Rozprawy doktorskie na temat „Optimization of protease”

Thesis (MSc)--Stellenbosch University, 2012.<br>ENGLISH ABSTRACT: This study describes: a) Characterization of traditional methodologies and testing methods used to purify and quantify trypsin and α-chymotrypsin b) Re-engineering / development of a new method for purifying trypsin and α-chymotrypsin that delivered higher product yields and improved control exercised over the process by investigating: i. Extraction methods ii. Centrifugation iii. Ultrafiltration iv. Chymotrypsinogen and trypsin crystallization v. Column chromatography vi. Investigation into different raw material sources for pancreatic enzyme production c) Development of kinetic and ELISA testing methodologies for in-process QC analysis.<br>AFRIKAANSE OPSOMMING: Hierdie Studie beskryf: a) Karakterisering van die ou prosessering metodes en toets metodes wat gebruik word om Tripsien en Alpha-chimotripsien te suiwer en te kwantifiseer. b) Herontwerp / ontwikkeling van 'n nuwe metode vir die suiwering Tripsien en Chimotripsien wat „n hoër opbrengs lewer en meer kontrole oor die proses uit oefen deur ondersoek in te stel na: i. Ekstraksie- metodes ii. Sentrifugering iii. Ultrafiltrasie iv. Chymotripsienogeen - en tripsien kristallisasie v. Kolom chromatografie vi. Ondersoek na verskillende rou materiaal bronne vir die produksie van pankreas ensieme. c) Die ontwikkeling van kinetiese- en ELISA toets metodes vir die in-proses kwaliteitkontrole.

Proteases ácidas pertencem a um importante grupo de enzimas industriais produzidas por fungos filamentosos, com aplicações na indústria de alimentos, de couro, farmacêutica e de cosméticos. O objetivo principal deste trabalho foi avaliar a produção de proteases ácidas extracelulares de fungos filamentosos isolados do solo do cerrado do centro-oeste brasileiro. Inicialmente, foi realizada uma triagem para avaliar a capacidade de 17 linhagens de fungos quanto à produção de protease em meio de cultura contendo Agar-leite. O fungo Aspergillus foetidus foi selecionado como melhor produtor de protease ácida extracelular. Visando à otimização da produção de proteases pelo fungo selecionado, avaliou-se a influência de diversos fatores no cultivo (pH, temperatura, agitação e diferentes fontes de nitrogênio e carbono). Após essa etapa, um planejamento experimental estatístico foi realizado com as variáveis independentes temperatura, pH inicial do meio e fonte de carbono e nitrogênio. A produção máxima de protease foi encontrada (63,7 U/mL) nas condições: pH inicial do meio igual a 7,0 a 28 ºC, 150 rpm em peptona 2% (p/v). Os estudos em biorreator demonstraram produção de protease nas condições de agitação e aeração iguais à 300 rpm e 1,0 vvm, após 120 h de cultivo. Os ensaios com diferentes temperaturas para a estimativa dos parâmetros termodinâmicos demonstraram que a protease ácida produzida pelo fungo é altamente estável apresentando máxima atividade em pH 5,0 e temperatura ótima igual a 55ºC. E, finalmente, para a purificação da enzima foi realizada cromatografia de gel-filtração. A enzima apresentou massa molecular de 50,6 kDa, e a análise do zimograma confirmou a atividade proteolítica. Além disso, a protease purificada foi inibida pelo composto pepstatina, indicando uma característica de protease ácida. Esses resultados obtidos demonstram um fungo filamentoso produtor de uma nova protease ácida com potencial aplicação para indústria farmacêutica e de cosméticos.