Rozprawy doktorskie na temat „PWG-1”

Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A mutant of B. licheniformis PWD-1, named B. licheniformis WBG-2, was developed that is deficient in sporulation. The mutation was created using the Splicing by Overlap-Extension PCR method (Gene SOE?ing) to create a 256bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In-vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise from the B. licheniformis chromosome. PCR and DNA sequencing were used to confirm the deletion, while heat-treatment assays and electron microscopy verified the absence of spores. The mutant, while completely asporogenic, is able to expresses normal levels of keratinase as compared with B. licheniformis PWD-1.

Aortic stiffness underlies systolic hypertension, promotes heart failure and is associated with increased cardiovascular morbidity and mortality. It is regarded as a primary driver of left ventricular hypertrophy and aortic aneurysms and is linked to the pathogenesis of cognitive impairment, stroke and renal failure. Like most cardiovascular traits, aortic stiffness is a complex trait and is moderately heritable, yet the precise molecular mechanisms that underpin the stiffening process remain poorly defined. This study aimed to employ multiple approaches to further identify the genetic basis of aortic stiffness in a large repository of human donor aortas that had undergone ex vivo pulse wave velocity (PWV) phenotyping. The first part of this work sought to investigate the molecular basis of Loeys-Dietz type 4 syndrome in a pedigree with multiple cases of aortic aneurysms and dissections. A missense variant p.(Arg320Cys) was identified in a highly evolutionary conserved region of TGFB2. There was striking upregulation of TGFB1, TGFB2 and pSMAD2/3 on imunocytochemical straining and western blotting of the aortic tissue from the index case confirming the functional importance of the variant. This case highlighted the striking paradox of predicted loss-of-function mutations in TGFB2 causing enhanced TGFβ signalling in this emerging familial aortopathy and underscored the significance of TGFβ signalling in aortic extracellular matrix biology. The second part of this work attempted to characterise the biological basis for the susceptibility locus identified in the most recent genome wide analysis of carotid-femoral PWV. While the locus lies within the 14q32.2 gene desert, it contains regulatory elements, with the transcriptional regulator B-cell CLL/lymphoma 11B (BCL11B) and non-coding RNA DB129663 representing potential targets for these enhancers. The association of five lead SNPs from the genome-wide association studies (GWAS) meta-analysis was examined for ex vivo aortic stiffness and BCL11B and DB129663 aortic mRNA expression. Three of the five SNPs associated significantly with PWV and showed allele-specific differences in BCL11B mRNA. The risk alleles associated with lower BCL11B suggesting a protective role for BCL11B. Despite the strong association, BCL11B protein was not detected in the human aorta; however, qPCR for CD markers showed that BCL11B transcript correlated strongly with markers for activated lymphocytes. In contrast, DB129663 transcripts were detected in 55% of the samples, and of the five SNPs only one showed allele-specific differences in aortic DB129663 transcripts. No significant differences were observed in PWV between samples expressing or lack- ing DB129663, and therefore the implication of this lncRNA in aortic stiffness remains elusive. The BCL11B transcript detected in the human aorta may reflect lymphocyte infiltration, suggesting that immune mechanisms contribute to the observed association with PWV. For the final part of this work genetic associations with aortic stiffness were explored in a candidate gene-based study utilising tagging SNPs to effectively capture the genetic information from linkage disequilibrium blocks. Association analyses were performed in young, healthy ENIGMA study par- ticipants selected for high and low PWV values then validated in the remaining ENIGMA cohorts. The association of four lead SNPs was then examined for ex vivo aortic stiffness in human donor aortas. The tissue expression of these SNPs and their encoded proteins was also explored. Neither the aggrecan nor the fibulin-1 SNPs showed significant associations with ex vivo PWV in the donor aortas. The exonic aggrecan tagSNP rs2882676 displayed differential transcript abundance between homozygous allele carriers but this did not translate at the protein level. Both aggrecan and fibulin-1 were found in the aortic wall, but with marked differences in the distribution and glycosylation of aggrecan, reflecting loss of chondroitin-sulphate binding domains. These differences were age-dependent but the striking finding was the acceleration of this process in stiff versus elastic young aortas. These findings suggest that aggrecan and fibulin-1 have critical roles in determining the biomechanics of the aorta and their modification with age could underpin age-related aortic stiffening.

