
List of Abbreviations --- p.ii
Abstract --- p.v
撮要 --- p.viii
List of Tables --- p.x
List of Figures --- p.xi
Contents --- p.xv
Chapter CHAPTER 1 --- INTRODUCTION
Chapter 1.1 --- The General Characteristics of Glial Cells --- p.1
Chapter 1.1.1 --- Astrocytes --- p.1
Chapter 1.1.2 --- Oligodendrocytes --- p.5
Chapter 1.1.3 --- Microglial --- p.6
Chapter 1.2 --- Brain Injury and Astrocyte Proliferation --- p.6
Chapter 1.3 --- Reactive Astrogliosis and Glial Scar Formation --- p.9
Chapter 1.4 --- Astrocytes and Immune Response --- p.10
Chapter 1.5 --- Cytokines --- p.10
Chapter 1.5.1 --- Cytokines and the Central Nervous System (CNS) --- p.12
Chapter 1.5.2 --- Cytokines and brain injury --- p.13
Chapter 1.5.3 --- Cytokines-activated astrocytes in brain injury --- p.13
Chapter 1.5.4 --- Tumour Necrosis Factor-a --- p.14
Chapter 1.5.4.1 --- Types of TNF-α receptor and their sturctures --- p.16
Chapter 1.5.4.2 --- Binding to TNF-α --- p.17
Chapter 1.5.4.3 --- Different Roles of the TNF-a Receptor Subtypes --- p.17
Chapter 1.5.4.4 --- Role of TNF-α and Brain Injury --- p.19
Chapter 1.5.4.5 --- TNF-α Stimulates Proliferation of Astrocytes and C6 Glioma Cells --- p.23
Chapter 1.5.5 --- Interleukin-1 (IL-1) --- p.26
Chapter 1.5.5.1 --- Interleukin-1 and Brain Injury --- p.27
Chapter 1.5.6 --- Interleukin-6 (IL-6) --- p.28
Chapter 1.5.6.1 --- Interleukin-6 and brain injury --- p.29
Chapter 1.5.7 --- γ-Interferon (γ-IFN) --- p.30
Chapter 1.5.7.1 --- γ-Interferon and Brain Injury --- p.30
Chapter 1.6 --- Ion Channels and Astrocytes --- p.31
Chapter 1.6.1 --- Roles of Sodium Channels in Astrocytes --- p.33
Chapter 1.6.2 --- Role of Potassium Channels in Astrocytes --- p.33
Chapter 1.6.3 --- Importance of Calcium Ion Channels in Astrocytes --- p.34
Chapter 1.6.3.1 --- Function of Cellular and Nuclear Calcium --- p.34
Chapter 1.6.3.2 --- Nuclear Calcium in Cell Proliferation --- p.36
Chapter 1.6.3.3 --- Nuclear Calcium in Gene Transcription --- p.36
Chapter 1.6.3.4 --- Nuclear Calcium in Apoptosis --- p.38
Chapter 1.6.3.5 --- Spatial and Temporal Changes of Calcium-Calcium Oscillation --- p.39
Chapter 1.6.3.6 --- Calcium Signalling in Glial Cells --- p.39
Chapter 1.6.3.7 --- Calcium Channels in Astrocytes --- p.41
Chapter 1.6.3.8 --- Relationship Between [Ca2+]i and Brain Injury --- p.43
Chapter 1.6.3.9 --- TNF-α and Astrocyte [Ca2+]i --- p.45
Chapter 1.6.3.10 --- Calcium-Sensing Receptor (CaSR) --- p.46
Chapter 1.7 --- Protein Kinase C (PKC) Pathways --- p.49
Chapter 1.7.1 --- PKC and Brain Injury --- p.50
Chapter 1.7.2 --- Role of Protein Kinase C Activity in TNF-α Gene Expression in Astrocytes --- p.51
Chapter 1.7.3 --- PKC and Calcium in Astrocytes --- p.52
Chapter 1.8 --- Intermediate Early Gene (IEGs) --- p.54
Chapter 1.8.1 --- IEGs Expression and Brain Injury --- p.54
Chapter 1.8.2 --- IEGs Expression and Calcium --- p.55
Chapter 1.9 --- The Rat C6 Clioma Cells --- p.56
Chapter 1.10 --- The Aim of This Project --- p.58
Chapter CHAPTER 2 --- MATERIALS AND METHODS
Chapter 2.1 --- Materials --- p.61
Chapter 2.1.1 --- Sources of the Chemicals --- p.61
Chapter 2.1.2 --- Materials Preparation --- p.65
Chapter 2.1.2.1 --- Rat C6 Glioma Cell Line --- p.65
Chapter 2.1.2.2 --- C6 Glioma Cell Culture --- p.65
Chapter 2.1.2.2.1 --- Complete Dulbecco's Modified Eagle Medium (CDMEM) --- p.65
Chapter 2.1.2.2.2 --- Serum-free Dulbecco's Modified Eagle Medium --- p.66
Chapter 2.1.2.3 --- Phosphate Buffered Saline (PBS) --- p.66
Chapter 2.1.2.4 --- Recombinant Cytokines --- p.67
Chapter 2.1.2.5 --- Antibodies --- p.67
Chapter 2.1.2.5.1 --- Anti-TNF-Receptor 1 (TNF-R1) Antibody --- p.67
Chapter 2.1.2.5.2 --- Anti-TNF-Receptor 2 (TNF-R2) Antibody --- p.67
Chapter 2.1.2.6 --- Chemicals for Signal Transduction Study --- p.68
Chapter 2.1.2.6.1 --- Calcium Ionophore and Calcium Channel Blocker --- p.68
Chapter 2.1.2.6.2 --- Calcium-Inducing Agents --- p.68
Chapter 2.1.2.6.3 --- Modulators of Protein Kinase C (PKC) --- p.69
Chapter 2.1.2.7 --- Reagents for Cell Proliferation --- p.69
Chapter 2.1.2.8 --- Reagents for Calcium Level Measurement --- p.70
Chapter 2.1.2.9 --- Reagents for RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.71
Chapter 2.1.2.10 --- Sense and Antisense Used --- p.72
Chapter 2.1.2.11 --- Reagents for Electrophoresis --- p.74
Chapter 2.2 --- Methods --- p.74
Chapter 2.2.1 --- Maintenance of the C6 Cell Line --- p.74
Chapter 2.2.2 --- Cell Preparation for Assays --- p.75
Chapter 2.2.3 --- Determination of Cell Proliferation --- p.76
Chapter 2.2.3.1 --- Determination of Cell Proliferation by [3H]- Thymidine Incorporation --- p.76
Chapter 2.2.3.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.77