<br>The acid proteases belong to the most important group of industrial enzymes produced by filamentous fungi, with applications in the food, leather, pharmaceutical and cosmetics industries. This study aimed the evaluation of extracellular acid proteases production from filamentous fungi isolated from different samples of the midwestern Brazil cerrado. Initially, a screening was performed to assess the ability of the 17 strains of yeast for production of protease-agar medium containing milk culture. The Aspergillus foetidus was selected as the best producer. Aimed at optimizing the production of proteases by the selected fungus, first evaluated the influence of various factors on the cultivation (pH, temperatura, agitation and different sources of nitrogen and carbon). After this step, a statistical experimental design was carried out with the independent variables temperatura, initial pH of the medium and source of carbon and nitrogen. The best conditions for protease production were (63.7 U / mL): initial pH values greater than 7.0, at 28 °C, 150 rpm peptone 2% (w/v). Aiming future production of this protease in industrial scale, studies have shown better in bioreactor protease production under the conditions of agitation and aeration equal to 300 rpm and 1.0 vvm, after 120 h of cultive. The tests at different temperaturas to estimate the thermodynamic parameters showed that the acid protease produced by the fungus is highly stable with maximum activity at pH 5.0 and optimum temperatura of 55 °C. And finally, for the purification of the enzyme were performed gel-filtration chromatography. The enzyme had a molecular mass of 50.6 kDa, and the analysis of the zymogram showed a proteolytic band. Furthermore, the purified protease was inhibited by pepstatin compound, indicating a feature of acid protease. These results demonstrate a new filamentous fungus producing acid protease with potential application to pharmaceuticals and cosmetics.

Made available in DSpace on 2017-07-21T18:53:04Z (GMT). No. of bitstreams: 1 CRISTIANE BOURSCHEID.pdf: 1448163 bytes, checksum: 21f79c9c1335f39830bfef99ee3f5f19 (MD5) Previous issue date: 2015-03-11<br>The chicken slaughterhouse generate co-products during slaughter, for example, feathers, bones, blood and guts, usually intended for animal feed with low added value. In bone part of the meat remains bound even after deboning and is a good source of substrate for the enzymatic hydrolysis. The protein hydrolysates may be applied as a nutritional supplement in foods. Given this context, the objective was to optimize the enzymatic hydrolysis process for obtaining protein hydrolyzate coproduct of chicken bones (Gallus gallus domesticus) and the addition of the hydrolyzate as a protein supplement in burger. The co-product selected to carry out the work was thigh and drumstick bone from the chicken bones in a slaughterhouse. The study was conducted in two stages. In the first one a central composite design (CCRD), a total of 17 tests was adopted to evaluate the influence of temperature, enzyme: substrate ratio and time on the degree of hydrolysis. Following the statistical optimization was performed to obtain the best conditions of enzymatic hydrolysis of co-product of chicken bones. The co-product of chicken bones and the optimized protein hydrolyzate were characterized in terms of chemical composition, total amino acids and pattern. In the second step, the hydrolyzate was added as a protein supplement in poultry burger, where two formulations were prepared, Hamburger control (no addition of protein hydrolyzate) and hamburger with hydrolyzed (with addition of 8% protein hydrolyzate). The burgers were characterized in terms of physical-chemical, microbiological and sensory analysis. The optimum conditions for the enzymatic hydrolysis were temperature (T) of 50 ° C, enzyme: substrate ratio (E:S) from 4.96% to time (t) of 110.16 minutes under these conditions the degree of hydrolysis was 24, 0.22 ± 21%. The protein hydrolyzate has the potential to supplementation in food, it is good source of essential amino acids, meeting the recommendations established by FAO / WHO, except for leucine, phenylalanine, and valine, and had a higher concentration of protein fractions from 14.437 kDa and 3,496 kDa. The results obtained for microbiological analysis and physical-chemical analysis of the burger control and burger with hydrolyzed, are in accordance