This thesis deals with the history of a method of plants cultivation without original substrate. The method is hydroponics and its subsequent development is called aeroponics. This work outlines the design of the whole aeroponic system including a control unit. The control unit has been designed to control the injection of nutrient solution, which is essential to the life of plants.Further, the control unit performs the control of lighting period, temperature, air flow and also regulation of nutrient solution pH. The control unit also keeps records of all data necessary for the monitoring process and for the evaluation of time differencies in the system.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The objective of this study was to evaluate the effect of adding the protease enzyme 0.05% (Cibenza DP 100) in diets for broilers based on corn and soybean meal with two nutritional matrices. Two experiments were conducted with 5 treatments with a basal corn and soybean meal diet: T1 – basal diet with valued protease matrix twice without the enzyme, T2 – basal diet with valued protease nutritional matrix once without enzyme, T3 – basal diet, T4 – basal diet with valued protease nutritional matrix twice with enzyme and T5 – basal diet with valued protease nutritional matrix once with enzyme. The first experiment was carried out with 1280 chicks Cobb500 strain of 1 to 42 days and the second experiment was carried out with 120 chicks housed in the first experiment, and the chicks were housed in battery cages from 17 to 21 days for digestibility study. The experimental design of the experiments was a randomized block, being in the first experiment the row in the experimental industrial shed the criteria for distribution of the blocks, with 5 treatments and 8 repetitions each containing 32 broilers per box. In the second experiment the floor of the cage was the criteria distribution of the blocks, with 5 treatments and 6 repetitions each containing 4 broilers per cage. The analyzed variables were: digestibility, carcass yield, rest, thighs, wings, abdominal fat and performance variables. In all treatments the data were subjected to analysis of variance, evaluated by the R program, and the means were compared by Tukey test at 10% significance. In the first experiment, it was observed a statistical difference (P <0.10) in the final mean weight, weight gain and feed intake in the pre-initial phase with beneficial results with the enzyme supplementation with valued protease nutritional matrix once. In the period from 1 to 21 days, there was a statistical difference (P <0.10) in feed intake and feed conversion, but with positive results for the control treatment. In the cut yield, there was no statistical difference (P> 0.10) in the most used cuts, similar results between the treatments and the control. Statistically different result (P <0.10) was found with the lowest proportion of abdominal fat in the diet fed with diet supplemented with the enzyme and nutritional matrix valued at one time. In the second experiment, statistical difference (P <0.10) was observed for nitrogen balance, which was positive, resulting in higher retention of nitrogen with lower excretion in the environment. The dry matter retention also showed a statistically different result (P <0.10), with a positive result for the supplementation with the enzyme together with greater metabolizability of the dry matter. Based on this study, the protease enzyme supplementation is recommended along with a valued protease nutritional matrix once, in the pre-initial phase of life of the birds, in order to have a better final weight, better weight gain, lower feed intake, lower proportion of abdominal fat, positive nitrogen balance and higher dry matter retention. Based on this work, it is recommended to supplement the protease enzyme with a valued protease nutritional matrix, especially in the pre-initial phase, in order to have a better final weight, better weight gain, lower feed intake, lower abdominal fat ratio, positive nitrogen balance and higher dry matter retention. In the other stages of life of the birds (initial, growth and final) the enzyme supplementation with a valued protease nutritional matrix was also beneficial, since the results in performance, cut yield and metabolizability were equivalent to control. The treatment diets are reduced in nutritional levels if compared to control diet with ideal dietary level for the broilers, resulting in less expense with the nutrients and maintaining a high yield.
Objetivou-se com a presente pesquisa avaliar o efeito da adição da enzima protease 0,05% (Cibenza DP 100) em dietas para frangos de corte a base de milho e farelo de soja. Foram conduzidos dois experimentos com 5 tratamentos cada. Todas as rações foram elaboradas com milho e farelo de soja, diferenciando no seguinte: T1 - ração basal com a matriz da protease valorizada em duas vezes sem a enzima, T2 - ração basal com a matriz da protease valorizada em uma vez sem a enzima, T3 - ração basal, T4 - ração basal com a matriz da protease valorizada em duas vezes com a enzima e T5 – ração basal com a matriz da protease valorizada em uma vez com a enzima. O primeiro experimento foi realizado com 1280 pintos da linhagem Cobb500 de 1 a 42 dias e o segundo com 120 pintos alojados em baterias de gaiolas de 17 a 21 dias para ensaio de digestibilidade. O delineamento experimental foi em blocos casualizados, sendo no experimento 1 a localização da parcela experimental dentro do galpão, com cinco tratamentos e oito repetições cada, contendo 32 aves por box. No experimento 2 o andar da gaiola foi o critério de distribuição dos blocos, com 5 tratamentos e 6 repetições cada, contendo 4 aves por gaiola. As variáveis analisadas nos experimentos 1 e 2 foram: digestibilidade, rendimento de carcaça, peito, coxas, asas, gordura abdominal e variáveis de desempenho. Os dados foram submetidos a analise de variância, avaliados por meio do programa R, e as médias comparadas pelo teste de Tukey a 10% de significância. No primeiro experimento, observou-se diferença estatística (P<0,10) no peso médio final, ganho de peso e consumo de ração na fase pré-inicial com resultados benéficos com a suplementação da enzima com valorização em uma vez da matriz nutricional. Já no período de 1 a 21 dias, houve diferença estatística (P<0,10) no consumo de ração e conversão alimentar, porém com resultados positivos para o tratamento controle. No rendimento de cortes, não houve nenhuma diferença estatística (P>0,10) nos cortes mais utilizados, resultados semelhantes entre os tratamentos e o controle. Resultado diferente estatisticamente (P<0,10) foi encontrado com a menor proporção de gordura abdominal na parcela alimentada com dieta suplementada com a enzima e matriz nutricional valorizada em uma vez. No segundo experimento, observou-se diferença estatística (P<0,10) para balanço de nitrogênio sendo este positivo, resultando em maior retenção de nitrogênio com menor excreção no ambiente. A retenção de matéria seca também mostrou um resultado diferente estatisticamente (P<0,10), com resultado positivo para a suplementação com a enzima juntamente com maior metabolizabilidade da matéria seca. Baseado nesse trabalho recomenda-se a suplementação da enzima protease juntamente com uma valorização da matriz nutricional, principalmente na fase pré-inicial, para que se tenha melhor peso médio final, melhor ganho de peso, menor consumo de ração, menor proporção de gordura abdominal, balanço positivo de nitrogênio e maior retenção de matéria seca. Nas outras fases de vida das aves (inicial, crescimento e final) a suplementação da enzima com a valorização da matriz nutricional também se mostrou benéfica, visto que os resultados em desempenho, rendimento de cortes e metabolizabilidade foram equivalentes ao do tratamento controle. As dietas tratamento são reduzidas em níveis nutricionais se comparadas com a dieta controle com o nível dietético ideal para as aves, isso acaba resultando em menor gasto com os nutrientes e mantendo uma alta produção.

