Dissertations / Theses on the topic 'Increased released'

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [ H]NE uptake via the NET and simultaneously increased [ H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca . Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [ H]NE uptake but did not block KCl-stimulated increases in [ H]NE uptake. In contrast, PMA increased [ H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca -activated signaling cascades with KN93 (a Ca calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [ H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [ H]NE release. KCl-stimulated increases in [ H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca , different intracellular mechanisms mediate these two events.

Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(人間・環境学)<br>甲第21176号<br>人博第848号<br>新制||人||203(附属図書館)<br>29||人博||848(吉田南総合図書館)<br>京都大学大学院人間・環境学研究科相関環境学専攻<br>(主査)教授 加藤 眞, 教授 市岡 孝朗, 教授 瀬戸口 浩彰<br>学位規則第4条第1項該当

Pathological studies on Parkinson's disease (PD) patients suggest that PD pathology progresses from the enteric nervous system (ENS) and the olfactory bulb into the central nervous system. We have previously shown that environmental toxins acting locally on the ENS mimic this PD-like pathology progression pattern in mice. Here, we show for the first time that the resection of the autonomic nerves stops this progression. Moreover, our results show that an environmental toxin (i.e. rotenone) promotes the release of alpha-synuclein by enteric neurons and that released enteric alpha-synuclein is up-taken by presynaptic sympathetic neurites and retrogradely transported to the soma, where it accumulates. These results strongly suggest that pesticides can initiate the progression of PD pathology and that this progression is based on the transneuronal and retrograde axonal transport of alpha-synuclein. If confirmed in patients, this study would have crucial implications in the strategies used to prevent and treat PD.

Pathological studies on Parkinson's disease (PD) patients suggest that PD pathology progresses from the enteric nervous system (ENS) and the olfactory bulb into the central nervous system. We have previously shown that environmental toxins acting locally on the ENS mimic this PD-like pathology progression pattern in mice. Here, we show for the first time that the resection of the autonomic nerves stops this progression. Moreover, our results show that an environmental toxin (i.e. rotenone) promotes the release of alpha-synuclein by enteric neurons and that released enteric alpha-synuclein is up-taken by presynaptic sympathetic neurites and retrogradely transported to the soma, where it accumulates. These results strongly suggest that pesticides can initiate the progression of PD pathology and that this progression is based on the transneuronal and retrograde axonal transport of alpha-synuclein. If confirmed in patients, this study would have crucial implications in the strategies used to prevent and treat PD.

<p>Using a model to calculate the life cycle inventory of solid waste management alternatives, this study quantifies the cost effectiveness and marginal damage of several solid waste management strategies that involve recycling. Although findings from this study are not valid for any specific city, they are intended to provide decision-makers with a template upon which to base future case studies. The air emissions tracked in this study include carbon dioxide from fossil and biomass sources (CO2), nitrogen oxides (NOx), and sulfur oxides (SOx). The research was conducted in two parts. First, the maximum potential tons avoided and marginal avoidance cost resulting from expanding recycling programs for two settings, an urban and a rural area, are compared to emission control costs at a hypothetical coal-fired power plant. Second, the marginal damage associated with each recycling program expansion was calculated using published marginal damage functions. The study's findings indicate that although solid waste management (SWM) strategy upgrades are not as cost effective as additional coal-fired power plant controls for reducing the specified pollutants, marginal benefits are incurred by upgrading most SWM strategies to include drop-off recycling of waste material because its collection costs are relatively low.<P>

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである<br>Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>医学博士<br>甲第4772号<br>医博第1273号<br>新制||医||500(附属図書館)<br>UT51-91-E143<br>京都大学大学院医学研究科内科系専攻<br>(主査)教授 三河 春樹, 教授 泉 孝英, 教授 大島 駿作<br>学位規則第5条第1項該当