with the respective standards set by law. The burger with hydrolyzate showed 1.02% more protein than the burger control, giving a protein supplementation to the product developed. In the sensorial analysis for flavor attribute evaluators preferred the burger with protein hydrolyzate. The burger with protein hydrolyzate and the burger control achieved 84.2% and 81.8% of general acceptance, respectively. The intention to buy for the burger with added protein hydrolyzate was 76.4% and for the burger control was 67%. In this light, the protein hydrolyzate obtained from chicken bones of the co-product is an alternative to supplementation in foods, as well as add value to the co-product of chicken dessosa and increase the competitiveness of slaughterhouse.<br>Os frigoríficos de frango geram coprodutos durante o abate, por exemplo, penas, ossos, sangue e vísceras, geralmente, destinados para ração animal com baixo valor agregado. Uma parte de carne permanece aderida aos ossos, mesmo após a desossa, sendo uma boa fonte de substrato para a hidrólise enzimática. Os hidrolisados proteicos podem ser aplicados como suplemento nutricional em alimentos. Diante desse contexto, o objetivo do trabalho foi otimizar o processo de hidrólise enzimática para obtenção de hidrolisado proteico de coproduto da desossa de frango (Gallus gallus domesticus) e a adição do hidrolisado como suplemento proteico em hambúrguer. O coproduto selecionado para realização do trabalho foi osso de coxa e sobrecoxa proveniente da desossa de frango em frigorífico. O trabalho foi realizado em duas etapas. Na primeira um delineamento composto central rotacional (DCCR), totalizando 17 ensaios foi adotado para avaliar a influência da temperatura, relação enzima:substrato e tempo sobre o grau de hidrólise. Na sequência, foi realizada a otimização estatística para obter as melhores condições da hidrólise enzimática. O coproduto da desossa de frango e o hidrolisado proteico otimizado foram caracterizados em termos de composição centesimal, aminoácidos totais e perfil eletroforético. Na segunda etapa, o hidrolisado foi adicionado como suplemento proteico em hambúrguer de frango. Foram elaboradas duas formulações, hambúrguer controle (sem adição de hidrolisado proteico) e hambúrguer com hidrolisado (com adição de 8% de hidrolisado proteico). Os hambúrgueres foram caracterizados em termos de análises físico-químicas, análises microbiológicas e análise sensorial. As condições ótimas para a hidrólise enzimática foram temperatura (T) de 50ºC, relação enzima:substrato (E:S) de 4,96% e tempo (t) de 110,16 minutos, nessas condições o grau de hidrólise foi de 24,21% ±0,22. O hidrolisado proteico apresentou potencial para suplementação em alimentos, pois é boa fonte de aminoácidos essenciais, atendendo as recomendações estabelecidas pela FAO/WHO, exceto para leucina, fenilalanina e valina, bem como apresentou maior concentração de frações proteicas entre 14,437 kDa e 3,496 kDa. Os resultados obtidos para a análise microbiológica e análises físico-químicas do hambúrguer controle e do hambúrguer com hidrolisado, estão de acordo com os respectivos padrões estabelecidos pela legislação vigente. O hambúrguer com hidrolisado apresentou 6% mais proteína do que o hambúrguer controle, conferindo uma suplementação proteica ao produto desenvolvido. Em relação à análise sensorial para o atributo sabor, os avaliadores preferiram o hambúrguer com hidrolisado proteico. O hambúrguer com hidrolisado proteico e o hambúrguer controle obtiveram 84,2% e 81,8% de aceitação geral, respectivamente. A intenção de compra para o hambúrguer com adição de hidrolisado proteico foi de 76,4% e para o hambúrguer controle foi de 67%. Diante do exposto, o hidrolisado proteico obtido a partir de coproduto da desossa de frango é uma alternativa para a suplementação em alimentos, além de agregar valor ao coproduto da dessosa de frango e aumentar a competitividade dos frigoríficos.

