Dissertations / Theses on the topic 'Trimeresurus'

Les venins de serpent des familles de viperidae et crotalidae sont riches en protéinases à sérine de type chymotrypsine agissant sur l'hémostase. Dans le but d'améliorer les agents thrombolytiques actuels, nous avons réalisé l'étude du TSV-PA (trimeresurus stejnegeri venom plasminogen activator) qui active le plasminogène en plasmine au meme site de clivage que les activateurs du plasminog7ne humains (t-PA et u-PA). Dans un premier temps, nous avons détermine le rôle des positions 192 et 193 dans la spécificité du TSV-PA. Nos travaux montrent que le résidu 192 ne semble pas important dans la reconnaissance de substrat ou d'inhibiteur du TSV-PA. Par contre, le mutant f193g active le plasminog7ne avec une efficacité catalytique 10 fois supérieure a celle du TSV-PA. De plus, ce mutant est inhibe par les inhibiteurs de type Kunitz (BPTI) et de la famille des serpins (PAI-1, 1-antitrypsine et 2-antiplasmine) quand le TSV-PA ne l'est pas, suggérant un rôle central du résidu en position 193 pour la spécificité du TSV-PA. Dans une seconde partie, nous nous sommes intéressés a la résistance du TSV-PA aux serpins. Le PAI-1 interagit avec le t-PA par l'intermédiaire de la région 36 chargée positivement. L'absence de cette boucle sur le TSV-PA lui assure en partie la résistance au PAI-1 puisque l'insertion des residus charges donne lieu a un mutant inhibe par le PAI-1. De plus, le double mutant comportant cette insertion et la mutation f193g est inhibe aussi bien par le PAI-1 que l'est le t-PA. A l'heure actuelle, nos résultats ne permettent pas d'inscrire le TSV-PA dans une nouvelle génération d'agents thrombolytiques, mais confirment son utilité dans l'amélioration des agents thérapeutiques existants. En effet, la construction d'un mutant du t-PA comportant la mutation g193f ainsi que la neutralisation des charges positives de la vr1, pourrait aboutir a l'obtention d'un mutant du t-PA résistant aux serpins et ayant de ce fait une demi-vie plus enlevée in vivo.

El accidente ofídico u ofidismo, es considerado una enfermedad tropical desatendida que afecta a las poblaciones rurales de los continentes de Asia, África y América. El único tratamiento científicamente válido para combatir el ofidismo es la administración de un antiveneno, el cual es una mezcla de anticuerpos producidos mediante la inmunización de animales con venenos de serpientes relevantes en la región y este posee la capacidad de neutralizar la actividad tóxica de los venenos con alta eficacia; sin embargo, existe poca disponibilidad de antivenenos en múltiples regiones del mundo, lo cual afecta principalmente a las poblaciones más vulnerables de Asia y África. Frente a esto, diversos estudios reportan que los antivenenos poseen la capacidad de reconocer componentes proteicos de venenos de especies que no han sido utilizadas en su producción, fenómeno conocido como reactividad cruzada y este proceso puede ser aprovechado para extender el alcance de los venenos disponibles, a regiones carentes de antivenenos. El Instituto Nacional de Salud (INS) produce tres antivenenos comerciales los cuales son utilizados para los géneros de serpientes más importantes del Perú. En el presente informe se presenta la utilización del antiveneno botrópico polivalente para la realización de ensayos de reactividad cruzada de los venenos de las serpientes foráneas Crotalus atrox y Trimeresurus puniceus mediante el diseño y aplicación de una metodología de cromatografía de afinidad, con el fin de crear una matriz de inmunoafinidad para estudiar la interacción entre los venenos y sus respectivos antivenenos.
Perú. Consejo Nacional de Ciencia y Tecnología. Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación (FONDECYT). Contrato N° 101-2018-FONDECYT-BM-IADT-AV

碩士
國立臺灣師範大學
生物研究所
90
There are three species of Trimeresurus snakes in Taiwan. T. gracilis is the endemic species and it can be found only at an altitude above 2000 meters in Taiwan. Whereas, the other species, T. mucrosquamatus and T. s. stejnegeri inhabit under 2000 meters. In Taiwan, the species that restrict at highland are sometimes the glacial relics of the Paleartic zoogeographical region. Reptiles are ectotherms and temperature may directly affect their physiology and behaviors. Therefore, temperature may become one of the major factors that set the distribution limit of Trimeresurus snakes along different altitude in Taiwan. I predict the critical thermal maximum (CTMax) of T. gracilis is lower than that of T. mucrosquamatus and T. s. stejnegeri. On the other hand, the critical thermal minimum (CTMin) of the latters should higher than that of the former. Critical temperatures are defined as the thermal points at which the locomotory activity of an animal becomes disorganized and it loses its ability to escape from conditions that will promptly lead to its death. From the ecological viewpoint, critical temperatures are the lethal temperatures. Before testing the CTMax and CTMin, all snakes are individually acclimated at 10℃, 20℃ and 30℃ for more than two weeks. The criterion of CTMax is panting, and that of CTMin is losing the righting responses. When acclimation temperature is higher, both CTMax and CTMin in all three species become higher. The CTMax of T. gracilis is lower than that of T. mucrosquamatus and T. s. stejnegeri in winter, but not in summer. The CTMin of T. s. stejnegeri is higher than that of T. gracilis in both winter and summer. However, the CTMin of T. mucrosquamatus is not always higher than that of T. gracilis. Consequently, my prediction is only partially supported. The CTMax (36.6℃) of T. gracilis when acclimated at 10℃ is higher than the highest mean temperature （35.2℃） in lowland nearby Chiai City. On the other hand, the CTMin acclimated at 10℃ of all three species are lower than the temperature (6.1℃) that under ground 1 meter nearby Tatachia, Nantou County. So, I conclude that thermal tolerance is not the main factor that set the distribution limit of Trimeresurus in Taiwan.

碩士
國立東華大學
自然資源與環境學系
105
Predators might switch foraging pattern due to prey availability. Trimeresurus stejnegeri switches frog species as a food resource between adult and subadult snakes. We surveyed snake ambush sites and presence of frogs. We suspected snakes to switch frog species as food resources during different seasons. Climate, and snake body mass also might affect a snake’s activity. Trimeresurus stejnegeri is a common sit and wait predator. In Taiwan, it mainly eats frogs. From February 2014 to March 2016 at the Chongguang Community, Xiulin Township, Hualien County, Taiwan. We recorded encounters with each snake and frog, recording snake age (adult and subadult) and ambush habitat: lotic, lentic, and temporary water. Snakes were sprayed and photographed for tracking. Trimeresurus stejnegeri was most active in spring and fall. Adults were also active throughout the winter, possibly gaining an advantage over competing snakes or escape from predation. Most adults used lentic surroundings as ambush site. Subadults used all three habitats. Subadults switched ambush sites based on numbers of Buergeria japonica, Buergeria robusta, and Bufo bankorensi. Adult switched sites based on numbers of Buergeria. japonica, Buergeria. robusta, Rhacophorus moltrechti and Bufo. bankorensi. Competition or predation relation between adult and subadult might exist based on different habitat utilization. Differences in habitat used by adult and subadult snakes may be due to diet seasonal changes. Adults ambush sites had abundant, large- sized prey. Foraging pattern varied with life stages of T. stejnegeri. Understanding shift of ambush habitat in different life stages of T. stejnegeri is central information for viper’s habitat conservation.