Zeolites have been used in agriculture since the 1960s, due to the effectiveness of these crystalline microporous solids as soil amendments for plant growth, their cation exchange capacity (CEC) and slow-release fertilizer properties. Most work on slow-release fertilizers has focused on natural Clinoptilolite, Phillipsite and Chabazite. The aim of this study was to synthesize zeolites, study their effectiveness as soil amendments and their ability to act as controlled release fertilizers to decrease nitrate leaching. Nitrate pollution of groundwater is a major agro-environmental concern. The zeolites Phillipsite and Linde-type F were synthesized from aluminosilicate gels; ion exchanged to introduce ammonium and characterized using X-ray diffraction (XRD), X-ray fluorescence (XRF), Thermo-gravimetric analysis (TGA) and Scanning electron microscopy (SEM) techniques, both before and after ion exchange. Ammoniumexchanged Phillipsites (natural and synthetic), ammonium-exchanged synthetic Linde-type F (the zeolite having highest affinity towards ammonium) and ammonium exchanged Phillipsites (high crystalline and high aluminium) were compared with conventional NPK fertilizer.Three glasshouse experiments were performed to study the effects of zeolite-amended soils on maize growth. Ion exchanged synthetic and natural Phillipsites were first used as soil amendments (w/w 2, 4, 8% zeolite to soil). Synthetic Phillipsite, at 2% loading, resulted in the most significant improvement in both plant growth and phased ammonium release. The synthetic ammonium-exchanged zeolites Phillipsite and Linde-type F (at w/w 1, 2, 4%) were then compared; synthetic Phillipsite, at 2% loading, again resulted in the most significant plant growth response with an increase (≥15%) in shoot dry weight and a decrease (≥30%) in nitrate leaching. Experiments using unexchanged synthetic Phillipsite (at w/w 2%), but with added NPK fertilizer, showed increased plant growth and decreased nitrate leaching, compared with parallel experiments containing unexchanged synthetic Linde-type F (at w/w 2%) and a conventional fertilizer amended soil. This revealed the beneficial effect of Phillipsite for soil amendment, even without ion exchange to the ammonium form. To study the physico-chemical properties affecting the release of ammonium from the Phillipsite framework; high crystalline/low aluminium and low crystalline/high aluminium forms were synthesized and ion exchanged. Both forms were introduced as soil amendments (at w/w 1 and 2%) and experiments showed that the lower zeolite crystallinity decreased cation exchange and therefore decreased nitrate leaching. Experimental results from the glasshouse experiments and cation exchange capacity (CEC) experiments suggest that synthetic Phillipsite, at lower loadings (1 and 2% w/w zeolite to soil) have most potential as soil amendments for both plant growth and controlled-release applications. This conclusion is supported by soil leachate and shoots dry weight analysis. Furthermore, Phillipsite, synthesized in a low crystalline and low ammonium form, may be an even better soil amendment for controlled release of ammonium, which will thereby further decrease nitrate pollution.