Developing microbial systems capable of converting low cost lipids into value added products depends on the ability to acquire substrates from the growth media. Saccharomyces cerevisiae can acquire free fatty acids from the growth media and a portion of these lipids can be converted into new lipid products. However, they cannot acquire complex lipids from the growth media unless a nonspecific lipase is included. To circumvent lipase addition, we are genetically engineering S. cerevisiae to secrete a lipase into the growth media. We selected the LIP2 gene from Yarrowia lipolytica, which encodes a nonspecific lipase. Several modifications were made to the LIP2 gene to improve processing. Results identified strains secreting the most lipase. From these results, high producing strains were inserted into an oil inducible vector. Halo assays confirmed lipase secretion, while measuring the fatty acid composition confirmed triacylglycerol breakdown, and yeast uptake of the free fatty acids released.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Physics, May, 2020<br>Cataloged from student-submitted PDF version of thesis.<br>Includes bibliographical references (pages 315-348).<br>The quantitative composition of proteomes results from biophysical and biochemical selective pressures acting under system-level resource allocation constraints. The nature and strength of these evolutionary driving forces remain obscure. Through the development of analytical tools and precision measurement platforms spanning biological scales, we found evidence of optimization in bacterial gene expression programs. We compared protein synthesis rates across distant lineages and found tight conservation of in-pathway enzyme expression stoichiometry, suggesting generic selective pressures on expression setpoints. Beyond conservation, we used high-resolution transcriptomics to identify numerous examples of stoichiometry preserving cis-elements compensation in pathway operons. Genome-wide mapping of transcription termination sites also led to the discovery of a phylogenetically widespread mode of bacterial gene expression, 'runaway transcription', whereby RNA polymerases are functionally uncoupled from pioneering ribosomes on mRNAs. To delineate biophysical rationales underlying these pressures, we formulated a parsimonious ribosome allocation model capturing the trade-off between reaction flux and protein production cost. The model correctly predicts the expression hierarchy of key translation factors. We then directly measured the quantitative relationship between expression and fitness for specific translation factors in the Gram-positive species Bacillus subtilis. These precision measurements confirmed that endogenous expression maximizes growth rate. Idiosyncratic transcriptional changes in regulons were however observed away from endogenous expression. The resulting physiological burdens sharpened the fitness landscapes. Spurious system-level responses to targeted expression perturbations, called 'regulatory entrenchment', thus exacerbate the requirement for precisely set expression stoichiometry.<br>by Jean-Benoît Lalanne.<br>Ph. D.<br>Ph.D. Massachusetts Institute of Technology, Department of Physics

A demanda por antioxidantes naturais vem aumentando devido à toxicidade de alguns antioxidantes sintéticos. Estudos vêm identificando antioxidantes de origem natural, como a proteína da soja, que é capaz de contribuir na melhoria de propriedades funcionais e biológicas de alimentos. A hidrólise enzimática da proteína de soja aumenta sua atividade antioxidante, assim como a capacidade emulsificante e a capacidade de formação de espuma. O objetivo deste trabalho foi o estudo da hidrólise da proteína de soja através de uma protease produzida por Chryseobacterium sp., a verificação da capacidade antioxidante e aplicação do hidrolisado em diferentes tipos de carnes para evitar a oxidação lipídica. A eficácia da hidrólise foi determinada através da proteína solúvel utilizando o método de Folin enquanto que a atividade antioxidante foi avaliada pelos métodos referentes à captura do radical DPPH e ABTS. Os hidrolisados foram adicionados em carne de porco e peixe e foi verificada a inibição da oxidação lipídica. A influência de três parâmetros (temperatura, pH, relação enzima/substrato) na hidrólise foi estudada através um experimento fatorial 23 . Como respostas foram avaliadas a atividade antioxidante (DPPH e ABTS), atividade quelante de ferro, proteína solúvel, capacidade de formação de espuma e capacidade emulsificante. Observou-se um aumento na concentração de proteína solúvel em função do tempo, sendo que os hidrolisados foram capazes de inibir tanto o radical DPPH quanto o ABTS. Os hidrolisados inibiram em parte a oxidação lipídica em carne suína e peixe. Ainda foi possível concluir que dependendo da finalidade para que se deseja o hidrolisado, diferentes condições devem ser utilizadas. Os resultados demonstram uma potencial aplicação da protease microbiana para gerar hidrolisados antioxidantes da proteína de soja.