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

The mechanical properties of the arterial wall are of great importance for blood pressure regulation and cardiac load. With increasing age, large arteries are affected by increased wall stiffness. Furthermore, atherosclerotic manifestations may increase the stiffness even further, both processes acting as independent cardiovascular risk factors affecting the arterial system in a heterogeneous way. The aims of this thesis was to characterize the local mechanical properties of brachial artery (BA) with the aid of ultrasound technique and to evaluate the influence of 1) age, gender, sympathetic stimulation and examination site; 2) type 1 diabetes (DM) and its association to circulatory biomarkers; and 3) to evaluate the general properties of the arterial system with the aid of pulse wave velocity (PWV) as well as pulse wave analysis (PWA) in systemic lupus erythematosus (SLE) and correlate the findings to disease activity and circulatory biomarkers. In the most proximal arterial segment of the upper arm a pronounced age-related decrease in wall distensibility, increase in intima-media thickness (IMT), and a slight increase in diameter were seen. Sympathetic stimulation had no influence on wall mechanics. More distally in BA, no change in diameter, and only minor increase in IMT and decrease in distensibility were seen. No gender differences were found. These findings suggest that the principle transit zone between elastic and muscular artery behaviour is located in the proximal part of the upper arm. Women with uncomplicated insulin-dependent DM had similar diameter, IMT and distensibility in their distal BA as controls, whereas flow-mediated dilatation (FMD) was slightly, and nitrate mediated dilatation (NMD) markedly reduced. NMD was negatively correlated with higher HbA1c levels. Vascular smooth muscle cell function seems to be an early manifestation of vascular disease in women with DM, influenced by long-term hyperglycaemia. Women with SLE had increased aortic PWV compared to controls, a finding positively associated with increased levels of complement factor 3 (C3), but not with disease activity. The increased stiffness of central arteries may be one factor contributing to the increased cardiovascular risk seen in SLE.