Coating urea to prepare controlled release N-fertilizer has been considered as an effective way to increase its nutrient use efficiency, thus reduce its waste and the consequent harmful environmental impacts. Inorganic sulphur and synthetic polymers have been used in the industry as coating materials together with utilization of various types of expensive coating equipment which commonly requires also complicated technical setup and controls. As development trends, biopolymers are attention-grabbing to replace the synthetic polymers. Alternative simple coating technique is also desired. So far, polylactic acid (PLA) has been reported as a more promising biopolymer than several synthetic polymers for coating. On the other hand, highly purified industrial softwood kraft lignin (SKL) produced after LignoBoost process is now available in a large quantity, which should also be a promising biopolymer for the coating application. Aiming at increase of the efficiency of PLA-coated urea and development of alternative coating technique to generally make the preparation of control-released fertilizer more effective, economic and environmentally sustainable, in this study, SKL has been used in a PLA-SKL blending form as complex coating material and simple dip-coating technique has been investigated and applied. In order to lower the wettability of PLA-SKL coat layer, four different anhydrides, namely acetic anhydride, palmitic anhydride, lauric anhydride and trifluoroacetic anhydride, were used to esterify SKL to form AcSKL, PaSKL, LauSKL and TFASKL respectively before its utilization. NMR and FTIR analyses showed that the esterification reactions have been completed for AcSKL and PaSKL. LauSKL was partly esterified due to the low charge of lauric anhydride regent, while TFASKL was not esterified expectantly due to the steric hindrance between the three F atoms and polymeric SKL. In order to obtain organically bound nitrogen structure to also create slow-release type of N-fertilizer, Mannich reaction on SKL using diethylamine was also conducted to prepare ManSKL. The reaction was completed as shown by NMR and FTIR spectroscopy. To bring further functionality of metal chelation to open the possibility to also bring essential trace element into the final fertilizer, ethylenediaminetriacetic acid (ED3A) was synthesized and further used via Mannich reaction to modify SKL to form ED3ASKL. ED3A is not commercially available and it was synthesized successfully with an environmentally friendly method from commercial EDTA and the structure was verified by NMR spectroscopy. However, the Mannich reaction using ED3A was not very successful as shown by product’s NMR and FTIR spectra. In a comparison experiment using vanillyl alcohol as a lignin model structure, ED3A was successfully coupled onto the vanillyl alcohol structure as shown by NMR and FTIR spectra. Apparently there was a severe steric hindrance from SKL for the Mannich reaction using the larger molecule of ED3A than diethylamine for Mannich reaction. For utilization of dip-coating technique, dichloromethane(DCM) and tetrahydrofuran (THF) were chosen to dissolve PLA and SKL or the modified SKL respectively. Cast films of PLA/modified lignin complex were prepared using Teflon Petri-dishes. The optimal concentration of PLA in DCM and the effect of DCM/THF ratios on the prepared cast film which expectantly represents the quality of the complex coat in the coated urea were compared with SEM images and contact angle determination. It has been found that a 30 wt% of PLA in DCM was the best and this solution mixed with modified lignin solution (6 % in THF) in a ratio of DCM/THF =3/2 (v/v) had the best film performances and water barrier properties. Generally, the cast films from PLA/modified lignin complexes showed better properties compared with the neat PLA cast film. No pores and cracks were found on the surface. Comparatively, the LauSKL film showed the most homogeneous surface. But the AcSKL film had the best water barrier properties. The PLA/modified lignin complex coated urea was then prepared by dip-coating process. The coat thickness and weight increase showed statistically positive correlations against the repeating times of the dip-coating process. The coating layer also showed one single layer structure. The speed of urea releasing for coated urea was tested and the results showed that it was much slower than the un-coated or PLA-coated urea. The single-layered PLA/AcSKL and PLA/ManSKL were both observed with sound properties in delaying the release of urea cores in water. Conclusively, the PLA/modified SKL coated urea fertilizers prepared by dip-coating technique demonstrated in this study have highly efficiency with better effects of water barrier, organically N slow release, and nitrification inhibiting (due to free phenolic functional groups) properties. Both SKL and the dip-coating technique are promising in the fertilizer applications.