博士
國立臺灣師範大學
生命科學研究所
94
Abstract Reproduction, growth and habitat selection of an organism can make a strong impact on its long-term fitness. Variation in fitness among individuals plays a primary force in natural selection. The Chinese green tree viper (Trimeresurus s. stejnegeri), a nocturnal, arboreal sit-and-wait predator, is one of the most common snakes in Taiwan. This serpent exhibits an obviously female-biased sexual-size dimorphism (SSD). It often retreats into vegetation during daytime and moves to ambush sites at dusk. Furthermore, a pattern of male-biased sex ratio was found in a population of Trimeresurus s. stejnegeri at Tsaochiao in the northern Taiwan. In this dissertation, I used demographic analysis from an over nine-year (1996-2005) mark-recapture data set of the population of Trimeresurus s. stejnegeri at Tasochiao, to explore the causes of observed male-biased sex ratio and female-biased SSD. Additionally, I applied an experimental approach to investigate the role of temperature, prey availability and vegetation density with respect to diurnal retreat site selection of the Chinese green tree vipers in outdoor enclosures. In Chapter I, the sex ratio and survivorship of the Chinese green tree viper Trimeresurus s. stejnegeri in Tsaochiao, Taiwan were investigated monthly by mark-recapture method from 1996 to 2005. A total of 577 individuals were captured and 2095 observations were made. They showed significant male-biased sex ratios with an average of 2.51 (male/females) for the half-year reproductive and postpartum periods. The male-biased sex ratio was not observed at the immature (newborn + juvenile) stage but at the adult stage. The average estimates of half-year survival rates and catch probability were 0.72 and 0.65, respectively. The survival rates were lower at the immature stage than adult stage, but they were not significantly different between immature males (mean survival rate, 0.62) and immature females (0.63). In contrast, the survival rate of adult females (0.65) was lower than that of adult males (0.75), but there was no significant difference in average capture probabilities between the two sexes (0.65 for males and 0.61 for female). Costs of reproduction in females, such as physical burden during gestation and emaciated condition after parturition are suggested as the primary causes for lower survivorship of adult females than adult males. In Chapter II, using the data of 577 individuals collected from the nine-year mark-recapture study, sexual differences in growth and body sizes were examined for the different life stages of the Chinese green tree viper Trimeresurus s. stejnegeri in Tsaiochio. The results showed that there were no significant sexual differences in SVL and body weight at the newborn stage but the differences were significant at the juvenile stage, young adult stage, and old adult stage. These indicated that the SSD of the Chinese green tree viper occurs at the post-newborn life stages after the first year of life. Growth rates were significantly faster for females (0.28 mm/day) than males (0.19 mm/day) at the immature stage but not at the adult stage. The Chinese green tree viper became reproductive maturity at the ages of 2.6 years for males and 1.9 years for females. Male-biased age distributions were observed for the old adult snakes; the average age of adult males was 5.2 years whereas females was 4.0 years, indicating that the females had lower survivorship and shorter life span than the males at the adult stage. The proximate mechanisms for SSD of the Chinese green tree viper are that females grow faster and larger than males at the immature stage. By doing so, the females reach sexual maturity faster and increase their reproductive fecundity. This may be evolved as a surviving strategy to compensate the lower survivorship and shorter life span at the adult stage of female Chinese green tree vipers. In Chapter III, I manipulated vegetation structure, distance to prey source, and monitoring the microhabitat temperature within the test enclosures. The results indicated that the height of daytime perch sites was influenced by ambient temperature. Snakes perched on lower layers of vegetation seeking cooler conditions when ambient enclosure temperature was high. In addition, when the ambient temperature rose above 25C, Trimeresurus s. stejnegeri retreated into denser vegetation, which provided significantly lower temperatures. In contrast, Trimeresurus s. stejnegeri in low temperature environments did not exhibit any preference for vegetation structures in terms of density, except for an apparent avoidance of vegetation with bare branches and no leaves. The distance to prey source did not appear to have any significant influence on the green tree viper’s selection of retreat site, this could be attributed to the fact that these snakes do not only hunt terrestrial preys, but also prey upon arboreal species. This study demonstrated that temperature was the most important factor influencing retreat site selection of Trimeresurus s. stejnegeri. The temporal variations and seasonal differences in the space utilization patterns may have been for the purpose of satisfying their thermoregulation needs.

碩士
國立臺灣師範大學
生命科學研究所
96
There are three species of Trimeresurus snakes in Taiwan. Taiwan mountain pit-viper (T. gracills) is an endemic species, and can be found only at an altitude over 2000 meters. The other species, Chinese green tree viper (T. stejnegeri stejnegeri) and Pointed-scaled pit-viper (T. mucrosquamatus) inhabit under 2000 meters in Taiwan. The reason why these snakes restrict at different altitudes is the question that we want to answer. We hypothesized Taiwan mountain pit-viper has lower optimal temperature than Chinese green tree viper and Pointed-scaled pit-viper. We tested strike maximum speed, reaction time, strike related behaviors, food taking rate and strike successful rate in 7 different temperatures; 7℃, 12℃, 17℃, 22℃, 27℃, 32℃, 36℃. The result did not support our hypothesis, Taiwan mountain pit-viper did not have a lower optimal temperature than the other two snakes. Nevertheless, in low temperatures, Taiwan mountain pit-viper had the best performance in strike maximum speed, reaction time, strike related behaviors and food taking rate in three species. Pointed-scaled pit-viper had the worst performance. In high temperatures, Taiwan mountain pit-viper still showed good performance and Pointed-scaled pit-viper had the worst performance again. Therefore, the results revealed a better adaptation of adult Taiwan mountain pit-viper than the other two species at high altitude mountains. But it could not tell why Taiwan mountain pit-viper does not distribute at lower altitude areas.

碩士
國防醫學院
解剖學研究所
83
Trimeresurus mucrosquamatus, a common venomous snake in Taiwan, belongs to the family Viperidae. In previous study we have obtained a partial cDNA clone by immunoscreening from venom gland cDNA library of T. mucrosquamatus. Partial cDNA sequence analysis indicate a strong homology with that of snake venom protein batroxobin. Batroxobin is a thrombin-like enzyme isolated from Bothrops atrox which are also member of the family Viperidae. Batroxobin has a defibrinogenerating effect and is clinically used for the treatment of thrombotic diseases. In this study, a full-length cDNA isolated from T. mucrosquamatus and designated as trimubin. The cDNA sequence and the structure and functional relationships of trimubin cDNA is characterized. The 5''-rapid amplification of cDNA end (RACE) are used in the isolation and construction of the full-length cDNA. Both RACE amplified cDNA and cloned partial cDNA detects a message with a similar size of 1.5 kb. Northern blot analysis by this result suggests that the RACE amplifi ed cDNA and cloned partial cDNA is in the same gene but on the opposite end. The full-length trimubin cDNA consists of 1576 bp with a 5''-noncoding region of 188 bp and a 3''-noncoding segment of 614 bp. Based on the sequence homology comparison and Kozak sequence analysis, the protein translation begins at 189 base and stops at 962 base. The encoding region in between is 771 bases and could code for 257 amino acids with a calculated molecular weight of 28.1 kDa. A typical poly (A) signal, AATAAA is located 15 bases in the upstream of the poly (A) tail. Amino acid sequence comparison shows strong homology with venombin A, ancrod and batroxobin. trimubin is a serine proteinase.

碩士
國防醫學院
解剖學研究所
82
Trimeresurus mucrosquamatus, one of the commonest land venomous snake in Taiwan, belongs to the Subfamily Crotalinae. The venom contains many valuable components including the thrombolytic enzymes which have therapeutic potential in stroke and human heart disease, and neurotransmitters used as tools in neurobiological research. However,it is hard to get quantitatively protein material for investigate.In this study, we constract a λ gt11 cDNA library from Trimeresurus mucrosquamatus venom glands and analyzing the cloned cDNAs.The optimal time forRNA expression in the venom gland were analyzed by histological observation using hematoxylin and eosin, and wheat germ agglutinin stained paraffin sections. Morphological evidence indicate that maximal RNA expression was 24 hours after venom milking were presumably in correlated with maximum RNA synthesis. Poly (A) RNAs purified at this period were used to construct a cDNA library. The phage titer of the cDNA library was 1.0 x 108 pfu/ml, and the recombinant efficiency was higher than 80%. Polyclonal antiserum against the whole venom were used to screen the cDNA library and identified two positive clones (TMV1 and TMV2). DNA sequence of TMV1b shared 90% homology with batroxobin, a thrombin-like snake venom protein and TMV2 exhibited 92% similarity with trigramin, a platelet aggregation inhibitor. Northern blot analysis revealed a tissue specific expression of the TMV1. The 1.4 Kb TMV1 mRNA was identified only in venom gland among 6 organs of the snake investigated. These results indicated that a high quality of constructed.

碩士
國立臺灣大學
藥理學研究所
90
By using ion-exchange column chromatography of CM-Sephadex C-50 and gel filtration chromatography of Superdex G-75 column, we purified the venom protein triflamp from Trimeresurus flavoviridis venom with a potent inhibitory activity on ristocetin-induced platelet aggregation. Judging from the SDS-PAGE profile, triflamp was a homogeneous monomer with an apparent molecular weight of 26 kDa under non-reducing and reducing conditions. In human platelet suspension, triflamp concentration-dependently inhibited ristocetin (1 mg/ml)-induced platelet aggregation with an IC50 of 2 mg/ml (0.08 μM), while it showed little effect on collagen (10 mg/ml) and thrombin (0.1 U/ml)-induced platelet aggregation even at the concentration of 10 mg/ml. Nevertheless, triflamp prolonged the latent period in triggering platelet aggregation and inhibited platelet aggregation caused by low concentration of thrombin (0.03 U/ml). Triflamp inhibited ristocetin (1 mg/ml)- or thrombin (0.03 U/ml)-induced platelet TXB2 formation in a dose-dependent manner. In human platelet-rich plasma (PRP), triflamp exhibited an inhibitory effect on ADP (20 mM)-induced platelet aggregation in a time-dependent manner, which may result from the plasma fibrinogen degradation by triflamp. It was demonstrated that triflamp-pretreated fibrinogen lost activity in triggering aggregation of elastase-treated platelets, in which the functional GPIIb/IIIa was exposed. However, further addition of intact fibrinogen reversed the impaired capacity of triflamp-treated fibrinogen, implying that triflamp has little cleaving activity on GPIIb/IIIa. Additionally, triflamp (5 mg/ml) degraded Aa-chain of fibrinogen in a time-dependent manner whereas the Bb- and g-chains of fibrinogen was apparently unaffected. Pretreatment of triflamp with EDTA (10 mM) or 1,10-o-phenanthroline (5 mM) abolished its fibrinogenolytic activity. In flow cytometric analysis, triflamp (10 μg/ml) treatment significantly decreased the binding of FITC-conjugated anti-CD42b mAb to platelets in a dose-dependent manner without affecting the binding of FITC-conjugated anti-CD42a, anti-CD41, anti-CD62P or 6F1 mAb, which was raised against GPIX, GPIIb/IIIa, P-selectin or GPIa/IIa, respectively. This result suggests that triflamp exerts a specific blocking action on GPIb expressed on human platelets. By Western blot analysis, the cleavage of GPIba on platelets by triflamp was demonstrated to be time- and dose-dependent and a cleavage fragment with a molecular mass of ~ 58 kDa appeared. This cleaving effect of triflamp was also bivalent metal cation-dependent. In conclusion, triflamp was a P-I class metalloproteinase which exerts a blocking action on GPIb-related activation pathway by its enzymatic activity. It inhibited ristocetin- or low concentration thrombin-induced platelet aggregation.