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Within scope of this work, double-fourier analysis method of rapid calculation and detailed simulation method, which are used to investigate DC bus current harmonics in two level and three level three-phase inverters systematically, will be emphasized and two methods will be compared via applying different modulation techniques. In addition, DC bus currents will be investigated visually for various working conditions and modulation methods. After that, analysis methods will be applied and harmonic spectrums will be determined. After all, it will be showed that calculated harmonic spectrums could be treated as unified harmonics around certain frequencies and these unified harmonics could be reached easily via looking at predetermined table. Moreover, it will also be showed that unified harmonic values could be used to determine harmonic current components that are necessary for sizing DC bus capacitor and could be used in various inverter analysis.

In conventional three-phase PWM (Pulse Width Modulation) current source converter based STATCOM (Static Synchronous Compensator) applications, DC link current is kept constant at a predefined value and the reactive power of STATCOM is controlled by varying modulation index. This control strategy causes unnecessary power losses especially when the reactive power of STATCOM is low. For this purpose, in order to reduce the active power drawn by STATCOM, the modulation index can be maximized by adjusting DC link current. Within the scope of this thesis, an adaptive control of DC link current will be designed and applied to a 0.4kV 50kVAr three phase current source converter based STATCOM so that the power losses can be reduced. The theoretical work will be compared and discussed with the experimental results.