<br>Att ytbelägga urea för att skapa kontrollerad frisläppning av kväverika gödningsmedel har ansetts vara en effektiv metod för att öka användandet av näringsämnena från urea och dessutom minska den möjliga miljöpåverkan urea har. Kommersiellt har oorganiskt svavel och syntetiska polymerer använts för att ytbelägga olika material och detta är kopplat till olika typer av dyra ytbeläggningsutrustningar som ofta kräver komplicerade tekniska lösningar och kontroller. För att förbättra dagens lösningar är en intressant trend användandet av biopolymerer och en annan viktig aspekt är att utveckla nya enklare ytbeläggningstekniker än vad som finns på marknaden idag. Polylaktid (PLA) har till exempel rapporterats som en mer lovande förnybar polymer än flera av de syntetiska polymererna för ytbeläggnig. En annan intressant förnybar polymer är lignin, som idag tillverkas med hög renhet industriellt som barrveds kraft lignin (SKL) ur LignoBoost processen. Med målet att öka effektiviteten hos urea belagd med PLA och att utveckla en alternativ ytbeläggningsmetod för att göra den generellt mer effektiv, ekonomisk och miljövänlig har SKL använts i en PLA-SKL blandning för att ytbelägga med urea och en enkel doppbeläggningsmetod utvecklats och applicerats. För att minska vätbarheten av PLA-SKL ytbeläggningen har fyra anhydrider, ättiksyraanhydrid, palmitisk anhydrid, lauric anhydrid, och trifluoroacetisk anydrid, använts för att esterifiera SKL och bilda AcSKL, PaSKL, LauSKL och TFASKL. NMR och FTIR användes för att verifiera esterifieringsreaktionerna. Fullständig reaktion kunde konstateras för AcSKL och PaSKL, LauSKL hade bara delvis esterifierat pga den låga mängd lauric anydrid som användes medan TFASKL inte ledde till den tilltänkta esterifieringen pga steriska hinder mellan de tre flor-atomerna och SKL polymeren. För att tillverka en organiskt bunden kvävestruktur, som dessutom har en långsam frisättning av N-rika gödningsmedel genomfördes en Mannich-reaktion på SKL med dietylamin som katalysator för att framställa ManSKL. Reaktion gick till full omsättning, enligt NMR- och FTIR-spektroskopi. För att få ytterligare funktionalitet såsom metallkelation, vilken öppnar möjligheten att tillföra väsentliga spårämnen till gödningsmedlet, syntetiserades och användes etylendiamintriättiksyra (ED3A) för att modifiera SKL för att bilda ED3ASKL. ED3A finns inte kommersiellt tillgängligt utan syntetiserades framgångsrikt med en miljövänlig metod från kommersiell EDTA, varefter strukturen verifierades genom NMR-spektroskopi. Mannich-reaktionen på SKL med ED3A var emellertid inte särskilt framgångsrik, vilket NMR- och FTIR-spektra av produkten visade, Som modellexperiment användes vaniljalkohol som en ligninmodellstruktur till vilken ED3A framgångsrikt kopplades. Orsaken till denna stora skillnad i reaktivitet tros vara steriska hinder från SKL. För att utveckla en doppbeläggningsteknik valdes diklormetan (DCM) och tetrahydrofuran (THF) som lösningsmedel för att lösa upp PLA, SKL och den modifierade SKL. Gjutna filmer av PLA/modifierat lignin tillverkades i Teflon Petri-skålar. Den optimala koncentrationen av PLA i DCM och effekten av DCM/THF-förhållandet på filmens morfologi förväntas representerar kvaliteten för den framtida ytbeläggningen på urea, därför jämfördes filmernas SEM-bilder och kontaktvinkel. Det kunde konstateras att en 30 vikt% PLA i DCM var optimal och att denna lösning blandad med modifierad ligninlösning (6% i THF) i ett förhållande av DCM/THF = 3/2 (v/v) hade den bästa filmprestanda och vattenbarriäregenskaper. Generellt visade filmer av PLA/modifierade lignin bättre egenskaper jämfört med den rena PLA-filmen då inga porer och sprickor hittades på ytan. LauSKL-filmen uppvisade den mest homogena ytan medan AcSKL-filmen hade de bästa vattenbarriäregenskaperna. Urea ytbelagdes med PLA och PLA/modifierade lignin genom en doppbeläggningsprocessen. Beläggningstjockleken och viktökningen visade statistiskt positiva korrelationer gentemot antalet upprepningar av doppbeläggningsprocessen. Beläggningsskiktet visade också en enda skiktstruktur. Hastigheten det tog för urea att frigöra sig från den ytbelagda urean undersöktes och resultaten visade att den var mycket långsammare än den obehandlade urean eller den PLA-belagda urean. Enkelskiktad PLA/AcSKL och PLA/ManSKL uppvisade båda goda fördröjningsegenskaper för frisättningen av urea i vatten. Sammanfattningsvis kan man säga att PLA/modifierade SKL-belagda ureagödningsmedel framställda genom en utvecklad doppbeläggningsteknik, uppvisar hög effektivitet med bättre egenskap som vattenbarriär, långsam frisättning av organiskt kväve och nitrifikationshämmande egenskaper (beroende på fria fenoliska funktionella grupper). Både SKL och doppbeläggningstekniken är lovande i för gödningsmedelstillämpningarna.