<br>The demand for natural antioxidants has been increasing due to the toxicity of some synthetic antioxidants. Studies have identified naturally occurring antioxidants, such as soy protein, which can contribute to improve functional and biological properties of food. Enzymatic hydrolysis of soy protein increases its antioxidant activity, as well as emulsifying capacity and foaming capacity. The purpose of this work was to study the hydrolysis of soy protein, verifying the antioxidant capacity, application of the hydrolysate in different types of meat and optimization of hydrolysis. The efficiency of hydrolysis was determined by the soluble protein by the method of Folin while the antioxidant activity was evaluated by the methods related to the capture of the radical DPPH and ABTS. The hydrolysates were added to pork and fish and the extent of lipid oxidation was determined by TBARS. In optimizing of the hydrolysis three parameters were varied (T, pH, enzyme substrate), it was applied to a surface response methodology for conducting trials using a 23 factorial experiment. As answers were evaluated antioxidant activity (DPPH and ABTS), iron chelating activity, Lowry, foaming capacity and emulsifying capacity. There was an increase in soluble protein concentration versus time, and the hydrolysates were able to inhibit both the ABTS and the DPPH radical. The hydrolysates were able of inhibit lipid oxidation in pork and fish. Was still possible to conclude that depending on the finality that will be given to hydrolysates different treatment conditions should be used. The results demonstrate a significant potential for application of microbial protease to generate antioxidants of hydrolyzed soy protein.

Pseudomonas fragi ATCC 4973 was grown in trypticase soy broth (TSB), on a trypticase soy broth + 1.5% agar (TSA) surface, and in a defined citrate broth. The citrate broth contained glutamine as the sole nitrogen source, Pseudomonas fragi grown in TSB started proteinase production at 24 h, during the late logarithmic early stationary growth phase. Pseudomonas fragi grown on TSA surfaces initiated proteinase production at 4 h, 20 hours earlier than in liquid medium. Electron micrographs of P. fragi grown on TSA revealed extracellular vesicles ca. 20 nm in diameter "blebbing" off the surface of the cells. These vesicles were absent from the surface of P. fragi cells grown in TSB, although vesicles could be isolated from the culture supernatant. Isolated extracellular vesicles were ca. 20 nm in diameter and contained a proteinase similar to that found in the supernatant. Electrophoretic analysis showed the vesicles and outer cell membrane of P. fragi to share similarities in their composition. Use of the centroid search technique of Aishima and Nakai, showed the optimum cultural conditions for proteinase production by P. fragi, in defined citrate broth to be: incubation temperature, 12.5 C; incubation time, 38 h; initial pH, 6.8; organic nitrogen concentration, 314 mmole nitrogen/L (glutamine); a gas mixture containing 16.4% oxygen flowing over the medium (7.42 ppm dissolved oxygen). Oxygen was the major factor influencing proteinase production by P. fragi. A comparison of optimization techniques suitable for microbiological experiments showed that the centroid search technique of Aishima and Nakai, the modified super simplex of Nakai and Kaneko and the simplex technique of Morgan and Deming all required similar time and experiment numbers to obtain the optimum point.<br>Land and Food Systems, Faculty of<br>Graduate

In this study, the aim was to investigate the effects of pH on therapeutically important protein, recombinant human growth hormone (rhGH), production by Pichia pastoris considering the expression levels of regulatory genes. In this frame, firstly the host microorganism was selected between two different methanol utilization phenotypes of P. pastoris, Mut+ and MutS on media containing glycerol/methanol or sorbitol/methanol. The highest rhGH production, 120 g L-1, and hGH gene expression, 9.84x109 copies mg-1 CDW, were achieved in the medium containing 30 g L-1 sorbitol and 1% (v/v) methanol by P. pastoris hGH-Mut+ strain. Thereafter, effects of pH on rhGH production and stability were investigated in laboratory scale bioreactors. RhGH was more stable at pH 5.0. Throughout the production, it is seen that medium of pH decreased. Thereafter, effects of pH on rhGH were investigated in pH controlled pilot-scale bioreactor. In addition to rhGH concentration, AOX intracellular enzyme activity, extracellular proteases concentrations<br>expression levels of hGH, AOX, pep4, prb1 and prc1 genes were determined. The highest cell concentration was obtained as 53 g L-1 at pH 6.0 but hGH concentration was found as 24 mg L-1 at t=24 h. The highest rhGH concentration was obtained as 271 g L-1 with 42 g L-1 cell density at pH 5.0 in medium containing sorbitol at t=24 h. At this condition, the overall product and cell yield on total substrate were found as 2.08 mg g-1 and 0.15 g g-1. Furthermore, the highest expression levels of hGH and AOX were attained at pH 5.0. Moreover, by keeping pH at 5.0, expression levels of three types of vacuolar proteases were minimized.