Anorexigenic gastrointestinal hormones as peptide YY (PYY), glucagon-like peptide 1 (GLP-1) and orexigenic hormone ghrelin influence the activity of the arcuate nucleus (ARC) neurons in the hypothalamous. The ARC plays a crucial role in the regulation of food intake and energy homeostasis. In contrast, the mesolimbic dopamine system encodes subjective ‘liking’ and ‘wanting’ of palatable foods, which is subjected to modulation by the hindbrain and the hypothalamic homeostatic circuits and by satiety and adiposity hormones. Reportedly, obesity reflects an energy imbalance in which genetically susceptible individuals become increasingly vulnerable to an ‘obesogenic’ environment. Dietary and behavioral approaches to severe obesity are frequently associated with limited persistent success over the time rendering bariatric surgery one of the more promising therapy for “super-obese” patients. Laparoscopic sleeve gastrectomy (LSG) is a purely “restrictive” operation applied in patients with body mass index (BMI) <50 kg/m2. Although early enthusiastic results on weight loss after LSG have been published, the underlying mechanism(s) of weight loss is still unknown and it could involve pos-LSG alterations of gastrointestinal hormones. Nowadays the number of overweight and obese people in the world are roughly 2 billion in 2013. The lifestyle of the modern civilization facilitates diffusion of fast feeding and, consequently, high energy intake. At the same time hedonic hunger may powerfully stimulate food intake in an environment where highly palatable foods are omnipresent and contribute to the diffusion of overweight and obesity. Therefore, understanding the physiological mechanisms underlying these eating behaviors may help to contrast it. The anorexigenic action of PYY is preserved in obese patients, although the circulating level of the peptide is low during fast and postprandial phase. obese individuals actually display low circulating ghrelin levels that could suggest greater sensitivity to the hormone. A pathogenic role for ghrelin has been suggested in individuals with genomic obesity as Prader–Willi syndrome, where hyperghrelinism precedes the development of obesity. On the contrary, only few studies have investigated postprandial GLP-1 and PYY in PWS, besides reporting conflicting results. Since the critical rule of gastrointestinal hormones in homeostatic and hedonic regulation of food intake and in the physiophatology of obesity we decided to study dynamically their secretion in association with eating behavior involved with the development of obesity. Particularly we conducted three studies: Study 1: evaluation of post-prandial anorexigenic gut peptide, appetite and glucometabolic responses at different eating rates in obese patients undergoing laparoscopic sleeve gastrectomy. Aim of this study was to determine whether eating the same meal at different rates (fast vs. slow feeding) evokes different post-prandial anorexigenic gut peptide responses in ten obese patients undergoing LSG. Circulating levels of GLP-1, PYY, glucose, insulin and triglycerides were measured before and 3 months after LSG. Visual analog scales were used to evaluate the subjective feelings of hunger and satiety. Irrespective of the operative state, either fast or slow feeding did not stimulate GLP-1 release (vs. 0 min); plasma levels of PYY were increased (vs. 0 min) by fast and slow feeding only after LSG. There were no differences in post-prandial levels of GLP-1 when comparing fast to slow feeding or pre- to post-operative state. Plasma levels of PYY after fast or slow feeding were higher in post- than preoperative state, with no differences when comparing PYY release after fast and slow feeding. Hunger and satiety were decreased and increased, respectively, (vs. 0 min) by food intake. Fast feeding evoked a higher satiety than slow feeding in either preor post-operative state, with no differences in hunger. In pre- or post-operative state there were similar responses for hunger and satiety after food intake. Finally, LSG improved insulin resistance after either fast or slow feeding. These (negative) findings would suggest a negligible contribution of the anorexigenic gut peptide responses in LSG-induced weight loss. Study 2: anticipatory and consummatory effects of (hedonic) chocolate intake on PYY, GLP-1, ghrelin and endocannabinoid secretion in obese adults previously satiated. To explore the role of some gastrointestinal orexigenic and anorexigenic peptides and endocannabinoids (and some related congeners) in chocolate consumption, we measured changes in circulating levels of ghrelin, glucagon-like peptide 1 (GLP-1), peptide YY (PYY), anandamide (AEA), 2-AG, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) in 10 satiated severely obese subjects after consumption of chocolate and, on a separate day, of a non-palatable isocaloric food with the same bromatologic composition. Evaluation of hunger and satiety was also performed by visual analogic scale. The anticipatory phase and the consumption of food for pleasure were associated with increased circulating levels of ghrelin, AEA, 2-AG, and OEA. In contrast, the levels of GLP-1, PYY, and PEA did not differ before and after the exposure/ingestion of either chocolate or non-palatable foods. Hunger and satiety were higher and lower, respectively, in the hedonic session than in the non-palatable one. When motivation to eat is generated by exposure to, and consumption of, chocolate a peripheral activation of specific endogenous rewarding chemical signals, including ghrelin, AEA, and 2-AG, is observed in obese subjects. Although preliminary, these findings predict the effectiveness of ghrelin and endocannabinoid antagonists in the treatment of obesity. Study 3: evaluation of post-prandial anorexigenic gut peptide, appetite and glucometaboplic responses at different eatin rates in adult PWS patients The effect of eating rate on the release of anorexigenic gut peptides in Prader-Willi syndrome (PWS), a neurogenetic disorder clinically characterized by hyperphagia and excessive obesity, has not been investigated so far.Postprandial PYY and GLP-1 levels to fast (5 min) and slow (30 min) ice cream consumption were measured in PWS adult patients and age-matched patients with simple obesity and normal-weighted subjects. Visual analog scales (VASs) were used to evaluate the subjective feelings of hunger and satiety. Fast ice cream consumption stimulated GLP-1 release in normal subjects, a greater increase being observed with slow feeding. Fast or slow feeding did not change circulating levels of GLP-1 in obese patients, while, unexpectedly, fast feeding (but not slow feeding) stimulated GLP-1 release in PWS patients. Plasma PYY concentrations increased in all groups, irrespective of the eating rate. Slow feeding was more effective in stimulating PYY release in normal subjects, while fast feeding was more effective in PWS patients. Slow feeding evoked a lower hunger and higher satiety compared with fast feeding in normal subjects, this finding being not evident in obese patients. Unexpectedly, fast feeding evoked a lower hunger and higher satiety in PWS patients in comparison with slow feeding. Fast feeding leads to higher concentrations of anorexigenic gut peptides and favours satiety in PWS adult patients, this pattern being not evident in age-matched patients with simple obesity, thus suggesting the existence of a different pathophysiological substrate in these two clinical conditions.