Our laboratory has reported neonatal quinpirole (D2/D3 agonist) treatment to rats increases dopamine D2 receptor sensitivity that persists throughout the animal's lifetime. This model appears to have clinical relevance to schizophrenia, and smoking is common in this population. Male and female Sprague-dawley rats were neonatally treated with quinpirole from postnatal (P) days 1–21. After habituation from P30 to 32, animals were administered saline or nicotine (0.3, 0.5, or 0.7mg/kg free base) every other day from P33 to 49 and locomotor activity was assessed. Generally, animals neonatally treated with quinpirole and administered nicotine during adolescence demonstrated increased behavioral activity and/or sensitization compared to animals neonatally given saline and sensitized to nicotine as well as controls. However, animals neonatally treated with quinpirole and given the 0.7mg/kg dose of nicotine demonstrated elevated activity throughout testing but did not show sensitization, and only mild sex differences were reported. Therefore, microdialysis was performed on male rats sensitized to the 0.5mg/kg dose of nicotine, and results revealed that neonatal quinpirole sensitized dopamine overflow in response to nicotine to 500% above animals neonatally given saline and sensitized to nicotine at peak levels. In addition, neonatal quinpirole increased the accumbal BDNF in response to nicotine compared to all other groups, and nicotine alone also produced significant increases in striatal and accumbal BDNF. This study reveals that neonatal quinpirole enhanced adolescent nicotine sensitization, accumbal dopamine overflow, and BDNF protein in response to nicotine, which may be related to changes in the brain's reward system.