Chapter 2.2.3.3 --- Measurement of Cell Proliferation by MTT Assay --- p.77
Chapter 2.2.3.4 --- Protein Assay --- p.78
Chapter 2.2.3.5 --- Data Analysis --- p.79
Chapter 2.2.3.5.1 --- The Measurement of Cell Proliferation by [3H]- Thymidine Incorporation --- p.79
Chapter 2.2.3.5.2 --- The Measurement of Cell growth in Neutral Red and MTT Assays --- p.79
Chapter 2.2.3.5.3 --- The Measurement of Cell Proliferationin Protein Assay --- p.79
Chapter 2.2.4 --- Determination of Intracellular Calcium Changes --- p.80
Chapter 2.2.4.1 --- Confocal Microscopy --- p.80
Chapter 2.2.4.1.1 --- Procedures for Detecting Cell Activity by CLSM --- p.81
Chapter 2.2.4.1.2 --- Precautions of CLSM --- p.82
Chapter 2.2.5 --- Determination of Gene Expression by Reverse- Transcription Polymerase Chain Reaction (RT-PCR) --- p.83
Chapter 2.2.5.1 --- RNA Preparation --- p.83
Chapter 2.2.5.1.1 --- RNA Extraction --- p.83
Chapter 2.2.5.1.2 --- Measurement of RNA Yield --- p.84
Chapter 2.2.5.2 --- Reverse Transcription (RT) --- p.84
Chapter 2.2.5.3 --- Polymerase Chain Reaction (PCR) --- p.85
Chapter 2.2.5.4 --- Separation of PCR Products by Agarose Gel Electrophoresis --- p.85
Chapter 2.2.5.5 --- Quantification of Band Density --- p.86
Chapter CHAPTER 3 --- RESULTS
Chapter 3.1 --- Effects of Different Drugs on C6 Cell Proliferation --- p.87
Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.87
Chapter 3.1.1.1 --- Effect of TNF-α on C6 Proliferation --- p.88
Chapter 3.1.1.2 --- Effects of Other Cytokines on C6 Cell Proliferation --- p.92
Chapter 3.1.2 --- The Signalling Pathway of TNF-α induced C6 Cell Proliferation --- p.92
Chapter 3.1.2.1 --- The Involvement of Calcium Ions in TNF-α-induced C6Cell Proliferation --- p.95
Chapter 3.1.2.2 --- The Involvement of Protein Kinase C in TNF-α- induced C6 Cell Proliferation --- p.96
Chapter 3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on C6 Cell Proliferation --- p.102
Chapter 3.2 --- The Effect of in Tumour Necrosis Factor-α on Changesin Intracellular Calcium Concentration --- p.102
Chapter 3.2.1 --- Release of Intracellular Calcium in TNF-α-Treated C6 Cells --- p.104
Chapter 3.2.2 --- Effects of Calcium Ionophore and Calcium Channel Blocker on TNF-α-induced [Ca2+]i Release --- p.107
Chapter 3.2.3 --- Effects of Other Cytokines on the Change in [Ca2+]i --- p.109
Chapter 3.2.4 --- The Role of PKC in [Ca2+]i release in C6 Glioma Cells --- p.109
Chapter 3.2.4.1 --- Effects of PKC Activators and Inhibitors on the Changes in [Ca2+]i --- p.114
Chapter 3.3 --- Determination of Gene Expression by RT-PCR --- p.114
Chapter 3.3.1 --- Studies on TNF Receptors Gene Expression --- p.117
Chapter 3.3.1.1 --- Effect of TNF-α on TNF Receptors Expression --- p.117
Chapter 3.3.1.2 --- Effects of Other Cytokines on the TNF Receptors Expression --- p.119
Chapter 3.3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on the TNF-a-induced Receptors Expression --- p.121
Chapter 3.3.1.4 --- Effect of Calcium Ions on TNF Receptors Expression --- p.121
Chapter 3.3.1.4.1 --- Effect of Calcium Ionophore on TNF Receptors Expression --- p.126
Chapter 3.3.1.4.2 --- Effect of TNF-α Combination with A23187 on the Expression of TNF Receptors --- p.128
Chapter 3.3.1.4.3 --- Effects of Calcium Ionophore and Channel Blocker on TNF Receptors Expression --- p.130
Chapter 3.3.1.4.4 --- Effects of Calcium-Inducing Agents on TNF Receptors Gene Expressions --- p.130
Chapter 3.3.1.5 --- Effects of PKC Activator and Inhibitor on TNF-α- induced TNF Receptors Expressions --- p.135
Chapter 3.3.1.6 --- Effect of PKC and Ro31-8220 on IL-l-induced TNF Receptors Expressions --- p.138
Chapter 3.3.2 --- Expression of Calcium-sensing Receptor upon Different Drug Treatments --- p.138
Chapter 3.3.2.1 --- Effect of TNF-α on the Calcium-sensing Receptor Expression --- p.141
Chapter 3.3.2.2 --- Effects of Other Cytokines on CaSR Expression --- p.143
Chapter 3.3.2.3 --- Effect of A23187 on CaSR Expression --- p.143
Chapter 3.3.2.4 --- Effect of TNF-α and A23187 Combined Treatment on CaSR Expression --- p.146
Chapter 3.3.2.5 --- Effects of Calcium-inducing Agents on CaSR Expression --- p.149
Chapter 3.3.2.6 --- Effects of PKC Activator and PKC Inhibitor on CaSR Expression --- p.149
Chapter 3.3.3 --- Expression of PKC Isoforms upon Treatment with Different Drugs --- p.153
Chapter 3.3.3.1 --- Effect of TNF-α on the PKC Isoforms Expression --- p.155
Chapter 3.3.3.2 --- Effect of A23187 on the PKC Isoforms Expression --- p.155
Chapter 3.3.3.3 --- Effect of TNF-α and Calcium Ionophore Combined Treatment on PKC Isoforms Expression --- p.157
Chapter 3.3.3.4 --- Effects of Calcium-inducing Agents on PKC Isoforms Expression --- p.159