碩士
國防醫學院
生物及解剖學研究所
89
英文摘要 Phospholipase A2 (PLA2 EC 3.1.1.4) is the most abundant enzyme in almost all snake venom. In addition to their abundance, over one hundred kinds of isoforms are identified. Despite they share high homology in their amino acid sequences, ranging about 40% to 90%, PLA2s in snake venom exhibit a wide variety of pharmacological effects by interfering in normal physiological process. Because of their similarity, purification of a PLA2 without isoforms becomes a hard work. This may be the reason why that distinct functional difference in the ability to induce various pharmacological effects among snake venom cannot be easily correlated with their structural differences. In the last two decades, attempts to reach the structure-function relationships of PLA2 and their mechanism by monoclonal antibody and chemical modification but still remains uncertainly. In 1992, the first snake venom PLA2 is cloned and expressed in E. coli, and successfully refolded into an active PLA2 in vitro. This provides a new tool to testify the structure-function relationships of PLA2. In this work, a PLA2 from Taiwan habu was subjected to refolding into a structure with enzymatic activity successfully.

博士
國立臺灣師範大學
生命科學研究所
93
The Chinese green tree vipers (Trimeresurus s. stejnegeri) are nocturnal and ambush prey in arboreal habitats. They feed mainly on amphibians in field, but rodents, shrews, lizards, geckos, and birds were also consumed. Several topics about this species have been studied, but no studies have focused on the behavior and physiology of feeding. In this dissertation, all subjects were associated with the feeding of this snake, and the relations of these subjects to arboreality, nocturnality, and ambush-feeding habitat were concerned. In Chapter 1, I found Trimeresurus s. stejnegeri held on the prey after capturing it, which should be adapted to the less aggressive prey (i.e., frog), lower prey size, and avoidance the inconvenience for tracing the released prey from a twig. I fed the snake with frog (Rana limnocharis) and mouse (Mus musculus) in the laboratory (22 ± 1 oC), under three sensory deprivation conditions (intact cues, visual cue blocked, visual and infrared thermal cues blocked). The feeding behavior was recorded by videotape-recording method or naked eyes. The snake withdrawal and pulled up the prey from the ground following catching it. The prey was hanged and the body tilted, like a lever with the snake-biting site as the fulcrum. Mostly, the snake gradually moved its jaw to the upper end of the prey body and ingested it. That is, the capturing site, which should be decided before attacking the prey, determined the ingestion direction The snake ingested mouse mainly from head, as in other snakes; but the pattern was not dominant in feeding frog. More proportion of frogs was stroked on the posterior end than that of mice. The ratio of prey ingested from anterior to posterior side was 55 : 19 and 29 : 22 for mouse and frog, respectively, under intact sensory condition. When the visual and infrared cues were blocked, the ingested direction ratio, 14 : 4 and 14 : 9 for mouse and frog respectively, did not shift significantly from above. The snakes spent more time feeding on frogs than on mice. On feeding mice, the spending time to launch successful strikes (T1) was lowest at intact cues groups and significantly increased when the visual and/or thermal cues were blocked. On feeding frogs, however, T1 did not differ significantly among the sensory deprivation conditions. Visual and thermosensory cues seem to be less important on feeding frogs (but not on feeding mice) for T. s. stejnegeri. Besides, on feeding frogs but not mice, the time from capturing prey to start moving the jaws (T2) was significantly longer when ingesting prey from the anterior end. On feeding mice but not frogs, the time from moving the jaws to start flicking the tongue following the ingestion (T3) was longer when feeding prey from the posterior end. The feeding time (T2, T3) decreased when the sensory cues were blocked. In Chapter 2 and 3, I measured the temperature selection of adult males and females in a linear thigmothermal gradient and checked the degree of instrumental interferences. I conducted three experiments to study the possible effect of thermocouples, the influence of seclusion, and the presence of water on the temperature-selecting behavior of the snake. Thermocouples might change a snake’s preferred temperature (Tp) by causing it to lift its prehensile tail from the gradient floor or affecting its movement. With the videotape-recording method, the snake presented postprandial thermopily only when seclusion sites and water were provided in the gradient. In the absence of seclusion sites and water, the fasting and postprandial body temperature (Tb) of males was 23.0 ± 1.2 oC and 24.7 ± 1.2 oC, respectively. With seclusion sites and water, the fasting and postprandial Tb of males was 22.5 ± 1.0 oC (the set point Tset = 20.3 ~ 24.3 oC) and 27.8 ± 0.6 oC (Tset = 26.5 ~ 28.8 oC), respectively. The fasting and postprandial Tb of non-reproductive females (N = 16) was 21.2 ± 1.4 oC (Tset = 20.6 ~ 23.8 oC) and 24.8 ± 1.5 oC (Tset = 25.0 ~ 26.3 oC), respectively. Preferred temperature of females was higher after feeding or during pregnancy. Preferred temperature of pregnant snakes (N = 5) was 27.4 ± 2.0 oC. In Chapter 4, I investigated the combined effect of meal size (below 30%) and temperature (15~35 oC) on the aerobic metabolism, digestive efficiency, and digestive rate in this study. As other sit-and-wait foraging species, the snake had lower resting metabolic rate (0.033 ± 0.002 ml O2/g/hr at 25 oC). But it showed less difference at 20 and 25 oC (Q10 = 1.58). Respiratory quotient significantly increased at the anterior part of digestion period except at 15 oC. Specific dynamic action (SDA), peak VO2 and scope of peak VO2 increased with meal size, while temperature had little effect on SDA and SDA coefficient. Similar with other crotalids, the SDA coefficient was lower (15.8 ± 0.6%) in this study. With regression analysis, I found SDA in T. s. stejnegeri responded latterly and less sharply than other sit-and-wait feeding snakes. The metabolizable energy coefficient (= 0.66~0.89) was lowest at 15 oC and tended to peak at the postprandial Tset of the snake. In addition, I investigated the digestive rate from three aspects, included gut passage time (gut movement), gastric digesting time (Tbone; chemical digestion), and the timing of SDA (digestive metabolism). The final defecation time (PTe) and Tbone, but not the first defecation time, increased with food ration. PTe was less than two weeks at above 25 oC, but was larger than one month at 15 oC. The time to peak VO2, Tbone, and duration of SDA represented about 20%, 50%, and 80% of total digestion process (PTe), respectively. In Chapter 5, I tested whether temperature selection makes maximal net energy gain in Chinese green tree viper. I used multiple linear regressions, which expressing the effects of snake mass, mouse mass as well as temperature on digestion-associated variables, to simulate the monthly maximal net energy gain (Enet) under certain feeding frequencies and activity levels. With the energy budget model, I found some dominant trends. Enet peaked at lower temperature when snakes had less food ration. At high feeding frequency, selecting high body temperature, which closes to the postprandial Tp of the snake, could get higher Enet. When feeding frequencies lower down, Enet could be negative at 10% meal size, and the superiority or necessity to select high postprandial temperature disappear because of broad range of B80 at 20 ~ 30% meal size. Owing to the potential low feeding frequency in wild, T. s. stejnegeri may not get relative maximal Enet by postprandial behaviors. I have suggested potential reasons about why the snake selects high postprandial temperatures still under less energetic benefits. In conclusion, many innovated ideas and investigations have been mentioned in this dissertation. Several characters (nocturnality, arboreal habitats and ambush-feeding behavior) of Chinese green tree vipers may have important influences on the results. Whether these attributes occurred in wild as well as in other arboreal and/or ambush-feeding snakes is worth to study in future researches.

碩士
國防醫學院
生物及解剖學研究所
92
The cDNA encoding cysteine-rich venom protein (CRVP) was isolated from a cDNA library of venom gland of Trimeresurus mucrosquamatus. CRVP was belonged to the family of cysteine-rich secretory proteins, CRISPs. The CRISPs have sixteen consensus cysteines in their C terminus which can be bridged to form eight disulfide bonds. There were several snake venom proteins belonging to the CRISPs been isolated, such as ablomin, pseudechetoxin, tigrin and so on. CRISPs displayed a specific commom activity in the inhibition of ion channels. When recombinant CRVP was given to mice, a Ca2+ channel blocker resemblance effects of lethargy, paralysis, hypothermia, and death were observed. In addition, we also found that CRVP decreased the blood pressure of the rats without disturbing their heart beats. Therefore, CRVP may be ideally for the development of hypotensive drugs. In this study, CRVP could led into hypothermia and htpotension following intravenous administration in vivo. However, there were no similar effects in vivo in intracereventricular administration. In the search of possible biological mechanisms, we have found that CRVP could degrade kininogen to produce bradykinin in vitro. It is similar to some snake venom serine protease that produces it’s hypotensive effect by kallikrain-like protease activities. In this study, we successfully produced a functional CRVP from a yeast expression system and found that the hypotensive effect of CRVP might be mediated through kininogenase activity.