This thesis deals with design and implementation of a versatile control unit, which is primarily designed for control of solar collectors. It describes the various stages of design. First, the system of solar collectors is introduced. Its input-output parts are analyzed and the requirements are determined for the control unit. The characteristics of temperature sensors are examined, the work deals with continuous variable speed circulators. The following part is dedicated to hardware and software implementation of the objectives of the work. The boards were designed in Eagle design environment, the firmware was written in C programming language. In conclusion, the evaluation of the results of the proposed system and possible ways of its further development are discussed. The versatile control unit can be deployed in practice realistically.

Modern arc welding machines utilize controllable high frequency DC/DC power supply with high dynamic and steady state current regulation performance. In the design robustness, small size and low weight, low complexity, and high efficiency are the defining criteria. The most suitable approach for a 5 kW arc welding machine power supply application is the high frequency Full-Bridge Phase-Shifted Zero Voltage Switching (FB-PS-ZVS) DC/DC converter with an isolation transformer. This converter not only gives the advantage of zero voltage switching for a wide load current range, it also provides reduced Electromagnetic Interference (EMI) and reduced component stress compared to standard PWM converters. In this thesis a FB-PS-ZVS DC/DC converter with 5 kW power rating is designed for modern arc welding machine applications. IGBTs are utilized at 50 kHz switching frequency for high efficiency and control bandwidth. The output current of the DC/DC converter is controlled via a Digital Signal Processor (DSP) control platform. The performance of the designed DC/DC converter is evaluated via the computer simulations and the experimental study of the constructed prototype.

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

The study designs and constructs a three-phase to three-phase direct AC&ndash
AC matrix converter based surface mounted permanent magnet synchronous motor (PMSM) drive system. First, the matrix converter topologies are analyzed and the state-space equations describing the system have been derived in terms of the input and output variables. After that, matrix converter commutation and modulation methods are investigated. A four-step commutation technique based on output current direction provides safe commutation between the matrix converter switches. Then, the matrix converter is simulated for both the open-loop and the closed-loop control. For the closed-loop control, a current regulator (PI controller) controls the output currents and their phase angles. Advanced pulse width modulation and control techniques, such as space vector pulse width modulation and field oriented control, have been used for the closed-loop control of the system. Next, a model of diode-rectified two-level voltage source inverter is developed for simulations. A comparative study of indirect space vector modulated direct matrix converter and space vector modulated diode-rectified two-level voltage source inverter is given in terms of input/output waveforms to verify that the matrix converter fulfills the two-level voltage source inverter operation. Following the verification of matrix converter operation comparing with the diode-rectified two-level voltage source inverter, the simulation model of permanent magnet motor drive system is implemented. Also, a direct matrix converter prototype is constructed for experimental verifications of the results. As a first step in experimental works, filter types are investigated and a three-phase input filter is constructed to reduce the harmonic pollution. Then, direct matrix converter power circuitry and gate-driver circuitry are designed and constructed. To control the matrix switches, the control algorithm is implemented using a DSP and a FPGA. This digital control system measures the output currents and the input voltages with the aid of sensors and controls the matrix converter switches to produce the required PWM pattern to synthesize the reference input current and output voltage vectors, as well. Finally, the simulation results are tested and supported by laboratory experiments involving both an R-L load and a permanent magnet synchronous motor load. During the tests, the line-to-line supply voltage is set to 26 V peak value and a 400 V/3.5 kW surface mounted permanent magnet motor is used.

