Validity index generation in distributed intelligent sensor component using frequency domain analysis for real time control applications

Paudel, Ananda Mani Gwangju Institute of Science and Technology 2005 국내석사

In vitro and in silico study of Cassia seeds-derived secondary metabolites on human monoamine oxidase and G-protein coupled receptors

Paudel, Pradeep Pukyong National University 2020 국내박사

Cassia obtusifolia L. seed is one of the most popular traditional Chinese medicine for mutagenicity, genotoxicity, hepatotoxicity, and acute inflammatory diseases. Recently, its neuroprotective activity has been reported mainly via antioxidant mechanisms. There are no reports of neuroprotection via human monoamine oxidase (hMAO) inhibition and G protein-coupled receptors (GPCRs) modulation. The main objective of this study is to delineate and accentuate the neuroprotective effect of Cassia seeds‒derived secondary metabolites via in vitro and in silico hMAO inhibition and GPCRs modulation. The seed extract exhibited good inhibition of hMAO-A isozyme with an IC50 value of 86.89 ± 3.80 μg/mL. Among the solvent‒soluble fractions, EtOAc fraction (IC50: 20.82 ± 5.04 μg/mL) was the most potent inhibitor of hMAO-A isozyme followed by CH2Cl2 (IC50: 40.06 ± 3.58 μg/mL) and n-BuOH fraction (IC50: 93.49 ± 0.27 μg/mL). In isozyme‒B, inhibition by seed extract and solvent‒fraction was mild with an IC50 value range of 56.28 ‒ 246.50 μg/mL. Bioassay-guided isolation led to thirty‒six known secondary metabolites along with two new naphthalenic lactone glycosides, (3R)-cassialactone 9-O-β-D-glucopyranoside (37) and (3S)-9,10-dihydroxy-7-methoxy-3-methyl-1-oxo-3,4-dihydro-1H-benzo[g]isochromene-3-carboxylic acid 9-O-β-D-glucopyranoside (38). Almost all metabolites showed selective inhibition of hMAO-A isozyme. In particular, aglycons obtusin (3), alaternin (8), aloe-emodin (9), emodin (10), questin (12), and rubrofusarin (13) and glycosides cassiasid (15), toralactone gentiobioside (26), and 38 showed promising inhibition of hMAO-A isozyme with IC50 values 0.17 to 11 μM. Compounds 8 and 12 were active against hMAO-B with IC50 values 4.55 ± 0.09 and 10.58 ± 0.28 μM, respectively. The enzyme kinetic study revealed the mode of enzyme inhibition, and an in silico molecular docking simulation predicted that Ile335, Tyr407, Phe208, and Ile180 are the determinant interacting residues for hMAO-A selectivity. Furthermore, the effect of MAO‒active major anthraquinones 8, 9, 10 and 12 on various GPCRs (hD1R, hD3R, hD4R, h5-HT1AR, and hV1AR) modulation was evaluated via cell-based functional assays. Among them, 10 and 8 showed a concentration-dependent agonist effect on hD3R and antagonist effect on hV1AR. The half-maximal effective concentration (EC50) of 10 and 8 for hD3R was 21.85 ± 2.66 and 56.85 ± 4.59 μM, respectively. The reference agonist dopamine had the EC50 value of 4 nM. Compound 12 showed moderate hD3R agonist effect with 34% of control agonist response at 50 uM, and 9 showed mild hD3R antagonist effect. On hV1AR, 10 and 8 showed potent antagonist effect with IC50 values of 10.25 ± 1.97 and 11.51 ± 1.08 μM, respectively. Interestingly, 9 and 12 did not have any observable effect on hV1AR. Only 8 was effective in antagonizing h5-HT1AR (IC50: 84.23 ± 4.12 μM). The results of computational study revealed that ‒ hydroxyl group at C1, C3, and C8 and a methyl group at C6 of anthraquinone structure are essential for hD3R agonist and hV1AR antagonist effect; the H‒bond interaction of 1‒OH group with Ser192 at a proximity of 2.0 Å which 8 lacked role for the higher stability of 10‒hD3R complex and higher potency of 10; and interaction of the 7‒OH group of 8 with Trp358 is a determinant for the observed antagonist effect of 8 on h5‒HT1AR. Overall, this study characterizes anthraquinones, especially alaternin (8) and emodin (10) as principal components in the management of neurodegenerative disorders, particularly anxiety and depression, and supports Cassia seeds as a functional food. However, investigating in vivo effects of these compounds in receptor knock-down, knock-out and/or knock-in mice models deserve particular attention shortly.