碩士
國防醫學院
生物及解剖學研究所
87
Mucrosobin was cloned and sequenced from the venom gland cDNA library of Trimeresurus mucrosquamatus (Taiwan habu), one of the common land venomous snakes in Taiwan and belonging to the subfamily of Crotalinae. Sequence comparison of the deduced amino acid shows high sequence homology between mucrosobin and batroxobin suggested that it might have fibrinogenolytic activity. In order to investigate the biological function of mucrosobin, E. coli BL21 (DE3) was chosen as the expression system for mucrosobin protein. After in-frame expression and purification, the results of Western blot and protein microsequenc show that the recombinant protein with an apparent molecular weight of 28 kDa and with antigenic specificity for Taiwan habu snake venom. However, the recombinant protein in inclusion bodies are the main forms of expressed protein in E. coli with no biological activity. In order to produce functional proteins, the expressed proteins in inclusion bodies was transform into a soluble form and reestablish its biological function by refolding. First the inclusion bodies was solubilized in denature solution (pH 8.0, 8M urea, 1mM oxidize glutathion and 2mM reduced glutathion), then dialysis in refolding buffer I (pH 8.0, 20% glycerol, 0.75M NaCl, 1mM oxidize glutathion and 2mM reduced glutathion) for 24 hrs and dialysis again in refolding buffer II (pH 7.5, 1% glycerol, 5mM NaCl) for another 24 hrs. Biological activities was characterized by fibrinogenolytic activity analysis. In this study, a successful refolding condition was established for the functional expression of a well-characterized recombinant mucrosobin protein, with specific beta-fibrinogenolytic activity.

碩士
國防醫學院
解剖學研究所
84
Trimeresurus mucrosquamatus, a common venomous snake in Taiwan, belongs to the family of Viperidae. In previous study, we have cloned a partial cDNA by immunoscreening from venom gland cDNA library. Sequence analysis of the tmv2 cDNA indicates 56% homology with a rat epididymal glycoprotein( AEG). The highly conserved metal binding domain between tmv2 and AEG indicated that tmv2 cDNA belongs to a metalloprotein gene of the Trimeresurus mucrosquamatus. In this study, both immunoscreening and 5'-rapid amplification of cDNA ends (RACE) are used in the construction of a full- length cDNA. The full-length sequence of tmv2 cDNA consists of 1318 bp with a 5'- noncoding region of 228 bp and 3'-noncoding segment of 538 bp. Based on homologous sequence comparison and Kozak sequence analysis, the encoding region begins at 229 base and stops at 777 base and could code for a polypeptide of 183 amino acids with a calculated molecular weight of 20 kDa. Northern blot analysis of the cDNA probes suggests the tissue-specific localization in venomous gland. The encoding region of tmv2 cDNA was then subcloned into pET21a(+) plasmid for protein overexpression. Venom protein expressed in Escherichia coli is partially purified by gel filtration chromatography. Both SDS-PAGE and Western bloting analysis were used as tracer in the identification of the expressed venom proteins.

碩士
國立臺灣師範大學
生命科學研究所
93
Abstract I checked the stomach contents of Taiwan mountain pit-viper (Trimeresurus gracilis), an endemic species, and found only 28 (18.4 %, N = 152 ) snakes contained preys. Among these, 88.5 % preys were swallowed head-first. Prey-predator mass ratios ranged from 0.084 - 0.807, and prey mass was positively correlated with predator snout-vent length. When I combined the stomach contents and fecal remains, I identified 107 preys including 33 % mammals, 32 % reptiles, 7 % amphibians, 25 % arthropods (probably secondary prey) and 3 % unidentified preys. Mature snakes often ate mammals while immature snakes ate lizards and amphibians. The snout-vent length of individuals that consumed endothermic preys were significantly larger than those that consumed ectotherms. There was an ontogenetic dietary shift. Adult snakes preyed primary on mammals (41.4 %). Adult females preyed most frequently on rodents (45.4 %) whereas adult males preyed most frequently on insectivores (59.3 %). There was a dietary difference between sexes. I used cotton swab smeared with prey scents to determine whether the newborn T. gracilis can discriminate prey chemical cues. I also checked whether these snakes preferred some of these prey scents than others. A greater chemosensory response to the scent of skink than to that of slug indicated that T. gracilis was able to discriminate the prey chemical cues. Newborn snakes also showed a stronger response to Sphenomorphus taiwanensis scent than other prey scents. In rattlesnakes, visual cue plays an important role in eliciting a successful predatory strike. I designed a moving cotton swab method and compared it with stationary cotton swab method. I found moving cotton swab method was a better way to test the prey chemical preference especially for adult snakes. Then, I used the moving swab method to test whether neonates, sub-adult and adult snakes preferred different prey chemical cues. I presented chemical stimuli of seven preys that coexist with these snakes including mammals: Apodemus semotus and Soriculus fumidus, amphibians: Rana sauteri and Hynobius arisanesis, lizards: Takydormus hsuehshanesis, Sphenomorphus taiwanensis and Eumeces elegans. I found adult snakes preferred scents of amphibians and mammals than scents of lizards, and they liked the scent of Hynobius arisanesis mostly. Sub-adult preferred the scent of Sphenomorphus taiwanensis mostly and secondly the scent of Hynobius arisanesis. Neonates preferred the scent of Sphenomorphus taiwanensis mostly and secondly the scent of Eumeces elegans. There was an ontogenetic prey chemical preference shift in T. gracilis. The prey scent preference of neonates corresponded to their natural diets, but the preference of adults and sub-adults didn’t.

碩士
國防醫學院
生物及解剖學研究所
87
Trimeresurus mucrosquamatus (Taiwan habu), a common land venomous snake in Taiwan, belongs to the subfamily of Crotalinae. In previous study, we have cloned a metalloproteinase (META) cDNA, designated as mucroslysin, from venom gland cDNA library. Sequence analysis of this cDNA shows 67.6 % homologous with trigramin from Trimereserus gramineus. The full-length sequence of mucroslysin gene consists of 2139 bp with a 5-noncoding region of 95 bp and a 3-noncoding segment of 598 bp. Based on sequence comparison and Kozak sequence analysis, the encoding region begins at 96 base and stops at 1541 base and could code for a polypeptide of 481 amino acids with a caculated molecular weight of 54 kDa. In addition, sequence comparison of mucroslysin cDNA represents a strong homology with trimucin, atrolysin, trigramin and halystatin. This result reveals a possible biological function of mucroslysin protein with specifc activity in the digestion of type IV collagen and fibrinogen and in the prevention of platelet aggregation. In this investigation, the coding region of mucroslysin cDNA was subcloned into pET21a (+) plasmid for the expression of recombinant protein. Recombinant venom protein expressed in Escherichia coli (E. coli) was partially purified by His-bind affinity agarose and then characterized by tricine SDS-PAGE, Western blotting and protein microsequence analysis. This study established a successful refolding condition for the recombinant metalloproteinase with a specific activity on both Aα- and Bβ- fibrinogen. This is the first successful report of a recombinant protein larger than 30 kDa in size with reconstituted biological function.

碩士
國立屏東科技大學
野生動物保育研究所
92
Abstract We examined 1) ambush site characteristics, 2) orientations and posture of ambushing snakes and 3) the correlation between abiotic factors, preys and snakes to study the ambush site selection by the sit-and-wait pit-viper, Trimeresurus s. stejnegeri, at the stream area at night. This study was conducted from November 2001 to February 2004, at Boi-li Stream in Ping-tung County. In the 30 working nights, we observed 94 times of ambushing snakes (N=63). The proportion of female ambushing snakes observed is higher than that of male. Most of the ambush sites located at the stream section with high frog density. The shortest distance between two ambush sites was 0.5m. Almost all ambush sites located at the positions including at least one of the following three conditions：1) the distance between ambush sites and water edge (DW) < (less than) 1m, 2) the distance between ambush sites and stream bank (DS) < 1m or 3) the distance between ambush sites and the ground (DG) < 0.5m. About 51.7％（N=46） of the ambush sites located at the positions with DW < 2m, DS < 6m, and DG < 1.2m. The DS of females was longer than that of males. Almost all of the snakes ambushed either on stones or on vegetation. Only few ambush sites were on the ground. Females usually ambushed on the stones with the size: 6.5~25.5㎝, and had higher ratio using stones than males. About 72.3％（N=68）of the ambushing snakes oriented to water area. About 42.2％（N=38）of the snakes ambushed with head down toward the ground, but 53.3％（N=48）kept their heads horizontally. The forebody of ambushing snakes performed in a concertina shape, whereas the posture of the other part of body varied with different sites. When snakes ambushed on the ground, they raised their heads and kept the snout-vents horizontally. When snakes ambushed at high and flat places, their heads might hang down, put closely on the site surface or raise but keep the snout-vents horizontally. The ambushing snakes were found throughout the year during the survey period. The correlation between the number of frogs and the number of ambushing snakes sampled was not significant. The correlation between the number of ambushing snakes and abiotic factors was not significant. However, the water cover, current velocity, and water depth of the stream were negatively correlated with DW, respectively. In summary, the green tree vipers primarily searched for the stream section both with high density of preys and ambush sites available, and secondarily located at the places close by the water, above the grounds with certain height, or not far away from the stream bank. At last, the head orientation of the ambushing snakes was dependent on the movement and stay of the preys. Consequently, the results of these ambush selections would increase the probability of encountering with the preys. Particularly, in contrast to the males, the females had higher ratio for ambushing and showed a more strict selection of ambush site, indicating a higher energy requirement for females than for males.