Chapter 3.3.4 --- Expression of the Transcription Factors c-fos and c-junin Drug Treatments --- p.161
Chapter 3.3.4.1 --- Effect of TNF-a on c-fos and c-jun Expression --- p.163
Chapter 3.3.4.2 --- Effect of A23187 on c-fos and c-jun Expression --- p.163
Chapter 3.3.4.3 --- Effect of TNF-a in Combination with A23187 on c- fos and c-jun Expression --- p.165
Chapter 3.3.4.4 --- Effects of Calcium-inducing Agents on c-fos and c- jun Expression --- p.167
Chapter 3.3.5 --- Effects of Different Drugs on Endogenous TNF-α Expression --- p.167
Chapter 3.3.5.1 --- Effect of TNF-α on Endogenous TNF-α Expression --- p.169
Chapter 3.3.5.2 --- Effect of A23187 on Endogenous TNF-α Expression --- p.169
Chapter 3.3.5.3 --- Effect of TNF-α in Combination with A23187 on the Expression of Endogenous TNF-α --- p.172
Chapter 3.3.5.4 --- Effects of Calcium-inducing Agents on Endogenous TNF-α Expression --- p.172
Chapter 3.3.6 --- Effect of Different Drugs on LL-1 Expression --- p.175
Chapter 3.3.6.1 --- Effect of TNF-a on IL-lα Expression --- p.177
Chapter 3.3.6.2 --- Effect of A23187 on the IL-lα Expression --- p.177
Chapter 3.3.6.3 --- Effect of Calcium Ionophore and TNF-α Combined Treatment on IL-1α Expression --- p.179
Chapter 3.3.6.4 --- Effects of Calcium-inducing Agents on IL-lα Expression --- p.179
Chapter 3.3.6.5 --- Effect of PKC Activator on the IL-1α Expression --- p.181
Chapter CHAPTER 4 --- DISCUSSIONS AND CONCLUSIONS
Chapter 4.1 --- "Effects of Cytokines, Ca2+ and PKC and Anti-TNF-α Antibodies on C6 Glioma Cells Proliferation" --- p.184
Chapter 4.2 --- The Role of Calcium in TNF-α-induced Signal Transduction Pathways --- p.186
Chapter 4.3 --- Gene Expressions in C6 Cells after TNF-a Stimulation --- p.187
Chapter 4.3.1 --- "Expression of TNF-α, TNF-Receptors and IL-1" --- p.187
Chapter 4.3.2 --- CaSR Expression --- p.190
Chapter 4.3.3 --- PKC Isoforms Expressions --- p.192
Chapter 4.3.4 --- Expression of c-fos and c-jun --- p.193
Chapter 4.4 --- Conclusion --- p.194
REFERENCES --- p.200

by Chan Yick Bun.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references (leaves 145-165).
Abstracts in English and Chinese.
Acknowledgement --- p.II
Abstract --- p.IV
Contents --- p.VIII
Abbreviations --- p.XIV
List of Figures --- p.XVI
List of Tables --- p.XVII
Chapter Chapter One --- General introduction
Chapter 1.1 --- Leukemia: an overview --- p.1
Chapter 1.1.1 --- Background --- p.1
Chapter 1.1.2 --- Classification of leukemia --- p.1
Chapter 1.1.3 --- Origin of leukemia --- p.3
Chapter 1.1.4 --- Treatment of leukemia --- p.5
Chapter 1.2 --- Introduction of leukemia cell re-differentiation --- p.8
Chapter 1.2.1 --- Introduction --- p.8
Chapter 1.2.2 --- Inducers of cell differentiation --- p.8
Chapter 1.2.3 --- Genes involved in myeloid leukemia cell differentiation --- p.11
Chapter 1.2.3.1 --- Transcription factors --- p.11
Chapter 1.2.3.2 --- Signal transduction cascades --- p.16
Chapter 1.2.3.3 --- Receptors --- p.18
Chapter 1.2.3.4 --- Cytokines --- p.19
Chapter 1.3 --- Tumor necrosis factor alpha induced WEHI 3B JCS cell differentiation --- p.21
Chapter 1.3.1 --- Introduction --- p.21
Chapter 1.3.2 --- Tumor necrosis factor alpha --- p.21
Chapter 1.3.3 --- WEHI 3B JCS cells --- p.23
Chapter 1.4 --- Aims of study --- p.25
Chapter Chapter Two --- Isolation of differentially expressed genes during TNF-α induced WEHI 3B JCS cell differentiation
Chapter 2.1 --- Introduction --- p.26
Chapter 2.1.1 --- Overview of differential genes screening methods --- p.26
Chapter 2.1.2 --- Differential hybridization for analysis of gene expression profiles --- p.29
Chapter 2.1.3 --- Factors affect differential hybridization --- p.33
Chapter 2.2 --- Materials --- p.35
Chapter 2.2.1 --- Cell line --- p.35
Chapter 2.2.2 --- Mouse brain cDNA library --- p.35
Chapter 2.2.3 --- E.coli strains --- p.35
Chapter 2.2.3 --- Kits --- p.35
Chapter 2.2.5 --- Chemicals --- p.35
Chapter 2.2.6 --- Solutions and buffers --- p.36
Chapter 2.2.7 --- Enzymes and reagents --- p.37
Chapter 2.3 --- Methods --- p.38
Chapter 2.3.1 --- Preparation of total RNA from TNF-a induced WEHI 3B JCS cells --- p.38
Chapter 2.3.1.1 --- Preparation of cell lysates --- p.38
Chapter 2.3.1.2 --- Extraction of total RNA --- p.38
Chapter 2.3.2 --- Preparation of cDNA clones from cDNA library --- p.39
Chapter 2.3.2.1 --- Rescue of phagemids from cDNA library --- p.39
Chapter 2.3.2.2 --- Preparation of plasmids --- p.39
Chapter 2.3.3 --- Primary differential hybridization --- p.40
Chapter 2.3.3.1 --- Preparation of cDNA blots --- p.40
Chapter 2.3.3.2 --- Preparation of cDNA probes --- p.40
Chapter 2.3.3.3 --- Primary differential hybridization --- p.41
Chapter 2.3.4 --- Subcloning of putative differential cDNA clones --- p.42