Microparticles-mediated vascular inflammation and its ameliorative action by baicalin : Microparticles에 의한 혈관염증과 baicalin의 효과

Paudel, Keshav Raj 목포대학교 대학원 2017 국내박사

Microparticles (MP) are extracellular vesicles (0.1 to 1.0 μm) released by any type of cells in response to cell activation or apoptosis. MP originated from various cells are raised in cardiovascular diseases (CVD) including atherosclerosis. The high level of circulating MP is one of the important indicators of altered vascular function and could lead to vascular inflammation. The prominent MP present in blood is platelet microparticles (P-MP) and endothelial cell microparticles (EC-MP). EC-MP, vascular smooth muscle cell microparticles (VSMC-MP) and macrophage microparticles (MØ-MP) are key hallmark of atherosclerosis progression. EC-MP exert anticoagulant and pro-inflammatory activity and may facilitate the cellular oxidative stress followed by vascular inflammation. It is well established that MP release by cell is mediated by cell activation or apoptosis inducer such as angiotensin II, lipopolysaccharide (LPS), hydrogen peroxide, tumour necrosis factor-α (TNF-α), leading to progression of atherosclerosis. Atherosclerotic plaques formation is initiated by endothelial dysfunction leading to rupture of carotid plaques in later step causing stroke. Recent study highlights the role of EC-MP in the pathogenesis of CVD, acting as a hallmark of vascular damage, either by worsening disease progression or by triggering a repair response. VSMC-MP released from apoptotic vascular smooth muscle cell (VSMC) can cause endothelial dysfunction by binding to EC and inhibit the nitric oxide (NO) production whereas the microparticles produce from activated macrophage can exaggerate vascular inflammation.. LPS induces MØ-MP release can in turn overexpress inflammatory mediators such as NO via inducible nitric oxide synthase (iNOS) pathway, cyclooxygenase (COX)-2, and reactive oxygen species (ROS) from macrophage itself. In this study, we investigated whether MP are involved in pathogenesis of vascular inflammation and checked if baicalin ameliorates the MP induced VSMC, EC dysfunction and inhibits inflammatory mediators production in macrophage (RAW264.7) as well as preventing vascular inflammation in LPS induced rat model of vascular injury. Our results showed that baicalin significantly suppressed the apoptosis, ROS, NO production and expression of iNOS and COX-2 protein in MØ-MP induced RAW264.7 cells. Moreover, the formation of MØ-MP induced foam cell was attenuated by baicalin in RAW264.7 cells. Also, VSMC migration induced by MØ-MP, EC-MP and VSMC-MP was dose dependently inhibited by baicalin. The anti-migratory effect of baicalin on VSMC was due to its inhibitory effect on metalloproteinase-9 (MMP-9) expression preventing the degradation of extracellular matrix. Furthermore, baicalin suppressed VSMC-MP induced VSMC proliferation by down-regulation of mitogen activated protein kinase (MAPK) and proliferating cell