碩士
中興大學
生命科學院碩士在職專班
95
Taiwan is located in the sub-tropical zone. There are six frequent venomous land snakes in Taiwan, the highest biting rate of which is Trimeresurus mucrosquamatus. The snake venom will result in three kinds of reaction: deadly, hemorrhage and protein digestion. Current antibody is made from horses to generate the antivenins, but the traditional process of making the antivenins takes 3 months or 5 months of snake venom injection. Some horses can not bear the snake venom and they will get thinner or even end in death. Thus, new snake venom is bound to be cultivated. Our experiment is aimed at making the monoclonal antibody against Trimeresurus mucrosquamatus from mice and comparing the differences, advantages and disadvantages. If the latter one is better than the former one, we can get another way to make the antibody and make the antivenins from cell culture to reduce or substitute the way of nurturing the immunity serum from animals, eliminating the pain of animals. In the experiment of serum neutralization titer, 1 ml of ninth antibody and tenth monoclonal antibody can only neutralize 28μg and 20μg of Trimeresurus mucrosquamatus venom. The serum against Trimeresurus stejnegeri and Trimeresurus mucrosquamatus of CDC (Center of Disease Control) can neutralize 134μg of venom. 1 ml of serum of No. 264 horses can neutralize 106μg of venom. In the hemorrhagic test, only ninth antibody can depress hemorrhage of the skin lightly. Every research result shows that uncondensed and unpurified monoclonal antibody can not have better effects against snake venom than the traditional venom from horses, but still have the neutralization effect. If a bigger cell culture system can be implemented to nurture more hybridoma cells, higher density monoclonal antibody can be produced to become another way of making the antivenins.

碩士
國防醫學院
生物及解剖學研究所
85
Trimeresurus mucrosquamatus, a common land venomous snake in Taiwan, belongs to the subfamily Crotalinae. In previous study, we have cloned a partial cDNA by immunoscreening from venom gland cDNA library. Sequence analysis of this partial cDNA shows 67.6 % homology with trigrarnin from Trimereserus gramineus. This analysis suggests that a metalloproteinase-like gene of the Trimereserus mucrosquamatus has been isolated, and designed as META cDNA. In this study, both immunoscreening and 5''-rapid amplification of cDNA ends (5''-RACE) are used in the construction of a full-length cDNA. The full-length sequence of META gene consists of 2139 bp with a 5''-noncoding region of 95 bp and a 3''-noncoding segment of 598 bp. Based on sequence homologous comparison and Kozak sequence analysis, the encoding region begins at 96 base and stops at 1541 base and could code for a polypeptide of 481 amino acids with a caculated molecular weight of 54 kDa. Northern blot analysis of META cDNA suggests a tissue specific transcription in venomous gland. In addition, sequence comparison of META cDNA represents a strong homology with trimucin, atrolysin, trigramin and halystatin. This result reveals a possible biological function of META protein in specifc digestion of type Ⅳ collagen and fibrinogen and in the prevention of platelet aggregation. The META protein might be the third metalloproteinase isoform presented in Trimeresurus mucrosquamatus. The coding region of META cDNA was then cloned into pET21a (+) plasmid for recombinant protein expression. Venom protein expressed in Escherichia coli (E. coli) is partially purified by Ni-NTA affinity agarose and then characterized by both SDS-PAGE and Western blotting analysis.

碩士
國立臺灣大學
藥理學研究所
100
Disintegrins are small moleculars and potent platelet inhibitors found in the snake venom. Disintegrins are GPIIb/IIIa antagonists and potential antithrombotic agents. By means of CM-Sephadex C-50, Sephadex G-75 gel filtration, FPLC Superdex 75 gel filtration and reverse phase HPLC, two disintegrins, TMV-2 and TMV-7, were purified from Trimeresurus mucrosquamatus snake venom. In this study, we investigated and compared the difference between TMV-2 and TMV-7. TMV-2 and TMV-7 had different isoelectric point (pI). The pI of TMV-2 was estimated to be around 4.5, whereas that of TMV-7 was estimated to be around 5-7. TMV-2 and TMV-7 were shown to be a single peptide chain. By MALDI-TOF, the molecular weigh of TMV-2 and TMV-7 were determined as 7663 and 7672 Da, respectively. The sequence of TMV-2 was 24% identical to batroxostatin, a disintegrin purified from the snake venom of Bothrops atrox, whereas the sequence of TMV-7 was 24% identical to batroxostatin and 79% identical to cotiarin, a disintegrin purified from the snake venom of Bothrops cotiara. Both TMV-2 and TMV-7 concentration-dependently inhibited platelet aggregation in human platelet-rich plasma, washed human platelet suspension and elastase-treated human platelets. Also, TMV-2 and TMV-7 did not interfere with the formation of thromboxane A2. However, both TMV-2 and TMV-7 enhanced the P-selectin expression induced by collagen and thrombin. In the indirect binding assay, TMV-2 significantly inhibited 7E3, a mAb raised against GPIIb/IIIa, binding to GPIIb/IIIa, but TMV-7 did not. Both TMV-2 and TMV-7 enhanced 10E5, a mAb raised against GPIIb/IIIa, binding to GPIIb/IIIa. In the animal models, TMV-2 and TMV-7 dose-dependently inhibited platelet aggregation in mice PRP. Furthemore, TMV-2 (0.25μg/g) prolonged the bleeding time more significantly than TMV-7 (0.25μg/g) as they were intravenously administered. However, both TMV-2 and TMV-7 did not alter the platelet counts. In conclusion, TMV-2 and TMV-7, two disintegrins with different isoelectric point, have the different characters. Although both TMV-2 and TMV-7 are GPIIb/IIIa inhibitor, the inhibitory effect of TMV-2 was more potent than TMV-7. Also, our data revealed that the binding site of TMV-7 on GPIIb/IIIa was different from TMV-2 and mAb 7E3. Moreover, combination of TMV-2 or TMV-7 with mAb 10E5 could lead to platelet activation. Finally, we found that TMV-7 had fewer side effects than TMV-2 at the same dosage. Therefore, the difference between TMV-2 and TMV-7 may provide valuable information for the development of GPIIb/IIIa inhibitors and the mechanisms involved in GPIIb/IIIa inhibitor-induced thrombocytopenia.

碩士
國立臺灣師範大學
生命科學研究所
99
Trimeresurus gracilis is the only species limited to the altitude over 2000 m mountain area among the five Crotalinae snakes in Taiwan. The goal of my study is to clarify whether low temperature in hibernation influence the reproductive cycle of T. gracilis. In tradition, gonad anatomy and serum hormones were used in reproductive physiological studies, but the former requires sacrifice and the latter usually causes stress, antifeedant, or bacterial infections. Fecal hormone is a non-invasive technique that has been used in many researches of reproductive cycles of other animals. With this new technique, we don’t need to sacrifice animals, and there’s no stress problem. Furthermore, we are able to do repeat sampling. My study starts with linking serum hormones to fecal hormones of T. gracilis. Then I will probe the effect of hibernation on fecal hormones and on the reproductive behavior of T. gracilis. All T. gracilis were assigned to two treatments of different temperature, specifying hibernation group (10℃) and nonhibernation group (20℃) in winter of both 2008 and 2009. Courtship and mating behavior were recorded by video from July to November of 2009, and April to September of 2010. Blood samples were collected once a month, and were only from females that didn’t show sign of stress, in March to August of 2010. And fecal samples were also taken once a month, from March of 2008 to August of 2010, excluding winter times (October to February). The results showed that the concentrations of fecal estradiol (E2) and progesterone (P4) could reflect the serum ones of female snakes. Fecal testosterone (T) of male snakes showed periodicity and was similar to other Crotalinae males, hence would be able to apply to reproductive research of snakes. Mating behavior wasn’t affected by hibernation, but was associated with body condition. Males having larger body condition had higher mating rate. Most females with body weight over 80g mated. Concentration of fecal E2 was influenced by hibernation, yet didn’t affect mating behavior. Females of hibernation group which underwent parturition, had significant higher fecal E2 in the following second spring. The raised concentration of E2 in spring was associated with secondary vitellogenesis. Low concentration of E2 of non-hibernated females, may indicate obstructed growth of follicles, and thus made the females unable to produce. Pregnant females had higher concentration of fecal P4, but there was no significant correlation between fecal P4 and hibernation, nor between fecal P4 and mating behavior. Although hibernation didn’t affect the cycle of fecal T, those male who successfully mated had higher fecal T.