In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow. These changes could be inhibited completely by α-receptor blockers. The possible involvement of inositol phosphates in the intracellular signal transmission was studied. 1. In cell-suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose-dependent and time-dependent increase of inositol mono, bis and trisphosphate. 2. In the perfused rat liver 1 μM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time-dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions. 3. In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate. These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions.

The effect of chronic cigarette smoking on endothelin modulation of vascular contraction and CYP enzyme levels was studied in 20 male Sprague-Dawley rats. The animals were divided equally into smoking and non-smoking groups. The smoking group was exposed to 6 research cigarettes per rat per day 5 days a week for 16 weeks. The control group was sham smoked. Functional contractile studies were performed in aortas and carotid arteries to determine the regulation of vascular tone by basal release of endothelin. Liver samples were analyzed for CYPIAI and CYPIA2 gene expression by RT-PCR. Plasma samples were assessed for endothelin-l (ET-l) level by enzyme immunoassay (EIA). Treatment of aortas and carotid arteries with bosentan, the dual endothelin receptor antagonist, caused a significant reduction in constrictor responses of smoking rats, indicating increased regulation of tone by endothelin in smoker rats as compared to controls. There was also a greater expression of the cytochrome P450-liver enzymes (CYPIAI and CYPIA2) in smoker rats. Body weight gain was also significantly decreased in smoker rats. We conclude that increased endothelin release in smoker rats contributes significantly to increased arterial tone and may therefore contribute to the cardiovascular pathophysiology associated with cigarette smoking, such as increased vascular muscularization, increased contraction, decreased dilation and possibly vasospasm.<br>Medicine, Faculty of<br>Anesthesiology, Pharmacology and Therapeutics, Department of<br>Graduate

碩士<br>長庚大學<br>天然藥物研究所<br>96<br>Neutrophils are important in host’s defenses against invasion by microorganisms and are extensively involved in inflammatory processes. It has been reported that neutrophils contain soluble guanylyl cyclase (sGC) and protein kinase G (PKG). Despite this fact, the precise function of guanosine 3’,5’-cyclic monophosphate (cGMP) in regulating neutrophil granule secretion is still controversial. In addition, mitogen-activated protein kinases (MAPKs) play an important role in regulating human neutrophil functions, including degranulation, chemotaxis, and respiratory burst. However, the relationship between cGMP and MAPK signaling pathways is not well understood. BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3, 4-b]pyridin-3-yl]-pyrimidin-4-ylamine) is a potent sGC stimulator in an nitric oxide (NO)-independent manner which stimulates the enzyme synergistically with NO. The aim of this study was to investigate the role of BAY 41-2272 on human neutrophil degranulation and its underlying mechanism. BAY 41-2272 not only increased cGMP levels but also greatly potentiated sodium nitroprusside (SNP)- and S-nitroso-N-acetyl -1,1-penicillamine (SNAP)-induced cGMP formations, which was inhibited by the sGC inhibitor ODQ. BAY 41-2272 concentration-dependently promoted phosphorylation of p38 MAPK and JNK, and elevated formyl-methionyl-leucyl-phen ylalanine (fMLP)-induced elastase release. However, p38 MAPK and JNK activations and elastase release caused by BAY 41-2272 were not inhibited by inhibitors of sGC and PKG, and enhanced by SNP and SNAP. These results suggested that cGMP pathway did not involve in BAY 41-2272 induced MAPK activations and elastase release. Consistent with this, the cell permeable cGMP analogue, 8-Br-cGMP, failed to induce phosphorylation of MAPKs and elastase release. In addition, BAY 41-2272 induced phosphorylation of mitogen-activated protein kinase kinase (MKK) 4, but not MKK1/2 and MKK3/6, in a concentration-dependent manner, which was not enhanced by SNP. Furthermore, BAY 41-2272 increased fMLP-induced elastase release was inhibited by inhibitors of p38 MAPK and JNK. In summary, these results demonstrate that BAY 41-2272 promoted p38 MAPK and JNK activation and elastase release via a cGMP-independent but MKK4-dependent signaling pathway in human neutrophils.