Chapter 2.3.4.1 --- Preparation of DH5a competent cells --- p.42
Chapter 2.3.4.2 --- Transformation of cDNA clones --- p.42
Chapter 2.3.5 --- Secondary differential hybridization --- p.42
Chapter 2.3.5.1 --- Preparation ofcDNA blots --- p.42
Chapter 2.3.5.2 --- Secondary differential hybridization --- p.43
Chapter 2.4 --- Results --- p.44
Chapter 2.4.1 --- Analysis of total RNA prepared from TNF-α induced WEHI 3B JCS cells --- p.44
Chapter 2.4.2 --- Spectrophotometric analysis of plasmid DNA --- p.46
Chapter 2.4.3 --- Primary differential hybridization --- p.48
Chapter 2.4.4 --- Secondary differential hybridization --- p.58
Chapter 2.4.5 --- Comparison of two rounds of differential hybridization --- p.61
Chapter 2.5 --- Discussions --- p.63
Chapter 2.5.1 --- Study of gene expression profile by differential hybridization --- p.63
Chapter 2.5.1.1 --- cDNA library --- p.63
Chapter 2.5.1.2 --- Blots --- p.64
Chapter 2.5.2 --- Two rounds of differential hybridization --- p.66
Chapter 2.5.3 --- Comparison of two rounds of differential hybridization --- p.68
Chapter Chapter Three --- Sequence analysis of putative differentially expressed genes
Chapter 3.1 --- Introduction --- p.70
Chapter 3.1.1 --- Basic structure of cDNA clones --- p.70
Chapter 3.1.2 --- Strategies for DNA sequencing --- p.71
Chapter 3.1.2.1 --- Primer walking --- p.71
Chapter 3.1.2.2 --- Restriction digestion and subcloning --- p.71
Chapter 3.1.2.3 --- Nested deletion sets --- p.72
Chapter 3.1.2.4 --- Shotgun sequencing --- p.72
Chapter 3.1.2.5 --- Other sequencing strategies --- p.73
Chapter 3.1.3 --- Sequence alignment and database search --- p.74
Chapter 3.1.3.1 --- Sequence database --- p.74
Chapter 3.1.3.2 --- Sequence alignment --- p.74
Chapter 3.1.3.3 --- BLAST algorithm --- p.75
Chapter 3.2 --- Materials --- p.76
Chapter 3.2.1 --- Kits --- p.76
Chapter 3.2.2 --- Restriction enzymes --- p.76
Chapter 3.2.3 --- Solutions and buffers --- p.76
Chapter 3.2.4 --- Enzymes and reagents --- p.77
Chapter 3.3 --- Methods --- p.78
Chapter 3.3.1 --- Restriction digestion --- p.78
Chapter 3.3.2 --- Subcloning --- p.79
Chapter 3.3.2.1 --- Gel purification --- p.79
Chapter 3.3.2.2 --- Ligation --- p.79
Chapter 3.3.2.3 --- Transformation --- p.80
Chapter 3.3.3 --- Shotgun sequencing --- p.80
Chapter 3.3.4 --- Sequencing reaction --- p.81
Chapter 3.3.4.1 --- Preparation of sequencing gel --- p.81
Chapter 3.3.4.2 --- Sequencing reaction --- p.81
Chapter 3.4 --- Results --- p.83
Chapter 3.4.1 --- Restriction mapping of cDNA inserts --- p.83
Chapter 3.4.2 --- Sequencing results --- p.85
Chapter 3.4.3 --- Sequence analysis --- p.90
Chapter 3.5 --- Discussions --- p.103
Chapter 3.5.1 --- Sequencing strategies --- p.103
Chapter 3.5.2 --- Sequence analysis --- p.104
Chapter Chapter Four --- Characterization of the putative differentially expressed genes
Chapter 4.1 --- Introduction --- p.107
Chapter 4.1.1 --- Midazolam induced WEHI 3B JCS cells differentiation --- p.107
Chapter 4.1.2 --- Gene expression profiles in embryogenesis --- p.108
Chapter 4.2 --- Materials --- p.110
Chapter 4.2.1 --- Mouse embryo multiple tissue Northern (MTN´ёØ) blot --- p.110
Chapter 4.2.2 --- Megaprime´ёØ DNA labelling system --- p.110
Chapter 4.2.3 --- Chemicals --- p.110
Chapter 4.2.3 --- Solutions and buffers --- p.111
Chapter 4.3 --- Methods --- p.112
Chapter 4.3.1 --- Preparation of Northern blots --- p.112
Chapter 4.3.1.1 --- Preparation of total RNA from midazolam induced WEHI 3B JCS cells --- p.112
Chapter 4.3.1.2 --- Preparation of Northern blots --- p.112
Chapter 4.3.2 --- Preparation of DNA probes --- p.113
Chapter 4.3.2.1 --- Preparation of DNA templates --- p.113
Chapter 4.3.2.2 --- Preparation of 32P labelled probes --- p.114
Chapter 4.3.3 --- Northern blot analysis --- p.115
Chapter 4.3.3.1 --- Northern hybridization --- p.115
Chapter 4.3.3.2 --- Stripping of Northern blot --- p.115
Chapter 4.4 --- Results --- p.117
Chapter 4.4.1 --- Analysis of midazolam induced JCS cells total RNA --- p.117
Chapter 4.4.2 --- Preparation of DNA templates for probe syntheses --- p.119
Chapter 4.4.3 --- Northern Hybridization --- p.121
Chapter 4.4.4 --- Comparison of the results of differential hybridization and Northern hybridization --- p.126
Chapter 4.5 --- Discussions --- p.127
Chapter 4.5.1 --- Northern hybridization --- p.127
Chapter 4.5.1.1 --- Gene expression patterns under TNF-α induction --- p.127
Chapter 4.5.1.2 --- Normalization of Northern hybridization --- p.129
Chapter 4.5.1.3 --- Gene expression patterns under midazolam induction --- p.130
Chapter 4.5.1.4 --- Gene expression pattern during embryo development --- p.133
Chapter Chapter Five --- General discussion