博士
國立臺灣大學
生化科學研究所
89
Several venom proteases with fibrinogenolytic activity were isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus), one major Crotalinae snake species in Taiwan. The purified enzymes showed strong kallikrein-like and b-fibrinogenolytic activities in vitro, cleaving kininogen and b-chain of fibrinogen molecules efficiently. Further characterization showed that they are a family of single-chain proteins with molecular weights of about 28,000-33,000, the higher molecular-weight form being shown to be a glycoprotein. They also exhibit both esterase activity against N-p-tosyl-L-arginine methyl ester and amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, which is a specific synthetic substrate for kallikrein-like proteases. Their stability at high temperatures was examined and the cleavage specificity was studied using oxidized insulin B-chains as protein substrate. N-Terminal sequence analysis revealed that they are similar to batroxobin and ancrod, which were previously reported to possess vastly contrasting fibrinogen-clotting and antithrombotic effects respectively. Immunoblotting analyses using polyclonal antibodies revealed the cross-reactivities of these proteases with homologous enzymes from snake venoms of various species in the crotalid but not viperid snake families. Polymerase chain reaction (PCR) was employed to facilitate cloning and sequencing of these novel kallikrein-like fibrinogenases. More than five positive clones were selected and sequenced, corresponding to the coding sequences of 257-260 amino acids in these venom proteases, which all include a highly similar signal peptide of 24 amino acids. The amino acid sequences of these enzymes show homology with some thrombin-like enzymes from snake venoms of different species. Based on sequence comparison and homology alignment, the key residues of catalytic triad of serine protease family, i.e., His, Asp and Ser are well conserved. Expression and purification of these enzymes in E.coli system further corroborated that expressed venom proteases possessed similar fibrinogenolytic and hypotensive activities to those authentic enzymes purified from the crude venom of Taiwan habu. Most noteworthy is the observation that the proteases can cleave angiotensin I and release bradykinin from plasma kininogen in vitro, which is a strong vasodilator and probably responsible for the in vivo hypotensive effect of these venom proteases. We also show a novel method which can be used for the generation of active protein by refolding the recombinant protease from inclusion bodies expressed in the E. coli system. The autolytic cleavage of venom protease in situ from the polyhistidine-tagged fusion protein by the fused protease itself results in an active recombinant form of proteases with identical properties to the authentic natural protease.

碩士
國立臺灣大學
藥理學研究所
95
Snake venom metalloproteinase (SVMP), which is structurally similar to ADAM ( a disintegrin and a metalloproteinase ), is Zn2+ -dependent proteinase and have a catalytic sequence, i.e. HEXXHXXGXXH. Based on their domain structures, SVMPs are divided into four classes, N-I to N-IV and P-I to P-IV, where N and P represent nucleotide and protein, respectively. The molecular weights of P-I SVMPs are about 20~30kDa. However, the reduced or non-reduced molecular weight of trimpase, a SVMP purified from Trimeresurus mucrosquamatus in this study, migrates as a molecule of 16kDa. Trimpase inhibits platelet aggregation induced by thrombin and collagen, and its inhibitory effect could be blocked by pretreating with EDTA. According to MTT assay, DNA fragmentation, and cell cycle analysis with flow cytometer, all results demonstrated that trimpase can induce endothelial cell apoptosis. Further experiments reveal that the mechanism of apoptosis induced by trimpase was through degradation of extracellular matrices and inhibiting endothelial cell adhesion and inducing endothelial cell detachment. Trimpase preferentially cleaved α chain of fibrinogen and cleaved β chain after a longer incubation or at a higher concentration. Trimpase inhibited endothelial cell adhesion to immobilized fibrinogen and induced endothelial cell detachment. The effects on endothelial cell were abolished when trimpase was pretreated with EDTA. However, trimpase did not inhibit endothelial cell adhesion to immobilized collagen. The adherens junctions protein including α-catenin and β-catenin were cleaved by trimpase, but VE-cadherin and γ-catenin were not affected. On the other hand, we also found that the expression of Bcl-2 decreased and that of Bax increased in trimpase treated HUVECs, suggesting induction of cell apoptosis. In summary, we found a P-I SVMP with a smaller molecular weight, named trimpase, and it can inhibit endothelial cell adhesion and cleave adherens junction to induce endothelial cell apoptosis.

碩士
國立臺灣師範大學
生物研究所
87
英文摘要 A male-biased sampling of Chinese green tree viper, Trimeresurus s. stejnegeri, was found at Tsao-chiao study site. Animal skewed sex ratio may induced by various reasons. Although unlikely, the skewed sex ratio at birth was still found in a few snake species. Sampling bias and/or differential mortality rate between sexes are more likely to be the causes of the observed skewed sex ratio. The aim of this study is to find out the cause of this skewed sex ratio. By marking each individual, which encountered in the study site for 3 years, I could identify a total of 169 males and 79 females. The overall population sex ratio is significantly biased toward males(χ2=31.94, df=1, P＜0.001). I checked whether females may become more abundant than males at certain seasons or time of a day. But the results didn''t support such speculation. If habitat preference is different between sexes, it may bias toward males by sampling at only one type of habitat. Other than the main transect located at the edge of the deciduous forest, I surveyed another transect in the forest for one year. Both transects had significantly more males than females. Besides, I checked the vicinities of the study site occasionally and found the same male-biased sex ratio. 17 gravid females were collected from Tsao-chiao area in 2 consecutive reproductive seasons. A total of 77 newborn snakes(45 males and 32 females) were obtained in the laboratory. At birth, though the males are slightly more than females, there was no significant biased sex ratio(χ2=2.2, df=1, P＞0.05). Clearly, neither sampling bias nor differential birth rate between sexes was responsible for the skewed sex ratio of T. s. stejnegeri in Tsao-chiao. A differential mortality between sexes is likely to be the cause of this skewed sex ratio. I further divided the marked individuals into 4 groups according to their Snout-vent length. It revealed only the last group, which included all the mature individuals was significantly biased toward male. This implies mature females seem to have higher mortality rate than males.

碩士
國立師範大學
生物學研究所
86
I studied the reproductive cycle of Chinese green tree vipers, Trimeresurus s. stejnegeri in northern Taiwan by examining seasonal changes in the morphology and histology of their reproductive organ and the hormonal activity. From March 1996 to August 1997, I collected a total of 288 snakes with an average of 7 female and 9 male snakes per month. Based on the size of follicles and the presence of corpora lutea, I classified mature female snakes into 7 stages. The snakes in the first stage had only primary follicles of which the maximal length is less than 5 mm. The snakes that belonged to stage 2 to stage 6 had secondary follicles with the length ranged between 5- 10 mm, 10- 15 mm, 15- 20 mm, 20- 25 mm and above 25 mm respectively. The last stage snakes had both primary follicles and corpora lutea. I found estrogen level was highest in the snakes of stage 5 while that in stage 6 had the peak concentration of progesterone in serum. The numbers of reproductive females which belong to stage 2 to 7 versus that of nonreproductive females, classified in first stage, was close to unity. This suggested that female T. s. stejnegeri reproduced biennially rather than annually. The mass of fat body correlated negatively with the process from first stage to last stage. Fat reserves may become the limiting factor of reproductive frequency. Female Chinese green tree vipers had seasonal cyclic changes in morphology and histology of reproducitve organs as well as hormonal activities. Their timing of vitellogenesis belongs to postnuptial type (type II) vitellogenesis, which is common in the viperids.According to the morphological difference of seminiferous epithelium, I classified male Chinese green tree vipers into five stages, which were early recrudescence, late recrudescence, spermiogenesis, early regression and late regression. Early recrudenscence stage started from March and could last till May. Spermiogenesis stage occurred mainly in summer and autum. While in winter, I found most male snakes belonged to regression stages. Other parameters, such as diameter of seminiferous tubule, renal sexual segment and fat body mass also revealed seasonal cylcic changes. However the peak of each cycling may not be the same. For example, the peak of renal sexual segment diameter, which correlate well with copulation was later than that of seminiferous tubule diameter. The same as many other viperids, male Chinese green tree vipers had postnuptial spermaotgenesis cycle.

碩士
國立臺灣大學
藥理學研究所
103
Snake venoms profoundly affect platelet aggregation and hemostasis. By using column chromatography of CM Sephadex C-50 cationic exchanger and Sephadex G-75 gel filtration, a novel TMV inducer was purified from Trimeresurus mucrosquamatus snake venom. Under reducing conditions, it migrates as a protein with a mass about 45 KDa on SDS-PAGE. TMV inducer-induced platelet aggregation of human washed platelets and platelet-rich plasma in a concentration-dependent manner with EC50, 54.36 ± 0.92 and 13.8 ± 0.3 ng/ml, respectively. Aggrastat, anti-integrin αIIbβ3 mAb 7E3 significantly inhibited platelet aggregation caused by TMV inducer. Anti-GPIb mAb 6D1 and anti-GPIa/IIa mAb 6F1 only slightly inhibited platelet aggregation caused by TMV-inducer. An additive inhibitory effect was observed when 6D1and 6F1 were added. However, anti-GPVI mAb 326E12 and 342D7 profoundly inhibited platelet aggregation. In pull-down assay, biotinylated TMV inducer specifically bound to GPVI, but not GPIb or integrin α2. TMV inducer did not elicit agglutination of fixed platelets. PGE1 and BAPTA/AM completely inhibited platelet aggregation induced by TMV inducer. EDTA and indomethacin also caused profound inhibition. Syk inhibitor, piceatannol had higher inhibitory effect (80%). PLC inhibitor U73122, MEK inhibitor PD98059 and PI3-K inhibitor LY294002 totally inhibited TMV-inducer induced platelet aggregation without inhibiting platelet shape change. Src inhibitor, PP2 delayed platelet shape change, and profoundly inhibited aggregation. To confirm the downstream signal transduction, TMV-inducer induced a time-dependent tyrosine phosphorylation of a number of proteins similar to those activated by convulxin, including PLCγ2, PI3K, Syk, Src, Fyn and LAT. In mice models, TMV inducer did not significantly prolonged mice tail bleeding time. However, TMV inducer caused a dose-dependent decrease of platelet counts. Taken together, TMV inducer, a monomeric platelet aggregating inducer, activates platelets mainly through GPVI, leading to phosphorylation of many signal molecules, including PLCγ2, PI3K, Syk, Src, LAT and Fcγ, finally inducing the exposure of functional αIIbβ3 and Ca2+- dependent platelet aggregation. This novel snake venom protein may provide an useful tool for studies of GPVI and signaling mechanisms involved as well as for comparative study with other C-type lectin GPVI agonist, like convulxin or trowaglerix, and its association of platelet receptors, such as GPIb and GPIa/IIa. These studies would provide new insights regarding the molecular interaction of these GPVI agonists, providing clues for the design of GPVI antagonists, a new class of antithrombotic agent.