碩士<br>國立陽明大學<br>解剖暨細胞生物學研究所<br>97<br>The structure and activation of membrane proteins play key regulatory roles in numerous intracellular signal transduction in eukaryotic cells. Caveolae, a 50 nanometer-sized invagination of the plasma membrane, is well-known microstructure for endocytosis and transcytosis in endothelial cells. Caveolin-1 (CAV1) is a major component of caveolae. Strong evidence showed that caveolin-1 acts as a linker for eNOS and plays an important role for NO signaling in endothelial cells. In this study, we explored the functional roles of CAV1 in endothelial MS1 cells with RNA interference by using VSV-G pseudotyped lentivirus system. By shCAV1-lentiviral infection technique, we downregulated CAV1 expression in endothelial MS1 cells which express high levels of CAV1 mRNA and caveolin-1 protein. The infection of shCAV1 to MS1 cells (MS1cav1- cells) successful reduced the levels of CAV1mRNA and its protein about 46% and 45% compared with mock cells, respectively. Moreover, MS1cav1- cells have no morphological change and alteration of cell growth. However, the knock-down of CAV1 expression resulted in the decrease of angiogenic factors, (VE-cadherin and Tie-2), leading to the failure of tubule-like formation, and the downregulation of cell adhesion molecules (JAM-1、Itgb5、VCAM-1), leading to the decrease of cell adhesion. On the other hand, the knock-down of CAV1 expression in MS1cav1- cells increases the nitric oxide release and promotes the cell migration ability.

Japanese encephalitis virus (JEV) causes viral encephalitis in new born and young adults that is prevalent in different parts of India and other parts of South East Asia with an estimated 6000 deaths per year. JEV is a single stranded RNA virus that belongs to the Flavivirusgenus of the family Flaviviridae. It is a neurotropic virus which infects the central nervous system (CNS). The virus follows a zoonotic life-cycle involving mosquitoes and vertebrates, chiefly pigs and ardeid birds, as amplifying hosts. Humans are dead end hosts. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral leukocytosis in the brain and damage to Blood Brain Barrier (BBB). The exact role of the endothelial cells during CNS infection is still unclear. However, disruption of this endothelial barrier has been shown to be an important step in entry of the virus into the brain. Humoral and cell mediated immune responses during JEV infection have been intensively investigated. Previous studies from our lab have shown the activation of cytotoxic T-cells (CTLs) upon JEV infection. MHC molecules play pivotal role in eliciting both adaptive (T-cells) and innate (NK cells) immune response against viral invasion. Many viruses such as HIV, MCMV, HCMV, AdV and EBV have been found to decrease MHC expression upon infection. On the contrary, flaviviruses like West Nile Virus (WNV) have been found to increase MHC-I and MHC-II expression. More recently, data from our lab has shown that JEV infection can lead to upregulation of mouse non-classical MHC class Ib molecules like Qb1, Qa1 and T-10 along with classical MHC molecules. Non-classical MHC molecules are important components of the innate and adaptive immune systems. Non-classical MHC molecules differ from their classical MHC class I counterparts by their limited polymorphism, restricted tissue distribution and lower levels of cell surface expression. Human classical MHC class I molecules are HLA-A, -B and –C while non-classical MHC Class Ib molecules are HLA-E, -G and –F. HLA-E, the human homologue of the mouse non-classical MHC molecule, Qa-1b has been shown to be the ligand for the inhibitory NK, NKG2A/CD94 and may bridge innate and adaptive immune responses. In this thesis, we have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecule, HLA-E in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial like cell line ECV304 (ECV), human glioblastoma cell line U87MG and human foreskin fibroblast cells (HFF). We observed an upregulation of classical HLA molecules and HLA-E mRNA in endothelial and fibroblast cells upon JEV infection. This mRNA increase also resulted in upregulation of cell surface classical HLA molecules and HLA-E in HFF cells but not in both the human endothelial cell lines, ECV and HBMECs. Release of soluble classical HLA molecules upon cytokine treatment has been a long known phenomenon. Recently HLA-E has also been shown to be released as a 37 kDa protein from endothelial cells upon cytokine treatments. Our study suggests that JEV mediated upregulation of classical HLA and HLA-E upregulation leads to release of both Classical HLA molecules and HLA-E as soluble forms in the human endothelial cell lines, ECV and HBMEC. This shedding of sHLA-E from human endothelial cells was found to be mediated by matrix metalloproteinase (MMP) proteolytic activity. MMP-9, a protease implicated in release of sHLA molecules was also found to be upregulated upon JEV infection only in endothelial cell lines but not in HFF cells. Our study provides evidence that the JEV mediated solubilisation of HLA-E could be mediated by MMP-9. Further, we have tried to understand the role of the MAPK pathway and NF-κB pathway in the process of HLA-E solubilisation by using specific inhibitors of these pathways during JEV infection of ECV cells. Our data suggests that release of sHLA-E is dependent on p38 and JNK pathways while ERK 1/2 and NF-κB pathway only had a minor role to play in this process. Treatment of endothelial cells with TNF-α, IL-1β and IFN-γ is known to result in release of sHLA-E. In addition to TNF-α and IFNtreatment, we observed that activating agents like poly (I:C), LPS and PMA also resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Treatment of endothelial cells with IFN-β, a type-I interferon also led to release of sHLA-E. IFN-γ, a type II interferon and TNF-α are known to show additive increase in solubilisation of HLA-E. We studied the interaction between type I interferon, IFN-β and TNF-α with regard to shedding of sHLA- E. Both IFNand TNF, when present together caused an additive increase in the shedding of sHLA-E. These two cytokines were also found to potentiate the HLA-E and MMP-9 mRNA expression. Hence, our data suggest that these two cytokines could be working conjunctly to release HLA-E, when these two cytokines are present together as in the case of virus infection of endothelial cells. HLA-E is known to be a ligand for NKG2A/CD94 inhibitory receptors present on NK and a subset of T cells. Previous reports have suggested that NKG2A/CD94 mediated signaling events could inhibit ERK 1/2 phosphorylation leading to inhibition of NK cell activation. IL-2 mediated ERK 1/2 phosphorylation is known to play a very important role in maintenance and activation of NK cells. We studied the effects of sHLA-E that was released, either by JEV infection or IFN-γ treatment on IL-2 mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. The soluble HLA-E that was released upon JEV infection was functionally active since it inhibited IL-2 and PMA induced phosphorylation of ERK 1/2 in NKL and Nishi cells. Virus infected or IFN-γ treated ECV cell culture supernatants containing sHLA-E was also found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. CD25 is a component of the high affinity IL-2 receptor and hence could play an important role in proliferation and activation of NK cells. sHLA-E was also found to inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit the proliferation and activation of NK cells. In summary, we found that establishment of JEV infection and production of cytokines like IFN-β, TNF-α, IL-6 along with MMP-9 in human endothelial cells. These cytokines may also indirectly lead to the reported damage and leukocyte infiltration across infected and uninfected vicinal endothelial cells. The increased surface expression of HLA-E in fibroblast and release of sHLA and sHLA-E molecules from endothelial cells may have an important immunoregulatory role. HLA-E is an inhibitory ligand for NKG2A/CD94 positive CD8+ T and NK cells. Hence our finding that sHLA-E can inhibit NK cell proliferation suggests an immune evasive strategy by JEV.