Cryocoolers are mechanical devices that produce cold at temperatures below -150oC. Work required to produce the cold is supplied by a Pressure Wave Generator (PWG). This thesis is concerned with the development and analysis of Stirling type pulse tube cooler (PTC) systems of low and medium capacities along with performance improvement of PWG and design of a novel oil free and low maintenance helium recondensation system based on Stirling type pulse tube coolers and Joule-Thomson expansion. Pressure Wave Generator is a crucial part of pulse tube cooler system. Performance of PWG depends on many parameters like the seal gap, piston diameter etc. E ects of these parameters have been studied in detail. Analysis was done to arrive at optimal values and a methodology is suggested to improve performance. The analysis was applied extensively to indigenously developed Pressure Wave Generator (PWG-1). A signi cant improvement, reduced mechanical losses and lowered input electric current, has been found by application of the suggested methodology. The development of pulse tube coolers of low and medium capacities was studied. In low capacity coolers, the available PV power is low; thus, requiring an ultra e cient design. In medium capacity coolers, the available PV power can pose problems like acoustic matching, streaming etc. A low capacity pulse tube cryocooler capable of 0.5 W at 80 K and acoustically matched to indigenous Pressure Wave Generator PWG-1 was designed, fabricated and tested. Inertance tube con guration which plays signi cant role in low input power coolers has been analyzed. A no load temperature of 74 K was achieved with input power of 59 W; corresponding to a cooling power of 0.22 W at 80 K. The amplitude of mass ow passing into the pulse tube cooler has been measured by using a hot wire anemometer calibrated in oscillatory ow condition. A medium capacity pulse tube cryocooler capable of 10 W at 80 K was designed and developed. It requires an estimated input PV power of 375 W. On the other hand, the only available large PWG for this work, PWG-2, has a rated PV power output of 900 W at the rated piston stroke, thus posing the problem of acoustic matching. Improvement of acoustic matching between the PTC and PWG was studied by varying lling pressure, inertance tube con guration, and by using a long transfer line. A no load temperature of 72 K corresponding to 1 W at 80 K was achieved. It is concluded that steps to improve acoustic matching are useful only to a limited extent and acoustic matching has to be ensured at the design stage itself. A medium capacity two stage pulse tube cooler capable of reaching 25 K is described. Analytical proof has been given to shown that a two stage pulse tube cooler is thermo-dynamically less e cient than a single stage pulse tube cooler. But a two stage PTC is required to reduce regenerator losses. A no load temperature of 40.4 K was achieved at 24.1 bar lling pressure with an input power of 750 W. The e ects of pulse tube volume and regenerator con guration were experimentally investigated. The results point out the interplay of pressure drop and regenerator ine ectiveness losses. It is concluded that high mass ow from the PWG-2 is the reason for ine ectiveness of regenerator. A twin cooler design to reduce the mass ow of PWG-2 reaching the coldest stage has been proposed. A novel oil free and low maintenance helium recondensation system has been designed with a liquefaction rate of 17.86 liters per day corresponding to a cooling power of 0.53 W at 4.2 K. The system reaches liquid helium temperature by precooling helium gas in tube-in-tube heat exchangers, assisted by Stirling type pulse tube coolers operating at 80 / 25 K and two stage JT expansion. The cooling powers required of the pulse tube coolers are 14.22 at 80 K and 1.6 W at 25 K respectively. The twin cooler is capable of meeting these cooling power requirements. Design and development of tube-in-tube heat exchangers and JT valves are discussed.

碩士
國立海洋大學
水產生物技術研究所
86
The Bacillus licheniformis PWD-1 keratinase gene was subcloned in pET-20b(+), an transfer it into several E. coil strains. The transformants were fatal. A recombinant keratinase was constructed with a thioredoxin extended on its N-terminus by genetic engineering. However, the ftision protein lost keratinase activity. This suggests that the keratinase activity could be a toxic to the E. coli. 　　The keratinase shares a high similarity (98.95%) with the subtilisin Carlsberg, whose three-dimantional structure has been resolved. According to the computer simulation, keratinase was .rationally modified by site-directed mutagenesis. A imitant with double mutation sites, Gly 61/Cys 61 and Ser 98/Cys 98, was constructed to improve keratinase thermostability by introducing a disulfide bond. 　　Both the wild type and mutant enzymes exhibited the same optimal temperature, 50. However, the mutant enzymes displayed higher enzyme activity at temperature (40-90 ℃) and pH (6-10) than the wild .type. 　　In thermostability study, a 2-3 ℃ improvement was observed in the mutant, and the improvement is lost after DTT treatment. It suggested that Cys 61 and Cys 98 successfilUy formed a disulfide bond. In thermoinactivation kinetic study, the Ea of the wild type and mutated keratinase were 57 and 100 kcal/mol, respectively. The half-life of the mutant was over two times longer than wild type keratinase. It concludes, we have demonstrated the feasibility of protein engineering to increase the heat stability and improve the biochemical properties of keratinase for industrial applications.