nuclear antigen (PCNA) signalling molecules. Baicalin also inhibited ROS production and apoptosis in VSMC. In endothelial cell, the marker of endothelial dysfunction (ROS production, down regulation of peNOS, pAkt and upregulation of intracellular adhesion molecule) induced by EC-MP was halted by baicalin treatment. Moreover, the adhesion of macrophage and platelets to endothelial cells were inhibited by baicalin in co-culture study. Our in vivo results revealed that baicalin inhibit the potent vascular inflammation marker; serum NOx, MPO and lipid peroxidation. Likewise, the protein expression of peNOS, pAkt was upregulated and MMP-9, ICAM, pERK1/2, pJNK, and pP38 were downregulated by baicalin. Histological examination revealed that baicalin protect the damage of endothelial layer and inhibit cell adhesion to activated endothelial cell of the artery. Thus, our results suggest that baicalin exerts beneficial activity to prevent vascular inflammation by restoring the function of damage endothelial cell, inhibiting VSMC migration and proliferation as well as suppressing inflammation mediators release in response to LPS (in vivo) or microparticles (in vitro). 다양한 세포에서 유래된 microparticles (MP)는 동맥경화증을 포함한 다양한 심혈관질환을(CVD)을 야기한다. 혈관에 많은 MP는 혈관 기능의 저하 및 혈관 염증을 유발한다. 혈액에 존재하는 혈소판 MP와 내피세포MP가 있으며, MP는 세포외소포 (0.1 ~ 1.0μm)에서 방출되며 세포의 활성화 또는 세포사멸을 일으킵니다. 내피세포-MP (EC-MP), 평활근-MP (VSMC-MP) 및 대식세포-MP (MØ-MP)의 발현은 동맥경화 진행에 있어 전형적인 특징입니다. EC-MP는 항응고 및 세포에 산화스트레스를 유발하여 혈관에 염증반응을 일으킬 수 있으며, 혈관세포에서 방출된 MP은 동맥경화증의 진행으로 이끄는 안지오텐신 II, 지질다당류, 과산화수소와 같은 세포활성화제 또는 세포자멸유발제로 잘 알려져 있습니다. 세포자멸혈관평활근세포에서 방출된 VSMC-MP는 혈관내피와 결합하여 내피 기능장애와 산화질소(NO)의 생성을 억제합니다. 동맥경화에서는 죽상반이 형성되게 되면 내피세포에 기능 이상 및 파열을 야기하게 되고 심장마비를 일으킵니다. 최근 연구에서는 EC-MP가 혈관손상으로 인한 질병진행을 악화시키거나 치료하는 과정에서 병인으로서 EC-MP의 역할이 강조되고 있습니다. 유사하게, MP는 혈관평활근세포(VSMC)의 이동 및 증식을 유발하며 활성화된 대식세포로부터 혈관염증을 유발합니다. MØ-MP는 지질다당류, 염증성사이토카인(TNF-α, IL-1β) 및 C반응성 단백질에 의한 자극에 반응하여 대식세포에 의해 생산됩니다. Endotoxin는 MØ-MP 방출을 유도하여 (iNOS) 경로, cyclooxygenase (COX-2) 및 활성산소종을 통해 NO와 같은 염증 매개체를 과다 발현시킵니다. 본 연구에서는 MP가 혈관염증의 병인에 관여하는지를 확인하고, baicalin이 MP에 의한 VSMC 및 EC 기능장애를 완화시키고 대식세포(RAW264.7)에서의 염증 매개체 생산을 억제하고 LPS로 유도된 rat모델에서 혈관염증억제을 억제하는지 확인하였다. 실험결과 baicalin은 RAW264.7에서 MØ-MP로 유도된 iNOS와 COX-2 단백질발현을 억제하였으며, MØ-MP 유도된 RAW264.7 세포에서 foam cell의 형성을 약화시켰다. 또한, EC-MP와 VSMC-MP에 의해 유도 된 VSMC에서 baicalin은 농도의존적으로 세포이동을 억제하였으며, VSMC에 대한 baicalin의 세포이동억제 효과를 알아보기 위해 금속단백분해효소-9 (MMP-9) 발현 억제를 확인하였으며, 그로 인한 세포외 기질(ECM)의 손상을 예방하였다. 또한 baicalin은 VSMC-MP에 의해 유도된 VSMC의 세포이동를 억제하였으며, mitogen activated protein kinase (MAPK)와 PCNA의 단백질발현을 억제하였다. Baicalin은 VSMC에서 활성산소종 생성과 세포사멸을 억제하였고, 내피세포에서는 EC-MP에 의해 유도된 내피 기능 장애와 peNOS의 감소, pAkt 및 ICAM-1 증가 및 활성산소종의 생성이 억제되었다. 또한, 내피세포에 대식세포와 혈소판의 부착도 억제되었다. in vivo 실험결과 baicalin이 혈관염증표지인NOx, MPO 및 지질과산화를 억제하는 것을 확인하였으며, 마찬가지로, baicalin 처리 결과 peNOS, pAkt의 발현을 증가시켰으며, MMP-9, ICAM-1, pERK1/2, pJNK, pP38의 발현을 억제하였다. Baicalin 은 내피층의 손상을 방지하고 내피세포에 대한 세포부착을 억제하였다. 따라서, baicalin은 LPS 또는 MP에 반응하여 손상된 내피세포의 기능을 회복시키고, VSMC 이동 및 증식을 억제함으로써 혈관 염증을 예방하는 것으로 사료된다.