碩士
國立臺灣大學
藥理學研究所
100
TMF, a platelet aggregation inducer, was purified from Trimeresurus mucrosquamatus venom. By means of cationic exchange, gel filtration and FPLC column chromatography, two bands were detected on SDS-PAGE with apparent molecular weights of 26 kDa and 23 kDa, respectively, under reducing condition. On 2-D PAGE under reduction, there were four bands with different pI ranges 5.8~6.5 withsimilar mass of 26 kDa; pI 6.7~7.2 with similar mass of 23 kDa. Through LC/MS/MS analysis, the 26 kDa bands exhibit high similarity to β-fibrinogenase and the 23 kDa bands was similar to metalloprotease (α-fibrinogenase). TMF concentration-dependently induced platelet aggregation both in platelet-rich plasma (PRP) and platelet suspension (PS), with EC50 0.3 and 1.8±0.32 μg/ml, respectively. TMF also concentration-dependently caused P-selectin expression, increase of intracellular Ca2+ concentration, and release of TxB2. PGE1, EDTA and BAPTA/AM effectively inhibited TMF induced platelet aggregation, but heparin, hirudin, aprotinin did not. TMF specifically inhibited the binding of 326E12, mAbs of GPVI to platelet measured by Flow cytometry. TMF activated the signaling molecules involved in GPVI signal pathway, including Syk, Src, PLCγ2, LAT, which were completely inhibited by PP2. Regarding enzymatic activity, TMF concentration-dependently degraded α- and β-chain of fibrinogen. After incubation with serine protease inhibitors, PMSF or metalloprotease inhibitor, EDTA, its activities of β-fibrinogenase and α-fibrinogenase were respectively inhibited while its aggregation was not affected. These results suggest that its platelet aggregating activity is not related to the intrinsic enzymatic activities. Taken together, TMF might exist as a complex under isotonic condition, consisting of two proteins, namely serine proteinase (i.e.β-fibrinogenase) and metalloprotease (i.e.α-fibrinogenase). It activates platelets and the downstream signaling molecules mainly through the specific biding of platelet GPVI. By phosphorylation of Syk, Src, LAT, Fyn, PLCγ2, PI3K and Akt, TMF induced expression of P-selectin and TxB2 biosynthesis, and finally activated integrin IIb-IIIa, resulting in platelet aggregation. However, its platelet aggregating activity is not related to the proteolytic activity. This snake venom protein may provide a useful tool for studying how it interacts with platelet GPVI at a molecular level. The information may provide insights for designing the novel small-mass antagonist of platelet GPVI, a new class of antithrombotic agent.

碩士
國立臺灣大學
藥理學研究所
81
The effects of waglerin-1, a toxin from Trimeresurus wagleri on the neuromuscular (NM) transmission were studied on the mouse phrenic nerve diaphragm and triangularis sterninerve-muscle pre- parations. Waglerin-1 (1.20-3.99 uM) reversibly inhibited the directly elicited twitch tension of the diaphragm. The toxin(3.99 uM ) also inhibited indirectly elicited action potential of the diaphragm. However, the directly elicited twitch tension and the resting membrane potential of the diaphragm were not affected by waglerin-1(3.99 uM). The results above suggested that waglerin-1 neurotoxic but did not have myotoxic effects. The toxin ( 1.20- 3.99 nM)reversibly inhibited the amplitude of endplate potential The toxin (0.52-1.20 uM) also reversibly inhibited the amplitude of miniature endplate potential,while it decreased the frequency miniature endplate potential at a lower concentration (0.12-0.40 uM). Waglerin-1 (1.20 nM -0.40 uM) decreased the quantal content of endplate potential,too. In chronically denervated diaphragm, waglerin-1 (1.20-3.99uM) decreased the acetylcholine induced mu- scle contracture. The perineural waveforms were recorded with an extra cellular electrode placed into the perineural sheaths of motor nerves of triangularis sterni. Waglerin-1 (3.99 uM) did not alter the waveforms of sodium, fast potassium,slow potassium and calcium activated potassium currents of the nerve terminal, but it decreased the fast calcium and slow calcium currents.It was concluded that waglerin-1 acted on both pre-synaptic and post- synaptic sites of the mouse motor endplate. The neuro- toxicity of waglerin-1 may be resulted from the synergistic effects of both actions.The effects of waglerin-1 on the calcium currents of motor nerve terminal may contribute to its action on the pre-synapic transmission.

The Indian subcontinent is a considered a mega biodiverse region with representative regions belonging to three of the 35 global biodiversity hotspots, including the Western Ghats in Peninsular India and the Eastern Himalaya. These areas are threatened with biodiversity decline due to habitat loss and other factors such as climate change. Many species and populations may be extirpated even before they have been discovered, as these areas have not been adequately surveyed, especially for lower vertebrates and invertebrates. The region has over 300 species of snakes, but they are poorly studied despite the fact that snakes play an important ecological role in ecosystems, human landscapes and in medical science. Therefore, my study focused on the distribution and diversification of select taxa belonging to different families of snakes in Peninsular India, with an emphasis on systematics and biogeography. The evolution and biogeography of various taxa in Peninsular India are of particular interest as this region, a Gondawanan fragment, is critical to our understanding of historical biogeography in the Oriental realm. Over the past decade, molecular tools have enabled testing of alternative historical scenarios of faunal exchange and consequent biogeographic patterns. The snakes of Peninsular India, despite their spectacular diversity, remain known poorly with regard to their biogeographic affinities. While most Indian snakes are considered to be Malayan relicts, this hypothesis remains unexplored. I therefore explored the systematics and biogeography of snakes in the region. In addition, I used a phylogeographic framework to understand the genetic structure of widely distributed taxa which may comprise multiple lineages. I explored historical patterns of dispersal and diversification within Peninsular India using two distantly related snakes with broad differences in ecology and biology; an arboreal, non-venomous genus, Ahaetulla (vine snakes), belonging to the family Colubridae, and the genus Trimeresurus (Asian pit vipers), a group of terrestrial and arboreal, venomous snakes belonging to the family Viperidae. My broad objectives were to elucidate the historical biogeography of both snake genera in Peninsular India, more specifically, the role of vicariance and dispersal at broad spatial and temporal scales on observed patterns of distribution and diversity. At a regional scale, I also tested for patterns of lineage diversification within the Western Ghats region. To achieve these objectives, I used an extensive taxon sampling of snakes from Peninsular India and adjoining Northeast India with a comprehensive coverage of habitats and environmental gradients. With this dataset, I first delimited species using a coalescent method and a multicriteria approach including genes, geography and morphology. Second, using mitochondrial and nuclear DNA, I reconstructed the phylogenies and obtained a dated tree. Third, I tested for congruence in patterns of diversification and dispersal using ancestral range reconstruction of geographical ranges and diversification analyses. My dissertation is organized into the following chapters. Chapter 1 In this section, I introduce the patterns of distribution of snakes in the context of the biogeography of the Indian subcontinent with special reference to disjunct distributions in the wet evergreen parts of the Western Ghats and Northeast India. I discuss various hypotheses proposed to explain these patterns of distribution and the biogeographic affinities of snakes in the region. Two main processes have been proposed, namely dispersal and vicariance. The dispersal model suggests that lineages colonized different parts of Peninsular India through one or more routes of dispersal which may have appeared or disappeared over a period of time due to expansion and contraction of humid forest cover. The routes of colonisation proposed are through the Vindhya-Satpura hill ranges (known as the Satpura hypothesis), Eastern Ghats route or other routes from the Malayan region to Peninsular India. An alternative model, namely vicariance, suggests that a widespread ancestor of a lineage diverged in response to successive subdivisions of its distribution range. In addition to these broad biogeographic patterns, the complexity of the environmental gradient and ecological selection pressure could drive morphological variation leading to diversification. I provide an overview of patterns of distributions of snakes in India and their morphological variation I relation to geography and genetic structure. Finally, based on the broad distribution and diversity of taxa, I justify the value of snakes as a model system to study evolution and biogeography. Chapter 2 Genus Ahaetulla (family Colubridae) is distributed widely in Asia with nine species, of which three are endemic to India and seven are widely distributed. Species diversity in the drier and arid parts of Peninsular India is low compared to wet and humid areas. This is an understudied snake group in India, which includes a number of ecomorphs and color variations. A better understanding of the taxonomy and systematics of the group is required for both future ecological studies as well as for conservation. In addition, complete taxon sampling is needed to resolve uncertainties regarding the phylogenetic positions of lineages within this clade, including that of endemic species. Therefore, in this chapter, I present the first species delimitation and molecular systematics of vine snakes, incorporating all the known species across the Oriental region. I carried out comparisons between sister lineages which were identified with confidence due to our systematic sampling approach. The analysis revealed many cryptic species in vine snakes, which were delimited using a combination of genetics (phylogeny, Bayesian clustering algorithms and genetic distance), morphology and geography. In addition, I discovered a deeply divergent lineage from the southern Western Ghats, which is sister to all remaining members of Ahaetulla, and described it as a new genus, Proahaetulla. Chapter 3 Asian pit vipers belonging to the genus Trimeresurus are amongst the most diverse groups of vipers, and are distributed throughout Asia. Their distribution pattern is broadly similar to the genus Ahaetulla in Peninsular India. Species richness is low in drier and arid lowland parts of Peninsular India compared to wet and humid areas. In addition to resolving the taxonomy and systematics of lineages in Peninsular India, delimitation of species boundaries in widely distributed species is necessary for conservation and management and for further studies. In earlier studies, members of this clade had been assigned to different species or even genera (i.e Peltopelor in Peninsular India; Popeia, Viridovipera, Cryptelytrops in Northeast India) based on morphological characters, which needed to be revalidated using molecular tools. A taxonomic revision of Trimeresurus lineages from India is necessary to delimit species and understand their phylogenetic relationships and systematic position, and to revalidate the taxonomic status of ecomorphs within widely distributed species. Therefore, I carried out a delimitation of species boundaries in the genus Trimeresurus in India using a multicriteria approach including genetics, geography and morphology. I resolved the systematics of the group and assigned all members of the clade to the genus Trimeresurus; other proposed genera are not valid. In addition, I delimited multiple cryptic species within this group in the Western Ghats clade. Chapter 4 Multiple colonization events of different taxa into and out of Peninsular India, and lineage diversification associated with past geographic and climatic events is expected to have shaped species richness in these snake genera, a pattern observed in many other taxa as well. To evaluate the pattern of colonization and diversification, I used independent evolutionary lineages from Chapters 2 and 3 for further phylogenetic and biogeographic analysis. For this, I used five genes to obtain a species tree; chronograms were reconstructed with available fossil calibrations. The resulting chronograms were used to reconstruct the biogeographic histories of both groups using ancestral area reconstructions. The patterns of dispersal show signatures of congruence and contrast between the clades, with the Western Ghats acting as a major source for colonisation of ancestral lineages into the arid regions in Peninsular India and adjoining Sri Lanka as well as Southeast Asian regions. I tested for alternative models of diversification with an emphasis on the Western Ghats. Ahaetulla supports a model of constant rate of speciation over time while Trimeresurus showed support for diversity dependent decline. In contrast to earlier hypotheses, Peninsular India emerged as a centre of snake diversification and Western Ghats as a major centre of in-situ radiation for both genera. Chapter 5 In this concluding section, I summarize the major findings from Chapters 2, 3 & 4. Both snake genera, Ahaetulla and Trimeresurus, consist of a number of cryptic lineages in Peninsular India. In the case of Ahaetulla, I discovered a deeply divergent lineage, sister to Ahaetulla, and described it as a new genus, Proahaetulla. The Western Ghats was found to be a major source for colonisation of ancestral lineages into the arid regions in Peninsular India and adjoining Sri Lanka as well as Southeast Asian regions. The region acts as a major centre of snake lineage diversification and in-situ radiation in Peninsular India.