Chapter 5.1 --- Identification of differentially expressed genes in TNF-α induced WEHI 3B JCS diffentiation --- p.135
Chapter 5.2 --- Differentially expressed genes and myeloid leukemia cell differentiation --- p.137
Chapter 5.3 --- Differentially expressed genes and embryogenesis --- p.142
Chapter 5.4 --- Further studies --- p.144
References --- p.145

by Ho Wai Fong.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 207-243).
Abstracts in English and Chinese.
Title --- p.i
Abstract --- p.ii
摘要 --- p.v
Acknowledgements --- p.viii
Table of Contents --- p.x
List of Abbreviations --- p.xviii
List of Figures --- p.xxiv
List of Tables --- p.xxix
Chapter Chapter 1 --- Introduction
Chapter 1.1 --- Traumatic brain injury --- p.1
Chapter 1.2 --- Ceils of the nervous system: glia --- p.1
Chapter 1.2.1 --- Astroglia -
Chapter 1.2.1.1 --- Molecular markers of astroglia --- p.3
Chapter 1.2.1.2 --- Functions of astroglia --- p.3
Chapter 1.2.2 --- Oligodendrocyte --- p.5
Chapter 1.2.2.1 --- Molecular markers of oligodendrocyte --- p.6
Chapter 1.2.2.2 --- Functions of oligodendrocyte --- p.6
Chapter 1.2.3 --- Microglia --- p.7
Chapter 1.2.3.1 --- Molecular markers of microglia --- p.7
Chapter 1.2.3.2 --- Functions of microglia --- p.8
Chapter 1.3 --- Cytokine and brain injury --- p.8
Chapter 1.4 --- Tumor necrosis factor alpha (TNF-α) --- p.9
Chapter 1.5 --- TNF-α receptor --- p.10
Chapter 1.6 --- Biological activities of TNF-α --- p.11
Chapter 1.7 --- Signaling mechanism --- p.13
Chapter 1.7.1 --- Protein kinase C --- p.13
Chapter 1.7.2 --- Protein kinase A --- p.14
Chapter 1.7.3 --- p38 mitogen-activated protein kinase (p38 MAPK) --- p.15
Chapter 1.7.3.1 --- Biological activities of p38 MAPK --- p.18
Chapter 1.7.4 --- Inducible nitric oxide synthase (iNOS) --- p.20
Chapter 1.7.5 --- cAMP responsive element binding protein (CREB) --- p.21
Chapter 1.7.6 --- Transcription factor c-fos --- p.23
Chapter 1.7.7 --- Nuclear factor kappa-B (NF-kB) --- p.24
Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.26
Chapter 1.9 --- β-adrenergic receptor (β-AR) --- p.28
Chapter 1.9.1 --- Functions of β-AR in astrocytes --- p.29
Chapter 1.10 --- Why do we use C6 glioma cell? --- p.31
Chapter 1.11 --- Fluorescent differential display (FDD) --- p.34
Chapter 1.12 --- Aims and Scopes of this project --- p.36
Chapter Chapter 2 --- MATERIALS AND METHODS
Chapter 2.1 --- Material --- p.40
Chapter 2.1.1 --- Cell line --- p.40
Chapter 2.1.2 --- Cell culture reagents --- p.40
Chapter 2.1.2.1 --- Complete Dulbecco's modified Eagle medium (CDMEM) --- p.40
Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.41
Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.41
Chapter 2.1.3 --- Recombinant cytokines --- p.41
Chapter 2.1.4 --- Chemicals for signal transduction study --- p.42
Chapter 2.1.4.1 --- Modulators of p38 mitogen-activated protein kinase (p38 MAPK) --- p.42
Chapter 2.1.4.2 --- Modulators of protein kinase C (PKC) --- p.42
Chapter 2.1.4.3 --- Modulators of protein kinase A (PKA) --- p.42
Chapter 2.1.4.4 --- β-Adrenergic agonist and antagonist --- p.43
Chapter 2.1.5 --- Antibodies --- p.44
Chapter 2.1.5.1 --- Anti-p38 mitogen-activated protein kinase (p38 MAPK) antibody --- p.44
Chapter 2.1.5.2 --- Anti-phosporylation p38 mitogen-activated protein kinase (p-p38 MAPK) antibody --- p.44
Chapter 2.1.5.3 --- Antibody conjugates --- p.44
Chapter 2.1.6 --- Reagents for RNA isolation --- p.45
Chapter 2.1.7 --- Reagents for DNase I treatment --- p.45
Chapter 2.1.8 --- Reagents for reverse transcription of mRNA and fluorescent PCR amplification --- p.45
Chapter 2.1.9 --- Reagents for fluorescent differential display --- p.46
Chapter 2.1.10 --- Materials for excision of differentially expressed cDNA fragments --- p.46
Chapter 2.1.11 --- Reagents for reamplification of differentially expressed cDNA fragments --- p.46
Chapter 2.1.12 --- Reagents for subcloning of reamplified cDNA fragments --- p.47
Chapter 2.1.13 --- Reagents for purification of plasmid DNA from recombinant clones --- p.47
Chapter 2.1.14 --- Reagents for DNA sequencing of differentially expressed cDNA fragments --- p.47
Chapter 2.1.15 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.48
Chapter 2.1.16 --- Reagents for electrophoresis --- p.50
Chapter 2.1.17 --- Reagents and buffers for Western blot --- p.50
Chapter 2.1.18 --- Other chemicals and reagents --- p.50
Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.51
Chapter 2.3 --- RNA isolation --- p.52
Chapter 2.3.1 --- Measurement of RNA yield --- p.53
Chapter 2.4 --- DNase I treatment --- p.53
Chapter 2.5 --- Reverse transcription of mRNA and fluorescent PCR amplification --- p.54