Insect-Plant Interactions in a Warming World: Effects of Elevated Temperature on Tomatoes and Their Insect Herbivores

Paudel, Sulav ProQuest Dissertations & Theses The Pennsylvania S 2019 해외박사(DDOD)

Predicting the impact of climate change is one of the leading challenges of current times. Despite the potential to substantially impact crops economically, overall impacts of elevated temperature on insect-plant interactions are poorly understood, especially in agricultural systems. The goal of this dissertation is to investigate the impact of climate warming on insect herbivores, on their host plants and the interactions between them using the case of the corn earworm on tomatoes.First, the interactive effects of elevated temperature on insect herbivory (Helicoverpa zea) and resistance/tolerance traits of tomato (Solanum lycopersicum var Better Boy) were evaluated using artificial warming. In addition to an asymmetric responses between plant and insects, novel mechanisms were identified explaining how varying temperature affected the biosynthesis of insect elicitors and the ability of insects to trigger plant defense responses; insects reared at a warmer temperatures produced significantly less glucose oxidase (GOX), which paralleled a lower level of induction of plant defensive proteins, polyphenol oxidase (PPO) and trypsin protease inhibitors (TPI). Similarly, induction of plant defenses and plant resistance to the insect herbivore was highest in plants grown at above optimum temperatures but varied between damaged and undamaged leaves; herbivore growth was significantly reduced when fed on damaged leaves compared to undamaged control. These findings add an exciting new dimension to how climate change may alter plant-insect interactions.Second, using elevation as a proxy for temperature change, a field study in Nepal and greenhouse experiments at Penn State on tomato accessions from the Andes were tested to evaluate changes to plant herbivore interactions approximating the impacts of climate warming. The field study was conducted at various elevations in the Himalayan Mountains of Nepal in farmers’ fields to simulate climate change. Temperature varied with elevation in the field and  significantly affected both insect populations and plant damage. At higher elevation, natural herbivore populations and plant damage from herbivory were significantly increased compared to low-elevation counterparts. In greenhouse experiments, changes in plant defense strategies and resistance to insect herbivory along an elevational gradient was also established by using tomato accessions adapted to a specific elevation range in South America. Plant resistance and defensive chemicals (e.g., total phenolics content) to insect herbivory was enhanced in accessions from higher elevation. Results from both field and greenhouse experiments indicated a great deal of plasticity and variability in plant defense responses to both biotic and abiotic stresses.Last, the variation in induced plant defensive traits and strategies between wild and cultivated tomato genotypes was also investigated. Three different tomato genotypes were used; Solanum pimpinellifolium L. (accession LA 2093), b) cherry tomato, S. lycopersicum L. var. cerasiforme (accession Matts Wild Cherry), and c) cultivated tomato, S. lycopersicum L. var. Better Boy). Multiple chemical (plant volatiles, phenolics, defense proteins) and physical defenses (trichomes) in the cultivated tomato and its closest progenitors were measured. As expected, the wild species of tomato show higher levels of constitutive defenses, but the novel finding is that the cultivated tomato demonstrated the highest level of induced defenses (Paudel et al., 2019).While crop losses are expected to increase with global warming, elevated temperatures in this study produced asymmetric responses between insects and plants, indicating a more complicated response of plants and their herbivores under a climate change scenario. A plasticity in plant defense mechanisms were observed in the elevational studies which may possibly determine the amount of plant damages with expected geographical shift of insect pests towards higher elevations. Similarly, a large variation in plant defense mechanisms were demonstrated between wild and domesticated tomato genotypes which could be exploited as a component of sustainable crop protection in the face of climate change. Moving forward, we cannot assume that all of these crop-pest relationships will change in the same way due to climate warming. Therefore, future studies should include a wide range of host plants, insect herbivores (using both individual plant/herbivore pairs and groupings) and tri-trophic interactions complemented by field studies to provide more realistic assessments.