碩士
國立清華大學
生物資訊與結構生物研究所
95
The crystal structure of TM-1, a small snake-venom metalloproteinase (SVMP) isolated from Taiwan habu (Trimeresurus mucrosquamatus), was determined at 1.84 Å resolution with resultant R and Rfree values of 0.179 and 0.216, respectively. The overall structure of TM-1 is an oblate ellipsoid that contains three disulfide crosslinks, Cys119-Cys198, Cys160-Cys182 and Cys162-Cys165. The overall structure of TM-1 contains one zinc ion which is bound to four ligands, including three conserved histidines and one water molecule, displaying a tetrahedral geometry. We find that the distinct active site structures between TM-1 and TM-3 may reflect the different sensitivities toward the endogenous inhibitors from structural comparison of TM-1 and TM-3. Moreover, we have modeled the three endogenous inhibitors, i.e., pyroGlu-Asn-Trp (pENW), pyroGlu-Gln-Trp (pEQW) and pyroGlu-Lys-Trp (pEKW), into the active site of TM-1 structure. Results from interaction analysis of TM-1 and inhibitors show that the P-2 site of inhibitors is important for binding to TM-1 via hydrogen bonds and hydrophobic interactions. By proteolysis experiments of TM-1 using oxidized insulin B-chain as substrate, we sugguest P-1 and P-2 sites of substrates are important for cleavage by TM-1. The preferential amino acids at P-1 site are Leu, Phe and His, while The preferential amino acids at P-2 site are Leu, Val and Tyr. In conclusions, our results indicate that the structural difference between TM-1 and TM-3 may influence their endogenous inhibitor sensitivities and substrate specificity. Additionally, we also suggest that the P-1 and P-2 sites of substrate are critical for cleavage by TM-1.

碩士
國立臺灣師範大學
生物研究所
87
Abstract Hibernation is usually adopted by animals to save energy in the cold winter. Some reptiles may select lower body temperature or depress metabolic rates when they are in hibernation condition. In northern Taiwan, it is much harder to find a Trimeresurus s. stejnegeri than in southern Taiwan during winter. Consequently, I predict the northern population of T. s. stejnegeri may reveal hibernation behaviors as many temperate snakes. Whereas, without a long-term cold pressure, the southern snake population may lack hibernation behaviors as most tropical reptiles. By comparing the thermopreferrenda and oxygen consumption of both snake population in the winter, I may clarify above prediction. Thermopreferrenda and oxygen consumption of 4 groups snakes, that were 1.northern population, 10℃ acclimation 2.northern population, 20℃ acclimation 3.southern population, 10℃ acclimation and 4.southern population, 20℃ acclimation were compared from December, 1998 to the beginning of March, 1999. Thermopreferrenda behaviors were tested in a thermogradient. No significant difference of thermopreferrenda was found among the four groups. Oxygen consumption was conducted in a walking chamber, of which the temperature was controlled the same as each group's acclimation temperature. Oxygen consumption was different only when acclimation or testing temperature is different. Therefore, the results contradicted my prediction. It seems the phenomenon, that the northern T. s. stejnegeri becomes more secretive than southern population in winter is simply due to a passive reaction to cold weather rather than hibernation behavior.

碩士
中臺科技大學
生命科學研究所
98
Trimeresursus mucrosquamatus (TM) was one of six major vipers in Taiwan and it has hemorrhagic toxicity with highly biting rate among the six vipers. Due to the low neutralization titers of antivenin when immunized the horse with TM crude venom, which caused difficult manufacture of commercial TM antivenin. For investigate this, we firstly were separated the TM crude venom by ion exchange chromatography then used the hemolytic test and LD50 of ICR mice for identify the toxicity portions with hemolytic and non-hemolytic fractions (phospholipase A2, PLA2) . Secondly, we designed four immunized strategies to immunize ICR mice, which including: (A) TM crude venom, (B) toxicity portions of TM crude venom, (C) two vaccinations with toxicity portions of TM crude venom before TM crude venom, and (D) two vaccinations with TM crude venom before toxicity portions of TM crude venom and we also had immunized horse with (A) to (C) strategies. For understand the antibody titers to TM venom among different immunized strategies, we develop capture ELISA and in vitro neutralization antibody titer assay of TM crude venom to assay the antivenin of mice and horses. These results shown that A, C and D strategies have better antibody titers than B and the horses antivenin of in vivo neutralization antibody titers ≧ 60 Tanaka units (TU) neutralized TM crude venom were 0.2-0.3 cm hemolytic zone at 9 hrs, 37°C in rabbit blood agar. We conclude the TM crude venom could cause good antibody titer in mice and horse and the in vitro neutralization antibody titer assay could easy to preliminary screen the neutralization antibody titers of horse antivenin in this study.

碩士
國立臺灣師範大學
生物研究所
88
Abstract The aims of this study are: (1) examining the suitability of tread bobbin and radiotelemetry in tracking Chinese green tree vipers; (2) check effect of different sampling intervals on estimating activity; (3) investigating their daily activity pattern and checking whether it shifts in different season (4) studying seasonal activity pattern and displaying it by different indexes; and (5) estimating home ranges of Chinese green tree vipers. Because male Chinese green tree vipers are more numbers than females and sexes may influence activity significantly, I used only males in this study. I estimated the snake moving distance, the index of activity by recording the position of snakes repeatedly. The activities of snakes, tracking by tread bobbin had no difference with that by directly observation (paired t-test, p=0.131, n=10). This implied thread bobbin is suitable for tracking snakes. But, the snakes died after surgery for subcutaneous implanted transmitters. When the sampling interval for snake position was prolonged from 1 to 6 hours, the estimated activity became 92.2% and 64.4% of real activity. The male Chinese green tree vipers began active in the dust, and kept active in the night but the magnitude was lower than that in the dusk. During the day, they remained inactive mostly. Above daily activity pattern did not change through different season except in winter. The activity was so low that no difference of movement was found between night and day in winter. The daily average activity in summer, autumn, winter, and spring was 36 ± 19、17 ± 8、4 ± 2、7 ± 2 cm/hr(Mean ± SE) respectively. The activity was highest in summer, and lowest in winter during which, the snakes didn’t aggregated for hibernation. Above seasonal activity pattern remained the same when examined by different activity indexes, such as the activity in first half night (19:00-1:00), frequency of movement, and longest inactive days. However, the pattern was different, if I used the capture rate of male snakes as the index of activity. The capture rate was highest in spring, secondary in summer, and lowest in winter. The home range size of male Chinese green tree viper was about 29,800 m3 in one month, and was below 195,000 m3 when recording period was longer than three months.