Chapter 2.6 --- Fluorescent differentia display --- p.55
Chapter 2.7 --- Excision of differentially expressed cDNA fragments --- p.59
Chapter 2.8 --- Reamplification of differentially expressed cDNA fragments --- p.59
Chapter 2.9 --- Subcloning of reamplified cDNA fragments --- p.60
Chapter 2.10 --- Purification of plasmid DNA from recombinant clones --- p.63
Chapter 2.11 --- DNA sequencing of differentially expressed cDNA fragments --- p.64
Chapter 2.12 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.66
Chapter 2.13 --- Western bolt analysis --- p.67
Chapter Chapter 3 --- RESULTS
Chapter 3.1 --- DNase I treatment --- p.71
Chapter 3.2 --- FDD RT-PCR and band excision --- p.71
Chapter 3.3 --- Reamplification of excised cDNA fragments --- p.74
Chapter 3.4 --- Subcloning of reamplified cDNA fragments --- p.77
Chapter 3.5 --- DNA sequencing of subcloned cDNA fragments --- p.77
Chapter 3.6 --- Confirmation of the differentially expressed cDNA fragments by RT-PCR and Western blotting --- p.84
Chapter 3.6.1 --- Effects of TNF-α on p38a mitogen protein kinase (p38 α MAPK) --- p.84
Chapter 3.6.2 --- Effects of TNF-α on p38 a MAPK and p-p38 α MAPK protein level --- p.86
Chapter 3.7 --- Effects of TNF-α on p38 MAPK --- p.88
Chapter 3.7.1 --- "Effects of TNF-α on p38 α, β,γ andδ MAPK" --- p.88
Chapter 3.7.2 --- Role of TNF-receptor (TNF-R) subtype in the TNF-α-induced p3 8 MAPK expression in C6 cells --- p.89
Chapter 3.7.3 --- The signaling system mediating TNF-α-induced p38 a MAPK expression in C6 cells --- p.92
Chapter 3.7.3.1 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.92
Chapter 3.7.3.2 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.98
Chapter 3.7.4 --- The relationship between p38 MAPK and β-adrenergic mechanisms in C6 cells --- p.99
Chapter 3.7.4.1 --- Effects of isoproterenol and propanol on p38 MAPK mRNA levels in C6 cells --- p.103
Chapter 3.7.4.2 --- Effects of β1-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.106
Chapter 3.7.4.3 --- Effects of β2-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.107
Chapter 3.8 --- The relationship between p3 8 MAPK and inducible nitric oxide synthase (iNOS) expression --- p.113
Chapter 3.8.1 --- Effects of TNF-α on the iNOS expression in C6 cells --- p.113
Chapter 3.8.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced iNOS expression in C6 cells --- p.115
Chapter 3.8.3 --- The signaling system mediating TNF-α-induced iNOS expression in C6 cells --- p.115
Chapter 3.8.3.1 --- The involvement of p38 MAPK in the TNF-α-induced iNOS expression in C6 cells --- p.117
Chapter 3.8.3.2 --- The involvement of PKA in the TNF-α-induced iNOS expression in C6 cells --- p.119
Chapter 3.9 --- The relationship between p38 MAPK and cAMP-responsive element binding protein (CREB) expression --- p.120
Chapter 3.9.1 --- Effects of TNF-α on the CREB expression in C6 cells --- p.120
Chapter 3.9.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced CREB expression in C6 cells --- p.124
Chapter 3.9.3 --- The signaling system mediating TNF-α-induced CREB expression in C6 cells --- p.126
Chapter 3.9.3.1 --- The involvement of p38 MAPK in the TNF-α-induced CREB expression in C6 cells --- p.126
Chapter 3.9.3.2 --- The involvement of PKC in the TNF-α-induced CREB expression in C6 cells --- p.128
Chapter 3.9.3.3 --- The involvement of PKA in TNF-α-induced CREB expression in C6 cells --- p.129
Chapter 3.9.4 --- The relationship between CREB and β-adrenergic mechanisms in C6 cells --- p.136
Chapter 3.9.4.1 --- Effects of isoproterenol and propanol on CREB mRNA levels in C6 cells --- p.136
Chapter 3.9.4.2 --- Effects of β1-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.139
Chapter 3.9.4.3 --- Effects of (32-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.142
Chapter 3.10 --- The relationship between p38 MAPK and transcription factor c-fos expression --- p.146
Chapter 3.10.1 --- Effects of TNF-α on the c-fos expression in C6 cells --- p.146
Chapter 3.10.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced c-fos expression in C6 cells --- p.146
Chapter 3.10.3 --- The signaling system mediating TNF-α-induced c-fos expression in C6 cells --- p.149
Chapter 3.10.3.1 --- The involvement of p38 MAPK in the TNF-α-induced c-fos expression in C6 cells --- p.149
Chapter 3.10.3.2 --- The involvement of PKC in the TNF-α-induced c-fos expression in C6 cells --- p.151
Chapter 3.10.3.3 --- The involvement of PKA in TNF-α-induced c-fos expression in C6 cells --- p.154
Chapter 3.10.4 --- The relationship between c-fos and β-adrenergic mechanisms in C6 cells --- p.157
Chapter 3.10.4.1 --- Effects of isoproterenol and propanolol on c-fos mRNA levels in C6 cells --- p.157