Isolation and characterization of antibacterial and antioxidant compounds from the antarctic lichen Ramalina terebrata

Paudel-Bhattarai, Babita Soonchunhyang Univ. 2009 국내박사

Factors affecting investment climate and industrial development in nepal : focusing on private sectot's perspective

Paudel, Shesh Narayan Graduate School of International Studies, Korea Un 2012 국내석사

The private sector is the engine of economic growth and development. The private sector’s investment on industries and businesses strengthens the national economy and accelerates the pace of economic development. However, the private sector needs a sound investment climate for increased investments. The adverse investment climate in a country makes the entrepreneurs to withdraw from the investment and businesses. The present research studies the perspective of private sector on the factors affecting the investment climate in Nepal. The research finds that non-economic factors such as insecurity, labor disputes, strikes, energy crisis, political instability and impunity are the main factors affecting private investment in Nepal. The root cause of those problems is the political instability which is continuing since the political change in 1990. The degrading investment climate has affected the overall economic situation of the country. The industrial sector is continuously degrading since past two decades. It has turned the livelihood of Nepalese people harder and harder day by day. The government should soon address the investment problems to save the country going from further economic hardships. The existing criminal laws should be amended to the provision of severe punishment to deter the crime. The unruly trade unions’ activities should be checked and the right to lawfully earn and own the private property should be protected. The unwanted politics in the hydro-power sector should come to an end. Politicians should now work to give stability, peace and development in the country.

Determinants factors of value added tax in Nepal

Paudel, Binod Graduate School of International Studies, Korea Un 2021 국내석사

Value Added Tax (VAT) is a tax that exhibits an exceptional feature of both indirect and direct taxes. It was first introduced to Nepal in 1996. The major objectives of the study is to identify the various macro-economic factors as the determinants of the VAT revenue in Nepal by using time series data for the period of 1996 to 2018. Both qualitative and quantitative methods Ordinary Least Squares with time series method have been adopted to analysis data and information. The linear regression between dependent variable VAT Amount and independent variable GPD, Public Expenditure, Private Consumption, Import, Remittance inflow, Inflation, and Rate of VAT was performed to identify the nature and strength of relation among the variable considered. The amount is normalized using log-transformation due to the large scale of the data sets. The first difference of each variable has been calculated for time series analysis. On performing descriptive statistics, it is found that all the determinants were in an increasing trend with the passage of the year.

Spatio-Temporal Patterns of Human-wildlife Conflict in Bardia National Park, Nepal