Japanese encephalitis virus (JEV) causes viral encephalitis in new born and young adults that is prevalent in different parts of India and other parts of South East Asia with an estimated 6000 deaths per year. JEV is a single stranded RNA virus that belongs to the Flavivirusgenus of the family Flaviviridae. It is a neurotropic virus which infects the central nervous system (CNS). The virus follows a zoonotic life-cycle involving mosquitoes and vertebrates, chiefly pigs and ardeid birds, as amplifying hosts. Humans are dead end hosts. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral leukocytosis in the brain and damage to Blood Brain Barrier (BBB). The exact role of the endothelial cells during CNS infection is still unclear. However, disruption of this endothelial barrier has been shown to be an important step in entry of the virus into the brain. Humoral and cell mediated immune responses during JEV infection have been intensively investigated. Previous studies from our lab have shown the activation of cytotoxic T-cells (CTLs) upon JEV infection. MHC molecules play pivotal role in eliciting both adaptive (T-cells) and innate (NK cells) immune response against viral invasion. Many viruses such as HIV, MCMV, HCMV, AdV and EBV have been found to decrease MHC expression upon infection. On the contrary, flaviviruses like West Nile Virus (WNV) have been found to increase MHC-I and MHC-II expression. More recently, data from our lab has shown that JEV infection can lead to upregulation of mouse non-classical MHC class Ib molecules like Qb1, Qa1 and T-10 along with classical MHC molecules. Non-classical MHC molecules are important components of the innate and adaptive immune systems. Non-classical MHC molecules differ from their classical MHC class I counterparts by their limited polymorphism, restricted tissue distribution and lower levels of cell surface expression. Human classical MHC class I molecules are HLA-A, -B and –C while non-classical MHC Class Ib molecules are HLA-E, -G and –F. HLA-E, the human homologue of the mouse non-classical MHC molecule, Qa-1b has been shown to be the ligand for the inhibitory NK, NKG2A/CD94 and may bridge innate and adaptive immune responses. In this thesis, we have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecule, HLA-E in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial like cell line ECV304 (ECV), human glioblastoma cell line U87MG and human foreskin fibroblast cells (HFF). We observed an upregulation of classical HLA molecules and HLA-E mRNA in endothelial and fibroblast cells upon JEV infection. This mRNA increase also resulted in upregulation of cell surface classical HLA molecules and HLA-E in HFF cells but not in both the human endothelial cell lines, ECV and HBMECs. Release of soluble classical HLA molecules upon cytokine treatment has been a long known phenomenon. Recently HLA-E has also been shown to be released as a 37 kDa protein from endothelial cells upon cytokine treatments. Our study suggests that JEV mediated upregulation of classical HLA and HLA-E upregulation leads to release of both Classical HLA molecules and HLA-E as soluble forms in the human endothelial cell lines, ECV and HBMEC. This shedding of sHLA-E from human endothelial cells was found to be mediated by matrix metalloproteinase (MMP) proteolytic activity. MMP-9, a protease implicated in release of sHLA molecules was also found to be upregulated upon JEV infection only in endothelial cell lines but not in HFF cells. Our study provides evidence that the JEV mediated solubilisation of HLA-E could be mediated by MMP-9. Further, we have tried to understand the role of the MAPK pathway and NF-κB pathway in the process of HLA-E solubilisation by using specific inhibitors of these pathways during JEV infection of ECV cells. Our data suggests that release of sHLA-E is dependent on p38 and JNK pathways while ERK 1/2 and NF-κB pathway only had a minor role to play in this process. Treatment of endothelial cells with TNF-α, IL-1β and IFN-γ is known to result in release of sHLA-E. In addition to TNF-α and IFNtreatment, we observed that activating agents like poly (I:C), LPS and PMA also resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Treatment of endothelial cells with IFN-β, a type-I interferon also led to release of sHLA-E. IFN-γ, a type II interferon and TNF-α are known to show additive increase in solubilisation of HLA-E. We studied the interaction between type I interferon, IFN-β and TNF-α with regard to shedding of sHLA- E. Both IFNand TNF, when present together caused an additive increase in the shedding of sHLA-E. These two cytokines were also found to potentiate the HLA-E and MMP-9 mRNA expression. Hence, our data suggest that these two cytokines could be working conjunctly to release HLA-E, when these two cytokines are present together as in the case of virus infection of endothelial cells. HLA-E is known to be a ligand for NKG2A/CD94 inhibitory receptors present on NK and a subset of T cells. Previous reports have suggested that NKG2A/CD94 mediated signaling events could inhibit ERK 1/2 phosphorylation leading to inhibition of NK cell activation. IL-2 mediated ERK 1/2 phosphorylation is known to play a very important role in maintenance and activation of NK cells. We studied the effects of sHLA-E that was released, either by JEV infection or IFN-γ treatment on IL-2 mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. The soluble HLA-E that was released upon JEV infection was functionally active since it inhibited IL-2 and PMA induced phosphorylation of ERK 1/2 in NKL and Nishi cells. Virus infected or IFN-γ treated ECV cell culture supernatants containing sHLA-E was also found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. CD25 is a component of the high affinity IL-2 receptor and hence could play an important role in proliferation and activation of NK cells. sHLA-E was also found to inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit the proliferation and activation of NK cells. In summary, we found that establishment of JEV infection and production of cytokines like IFN-β, TNF-α, IL-6 along with MMP-9 in human endothelial cells. These cytokines may also indirectly lead to the reported damage and leukocyte infiltration across infected and uninfected vicinal endothelial cells. The increased surface expression of HLA-E in fibroblast and release of sHLA and sHLA-E molecules from endothelial cells may have an important immunoregulatory role. HLA-E is an inhibitory ligand for NKG2A/CD94 positive CD8+ T and NK cells. Hence our finding that sHLA-E can inhibit NK cell proliferation suggests an immune evasive strategy by JEV.