Chapter 3.10.4.2 --- Effects of β1-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.160
Chapter 3.10.4.3 --- Effects of β2-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.164
Chapter 3.11 --- The relationship between p38 MAPK and transcription factor NF-kB expression --- p.168
Chapter 3.11.1 --- Effects of TNF-α on the NF-kB expression in C6 cells --- p.168
Chapter 3.11.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced NF-kB expression in C6 cells --- p.168
Chapter 3.11.3 --- The signaling system mediating TNF-α-induced NF-kB expression in C6 cells --- p.171
Chapter 3.11.3.1 --- The involvement of p38 MAPK in the TNF-α-induced NF-kB expression in C6 cells --- p.171
Chapter 3.11.3.2 --- The involvement of PKC in the TNF-α-induced NF-kB expression in C6 cells --- p.173
Chapter Chapter 4 --- DISCUSSION AND CONCLUSION
Chapter 4.1 --- Effects of tumor-necrosis factor-alpha (TNF-α) on C6 cell proliferations --- p.176
Chapter 4.2 --- The Signaling System Involved in TNF-α-Induced p38 MAPK Expression in C6 cells --- p.178
Chapter 4.3 --- The Signaling System Involved in TNF-α-Induced iNOS Expression in C6 cells --- p.184
Chapter 4.4 --- The Signaling System Involved in TNF-α-Induced CREB Expression in C6 cells --- p.186
Chapter 4.5 --- The Signaling System Involved in TNF-α-Induced c-fos Expressionin in C6 cells --- p.190
Chapter 4.6 --- The Signaling System Involved in TNF-α-Induced NF-kB Expression in C6 cells --- p.193
Chapter 4.7 --- Conclusions --- p.195
Chapter 4.8 --- Possible application
References

碩士
國立成功大學
生物科技研究所碩博士班
96
Epinephelus coioides (E. coioides, orange-spotted grouper) is an important farmed fish in Taiwan; there is a paramount problem of raring fish is pathogen infection, especially it occurs at larval stage. Fish larvae were not able to against the pathogen invasion because its immune system was incompletely developed. Innate immune response is the first line to defense pathogens invasion by inducing inflammatory reaction, numerous of mediator and immunocytes are intervening in it, TNF-α is the most important cytokines and is secreted in the earliest inflammation reaction, TNF-α gene was found in some of bony fish; however, the gene of most fish is still unknown. In this study, a TNF-α gene of E. coioides was cloned, the gene was 1516 base pairs (bp) long with 4 exons and 3 introns. The length of open reading frame is 762 bp encoding 253 amino acids with the molecular weight of putative protein was about 27 kDa, the TNF family signature, transmembrane domain, TACE cutting site and two cysteins residues which involved in tertiary structure formation were predicted. Subsequently, the TNF-α mRNA expression level in ontogeny of fish and different organs was evaluated by quantitative reverse transcriptase real time PCR (qRT-PCR). The result shown the expression level of TNF-α mRNA raised after 19 dpf of fries. It indicated that the innate immune system was activated at that stage. The development and functions of immunity on larval fish will be comprehended in this study expectantly, furthermore, designing a proper and efficient strategy to prevent infection of larval stage of fish form the information of this study.

Besides the molecular mechanisms regulating activation of FLS mentioned above, we also investigated anti-inflammatory activities of Chinese herbal medicine sinomenine and Liang Miao San on activated human FLS in RA. Sinomenine, an alkaloid isolated from the root of Sinomenium acutum, has been used to alleviate the symptoms of rheumatic diseases. Liang Miao San (LMS), composed of the herbs Rhizoma Atractylodis (Cangzhu) and Cotex Phellodendri (Huangbai), is another traditional Chinese medicine formula for RA treatment. Since the potential anti-inflammatory activities of sinomenine and LMS have been demonstrated, we investigated the in vitro anti-inflammatory effects of sinomenine and LMS on inflammatory cytokine TNF-alpha activation of human normal and RA-FLS and the underlying intracellular mechanisms. In the present study, sinomenine was found to significantly inhibit TNF-alpha induced cell surface expression of VCAM-1 and release of inflammatory cytokine and chemokine IL-6, CCL2 and CXCL8 from both normal and RA-FLS (all p < 0.05). Our results provide a new insight into the differential anti-inflammatory activities of sinomenine and LMS through the suppression of TNF-alpha activated FLS by modulating distinct intracellular signaling pathways in RA, and help to provide a biochemical basis for the development of a cost-effective human synoviocyte model for the future screening of traditional Chinese medicine (TCM) possessing potential anti-rheumatic activities. (Abstract shortened by UMI.)