PAUDEL, Prabin University of Seoul, International School of Urban 2021 국내석사

지구 생태계의 균형을 이루기 위해서는 야생 생물 보존이 필수적이다. 하지만 야생 동물이 인간에게 피해를 입히면 인간은 그에 대한 복수로 야생 동물을 살상한다. 따라서 생태계 및 인간이 영위하는 생활의 지속 가능성을 위해서는 충돌을 최소화해야 한다. 인간과 야생 동물의 충돌은 바르디아 국립 공원(Bardia National Park)의 주요한 해결 과제이다. 효과적인 충돌 관리를 위해서는 구체적인 시공간 정보가 필수적이다. 그렇기 때문에, 본 연구에서는 바르디아 국립 공원의 완충 지대에서 11년(2010~2020) 동안 발생하여 기록된 충돌의 시공간적 분포를 조사하였다. 데이터 분석에는 기술 및 추론 통계를 사용하였다. 분석 결과에 따르면 기록된 충돌 횟수가 매년 증가하고 있다. 평균적으로 연간 806 건의 충돌이 기록되었다. 이 보호 구역에서 발생한 충돌의 주요 대상 종은 코끼리와 표범인데, 충돌의 59%가 남부 지역에서 기록되었다. 인간과 코끼리의 충돌 중 60% 이상이 8월, 9월, 10월 및 11월에 발생했다. 충돌이 가장 빈번한 계절은 종마다 다르지만, 코끼리, 표범, 호랑이 및 멧돼지의 경우 충돌의 계절적 분포가 유의미하다. 조사 대상 기간 동안의 손실 평가액 중 37%만이 보조금 형태로 지급되었다. 보조금을 받기까지의 과정도 길다. 따라서 지역을 바탕으로 주요 충돌 종을 관리하기 위한 월간 일정이 필수적이다. 초식 동물의 입맛에 덜 맞는 대체 작물을 재배하고 육식 동물의 침입을 막을 수 있는 축사를 장려하는 것이 필요하다. 전기 울타리를 설치하고 유지하며 대체 생계 수단을 제공함으로써 사람들의 삼림 출입을 줄이는 것 또한 중요하다. 이에 더해, 지역 사회를 대상으로 야생 동물의 행동에 대한 지식을 제공하고 교육을 실시하는 또한 바르디아 국립 공원 내에서의 충돌 사고를 축소하는 데 도움이 될 것이다. 주요어: 인간과 야생 동물의 충돌, 시공간 분포, 바르디아 국립 공권, 완충 지대 Human-wildlife conflict is one of the major conservation challenges. Detail spatial and temporal information is essential for effective conflict management. Therefore, this study examines the distribution of conflicts based on space and time for eleven years (2010 to 2020) in the buffer zone of Bardia National Park. Descriptive and inferential statistics were used to analyze the data. The result shows that the number of human-wildlife conflicts is increasing annually. Where on average, 806 conflicts per year were recorded. Elephant and leopard are the major conflicting species in this protected area, and 59 % of the conflict was recorded in the southern regions. More than sixty percent of human-elephant conflicts are recorded in August, September, October, and November. Major conflicting seasons vary with the species, where the distribution of conflict is significantly varied with the seasons for elephant, leopard, tiger, and wild boar. Attack due to carnivore is significantly higher in the new moon phase periods. Only 37 % of the evaluated loss was paid to the victim as a relief. A monthly calendar for the management of major conflicting species is essential. Farming of alternative crops that are less palatable for the herbivores and promoting predator-proof livestock shelter is needed. Where construction and maintenance of electric fences and reduction of dependency over forest resources by providing alternative sources of livelihood are crucial. Furthermore, providing knowledge and training about wildlife behavior to the local community will reduce the conflict and increase the harmony between people and wildlife.

Morphological and anatomical study of Calycanthaceae

Paudel, Niroj Kangwon National University 2019 국내박사

The accurate classification of four genera of Calycanthaceae was not completely resolved. They are closely related in vegetative characters and growth habits each other. Several contradicting taxonomic issues have been remained. However, there is still unable establishing a natural classification of the family and especially in proposing a relevant treatment of relationship within the genera. Therefore, the comparative morphological and anatomical study of leaf, fruits and seed, stem, and pollen was carried out. Cladistic analysis was also conduct based on morphological and anatomical characters. Out of the four genera, the vascular bundle of Sinocalycanthus and Calycanthus was the V-shaped, whereas Idiospermum and Chimonanthus was the U-shaped. The distinctive leaf morphological features were presence of trichome, stomata and variation of the density of trichome, stomata density, and leaf surface. In this study, the density of trichome was observed higher in Calycanthus than other genera. The abaxial surface of Idiospermum and Sinocalycanthus was smooth but Chimonanthus and Calycanthus was rough. Presence of crystal was notified in Sinocalycanthus, Calycanthus and Chimonanthus but absent in Idiospermum. The interesting structure of cuticle was observed in Idiospermum. The unicellular trichome is common in all genera. The vascular bundle contains four cortical vascular bundles in the stem. Calycanthus possess bordered-pit with circular aperture while Chimonanthus possess elliptic aperture. Tracheid is a characteristic feature of the wider spiral band in Chimonanthus than that of Calycanthus and Sinocalycanthus. Sclerenchyma cells were aggregated in Sinocalycanthus and Calycanthus but in case of Chimonanthus, it was the long chain with the layer of the sclerenchyma cell. Collenchyma cells were circular shape with an intercellular space in Calycanthus, ovoid shape in Chimonanthus but Sinocalycanthus was elongated with minor regular shape. Pith cells were also found in hexagonal and circular shape which was the different character distinguished within genera. The pollen grain is elliptic with single furrow, and ornamentation smooth. The basic shape of the pollen grains is elliptical. Observation in equatorial views showed spheroid shape in Calycanthaceae except for Idiospermum, which represent boat-shaped. The polar view is circular in Chimonanthus but equatorial view is elliptic in Sinocalycanthus. Exine surface of Idiospermum and Chimonanthus is smooth with tectate-perforate. Rugose exine is distinguished characters for Sinocalycanthus and Chimonanthus. All pollen was heteropolar. Morphological and anatomical information from all aspects were considered for the cladistic analysis. Siparuna (Siparunaceae) and Cinnamomum (Lauraceae) were used for outgroup. In conclusion, Calycanthaceae should be redefined wide circumscriptions of the morphological characters. Detailed investigation of the cladistic analysis revealed that the Sinocalycanthus and Calycanthus should be placed in sole genus each other. In addition, Idiospermum should be included in Calycanthaceae. These morphological characters display great features of taxonomic value in Calycanthaceae. The accurate classification of four genera of Calycanthaceae was not completely resolved. They are closely related in vegetative characters and growth habits each other. Several contradicting taxonomic issues have been remained. However, there is still unable establishing a natural classification of the family and especially in proposing a relevant treatment of relationship within the genera. Therefore, the comparative morphological and anatomical study of leaf, fruits and seed, stem, and pollen was carried out. Cladistic analysis was also conduct based on morphological and anatomical characters. Out of the four genera, the vascular bundle of Sinocalycanthus and Calycanthus was the V-shaped, whereas Idiospermum and Chimonanthus was the U-shaped. The distinctive leaf morphological features were presence of trichome, stomata and variation of the density of trichome, stomata density, and leaf surface. In this study, the density of trichome was observed higher in Calycanthus than other genera. The abaxial surface of Idiospermum and Sinocalycanthus was smooth but Chimonanthus and Calycanthus was rough. Presence of crystal was notified in Sinocalycanthus, Calycanthus and Chimonanthus but absent in Idiospermum. The interesting structure of cuticle was observed in Idiospermum. The unicellular trichome is common in all genera. The vascular bundle contains four cortical vascular bundles in the stem. Calycanthus possess bordered-pit with circular aperture while Chimonanthus possess elliptic aperture. Tracheid is a characteristic feature of the wider spiral band in Chimonanthus than that of Calycanthus and Sinocalycanthus. Sclerenchyma cells were aggregated in Sinocalycanthus and Calycanthus but in case of Chimonanthus, it was the long chain with the layer of the sclerenchyma cell. Collenchyma cells were circular shape with an intercellular space in Calycanthus, ovoid shape in Chimonanthus but Sinocalycanthus was elongated with minor regular shape. Pith cells were also found in hexagonal and circular shape which was the different character distinguished within genera. The pollen grain is elliptic with single furrow, and ornamentation smooth. The basic shape of the pollen grains is elliptical. Observation in equatorial views showed spheroid shape in Calycanthaceae except for Idiospermum, which represent boat-shaped. The polar view is circular in Chimonanthus but equatorial view is elliptic in Sinocalycanthus. Exine surface of Idiospermum and Chimonanthus is smooth with tectate-perforate. Rugose exine is distinguished characters for Sinocalycanthus and Chimonanthus. All pollen was heteropolar. Morphological and anatomical information from all aspects were considered for the cladistic analysis. Siparuna (Siparunaceae) and Cinnamomum (Lauraceae) were used for outgroup. In conclusion, Calycanthaceae should be redefined wide circumscriptions of the morphological characters. Detailed investigation of the cladistic analysis revealed that the Sinocalycanthus and Calycanthus should be placed in sole genus each other. In addition, Idiospermum should be included in Calycanthaceae. These morphological characters display great features of taxonomic value in Calycanthaceae.