IL-27, a novel member of the IL-12 family that is produced early by antigen-presenting cells (APCs), can promote T cell proliferation as well as the production of interferon-gamma by naive T lymphocytes. Recent studies have found that elevated expression of IL-27 has been detected in the synovial membranes and fluid of RA. Herein we investigated the in vitro effects of IL-27, alone or in combination with inflammatory cytokine TNF-alpha or IL-Ibeta on the pro-inflammatory activation of human primary FLS isolated from RA patients and normal control subjects, and the underlying intracellular signaling molecules were also studied. We found that the plasma concentration of IL-27 in RA patients (n=112) was significantly higher than that in control subjects (n=46). Both normal and RA-FLS constitutively express functional IL-27 receptor heterodimer, gp130 and WSX-1, with more potent IL-27-mediated activation of signal transducers and activators of transcription (STAT)1 in RA-FLS. IL-27 was found to induce significantly higher cell surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 and release of inflammatory cytokine IL-6, chemokine CCL2, CXCL9, CXCL10 and matrix metalloproteinase (MMP)-1 of RA-FLS than that of normal FLS (all p < 0.05). The above findings therefore provide a new insight into the IL-27-activated immunopathological mechanisms mediated by distinct intracellular signal transductions in joint inflammation of RA and may have important therapeutic implications.
In the present study, we have investigated the mechanisms of the activation of human fibroblast-like synoviocytes (FLS) induced by various stimuli including interleukin (IL)-27, tumor necrosis factor (TNF)-alpha and IL-beta. The activation of human FLS was studied in terms of the release of cytokines and chemokines and the expression of adhesion molecules.
We investigated the in vitro effects of uric acid crystals, alone or in combination with inflammatory cytokine TNF-alpha or IL-beta on the pro-inflammatory activation of human FLS from RA patients and normal control subjects, and the underlying intracellular signaling molecules were also determined. In the present study, uric acid crystals were found to result in a significant increase of inflammatory cytokine IL-6, chemokine CXCL8 and MMP-1 from both normal and RA-FLS (all p < 0.05). Moreover, additive or synergistic effect was observed in the combined treatment of uric acid crystals and TNF-alpha or IL-1beta on the release of IL-6, CXCL8 and MMP-1 from both normal and RA-FLS. Further investigations showed that the release of inflammatory cytokine, chemokine and matrix metalloproteinase stimulated by uric acid crystals was differentially regulated by intracellular activation of extracellular signal-regulated kinase (ERK) and JNK pathways. Our results therefore provide a new insight into the endogenous danger signal uric acid crystals-activated immunopathological mechanisms mediated by distinct intracellular signal transductions in joint inflammation, and also provide biochemical basis for the development of new modality for inflammatory rheumatic diseases.
Chen, Dapeng.
Adviser: Wong Chun Kwok.
Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 203-240).
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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Background: Uterine fibriod (UF) or leiomyoma is the most frequent benign tumour upon lower genital tract and represents the most frequent indication for hysterectomy. The aetiology remains still unknown. The genetic factors contributing for the development of UF are being intensively investigated. The aim of our study was to look for possible genetic markers which could be used as prognostic tools for evaluation of an increased risk for development of UF. Methods: The study group enrolled 102 patients diagnosed with UF and 145 healthy controls. Ultrasonographic examination of the pelvis was performed and a single blood sample was taken in all women. Histological verification followed the surgery in the patient group. The principal of the cytokine gene polymorphisms detection is based on PCR reaction with sequence-specific primers. Results: A large spectrum of Th1/Th2 cytokine gene polymorphisms in patients with uterine fibroid was compared with control group. The frequencies of the majority of tested cytokine gene SNP in the patient cohort were not statistically different from the cytokine SNP in the control group. However, an intriguing association between polymorphisms of the IL-4 gene promotor at positions -590 C/T and -33 C/T, and the risk of leiomyoma was observed. The CC genotype of IL-4 at position...

The involvement of the pro-inflammatory cytokine, tumour necrosis factor alpha (TNF-α) and the sympathetic nervous system in the development of delayed-onset muscle soreness has not been established. I assessed the effect of etanercept, a TNF- α inhibitor, and atenolol, a β1-receptor antagonist, on DOMS induced in the quadriceps muscle. Thirteen male subjects reported to the exercise laboratory on three separate occasions, 6-15 weeks apart. In a randomised, double-blind cross-over format, I administered etanercept (25mg), atenolol (25mg) or placebo, one hour before the exercise. Subjects then completed four sets of 15 repetitions at 80% of their one repetition maximum (1RM) on a 45° inclined leg press machine. Muscle strength changes were detected by remeasuring the subject’s 1RM 24h, 48h and 72h after the exercise. Sensitivity to pressure of the quadriceps muscle was measured using a pressure algometer before and 24h, 48h and 72h after exercise. The subject’s perception of the pain was measured with the visual analogue scale and McGill Pain Questionnaire. Muscle tumour necrosis factor-alpha concentration was measured before exercise and then 2h and 24h after exercise in four subjects. Muscle strength was impaired 24h and 48h after exercise regardless of agent administered (P < 0.001). At 72h after exercise, muscle strength was significantly improved (P < 0.01) in subjects receiving etanercept and atenolol compared to those receiving placebo. The subject’s were significantly more sensitive to pressure applied to the quadriceps 24h, 48h and 72h after exercise compared to before exercise, regardless of agent administered (P < 0.001). The VAS was elevated significantly at all three time intervals, with no difference after etanercept or atenolol administration compared to that of placebo. There was no significant difference in the muscle TNF-α concentration between any of the time intervals or between subjects receiving placebo and etanercept (P=0.065). The administration of atenolol and etanercept, at the regimen used, had no effect on the soreness associated with DOMS.