Entry Mechanism and the Development of Vaccine Candidate Harboring Chicken Fc Molecule for Avian Metapneumovirus (aMPV)

Paudel, Sarita 충남대학교 대학원 2015 국내박사

Avian metapneumovirus (aMPV) is a member of the subfamily Pneumovirinae under family Paramyxoviridae, and is the major pathogen of turkeys and chickens. aMPV has a single-stranded, non-segmented, negative-sense RNA genome. Only one serotype of aMPV has been identified so far. Four different subtypes of aMPV have been differentiated using monoclonal antibodies by nucleotide sequence analysis on the basis of the attachment (G) protein and neutralization test. Subtype A and B viruses are mainly prevalent in Europe, Asia and the Middle East, subtype C is most prevalent in the United States and it has been subsequently isolated from farmed ducks in France, pheasants in South Korea and chicken in China. An additional subtype D has been first reported in France. aMPV causes turkey rhinotracheitis (TRT), an acute respiratory tract infection in turkeys of all ages. The virus is also associated with swollen head syndrome (SHS) in broilers and broiler breeders (and egg production losses in layers. Replication of paramyxoviruses begins with entry into the cell and then proceeds in the cytoplasm where virus progeny are formed and later released from the plasma membrane of infected cells by budding. Generally the two viral glycoproteins promote most of the direct fusion events, whereby the attachment protein facilitates primary receptor binding of the virus to the target cell and the F protein promotes the subsequent membrane fusion events. The cleavage of the F protein is an essential step for pathogenicity of the virus. To be fusogenically active, paramyxovirus F proteins must be proteolytically cleaved from the precursor F0 form to a disulfide-linked F1+F2 heterodimer. So, we tried to investigate the role of trypsin in the aMPV replication and entry mechanism of avian metapneumovirus. To evaluate the role of trypsin in aMPV, trypsin was added in different stages of infection: before aMPV entry, during the entry stage and at the budding stage. To investigate the effects of surface proteins on Vero cells and/or free virions, Vero cells and aMPV were pretreated with trypsin prior to infection. Trypsin treatment on either cells or viruses prior to virus infection did not enhance infection. However, trypsin treatment at the later stage of virus enhanced virus replication. Our results showed that trypsin is not required for the aMPV entry in Vero cells. But, the role of trypsin is relatively more important in the later stage of infection, so it might be important when aMPV infects neighboring cells. Furthermore we found that various protease inhibitors, including E-64, pepstatin A, phosphoramidon has no effect in the virus replication but only AEBSF-HCl; the serine protease inhibitors inhibited aMPV replication. As we found trypsin is not required for aMPV infection. So, we hypothesized that aMPV may enter Vero cells by either of the pathways; direct membrane fusion on the cell surface or endocytosis. After treating the cells with various inhibitors of clathrin-and caveolae-mediated endocytosis, aMPV infection was similar in both treated and non-treated cells and similar to the control cells. The lysomotropic agents ammonium chloride and bafilomycin-A1, also did not inhibit the replication of aMPV or the production of aMPV progeny. Even when the concentration of both inhibitors was doubled, there was no difference in the virus replication. These results clearly showed that aMPV does not require the low-pH environment found in endosomes, and further supports our hypothesis that a non-endocytic pathway is involved in its entry. To reduce the economical loss in the poultry, different vaccines are in use. Mainly attenuated live and inactivated vaccines are used to protect against aMPV type A and B field infections in Europe. Also, a cold-adapted strain of aMPV C resulted in good protection against virulent aMPV C infection in turkeys. In addition it was reported that live aMPV vaccines are known to revert to virulence and became persistent in the field. In spite of these vaccines, still there is a need of the development of efficient vaccine. We tried to construct genetically modified cells that express chicken immunoglobulin G (IgG) Fc on the cell surface, as a fusion protein, in a reverse orientation mimicking natural IgG opsonization. Propagation of aMPV virus in Vero cells expressing chicken Fc on the surface results in the transfer of the Fc portion to virus progeny during budding process. These Fc bearing virus particles will form a highly efficacious strain enhancing its immunogenicity. First the plasmid vector expressing the Fc chimera, pcFc expressing Fc domain of IgG1 was constructed. Vero cells expressing Fc chimera on its surface was established and we found that aMPV grown in Vero-cFc incorporated host derived chimera Fc, pcFc, into the virions. Immunization of chicken with killed aMPV harboring Fc, which had been propagated in the cells expressing Fc on its surface, induced higher IgG and IgA and inflammatory cytokines IFN-Ƴ and IL-1β compared to that of killed aMPV and control and is statistically significant. The aMPV Fc group also presented the highest neutralizing antibody response and is also statistically significant to that of aMPV. The greater ability of aMPV harboring Fc to that of killed aMPV presented it as a possible candidate for vaccine.