The aim of this thesis is to investigate the signatures of evolutionary optimization in biological systems, such as in proteins, human behaviours and transport tissues in vascular plants (xylems), by means of the Pareto optimality analysis and the calculus of variations. In the first part of this thesis, we address multi-objective optimization problems with tradeoffs through the Pareto optimality analysis ( [132],[69]), according which the best tradeoff solutions correspond to the optimal species, enclosed onto low-dimensional geometrical polytopes, defined as Pareto optimal fronts, in the space of physical traits, called morphospace. Chapter 3 is devoted to the Pareto optimality analysis in the Escherichia coli proteome by projecting proteins onto the space of solubility and hydrophobicity. In chapter 4 we analyze the HCP dataset of cognitive and behavioral scores in 1206 humans, in order to identify any signature of Pareto optimization in the space of Delay Discounting Task (DDT), which measures the tendency for people to prefer smaller, immediate monetary rewards over larger, delayed rewards. The second part of this thesis is devoted to solving an optimization problem regarding xylems, which are the internal conduits in angiosperms that deliver water and other nutrients from roots to petioles in plants. Based on the optimization criteria of minimizing the energy dissipated in a fluid flow, we propose in chapter 5 a biophysical model with the goal of explaining the underlying physical mechanism that affects the structure of xylem conduits in vascular plants, which results in tapered xylem profiles [104, 105, 117, 164]. We address this optimization problem by formulating the model in the context of the calculus of variations. The results of these investigations, besides providing quantitative support to previous theories of natural selection, demonstrate how processes of optimization can be identified in different biological systems by applying statistical methods such as the Pareto optimality and the variational one, showing the relevance of employing these statistical approaches to various biological systems.<br>Lo scopo di questa tesi è quello di identificare le impronte che l’evoluzione ha avuto nei sistemi biologici, come ad esempio nelle proteine, nei comportamenti umani e nei tessuti trasportatori delle piante vascolari (xilemi), attraverso un’analisi di ottimizzazione di Pareto ed il calcolo delle variazioni. Nella prima parte della tesi, affrontiamo l’ottimizzazione di problemi multi-obiettivo con competizione, attraverso l’analisi di ottimizzazione di Pareto, in base alla quale le migliori soluzioni di compromesso corrispondono alle specie ottimali, le quali vengono racchiuse in politopi geometrici, definiti come fronti ottimali di Pareto, nello spazio dei tratti fisici. Il capitolo 3 è dedicato all’analisi dell’ottimizzazione di Pareto nel proteoma dell’Escherichia coli, proiettando le proteine nello spazio della solubilitá ed idrofobicitá. Nel capitolo 4 analizziamo il set di dati HCP cognitivi e comportamentali in 1206 umani, al fine di identificare qualsiasi traccia di ottimizzazione alla Pareto nello spazio del “Delay Discounting Task” (DDT), che misura la tendenza per le persone a preferire ritorni economici piú piccoli e immediati rispetto a ricompense di premi piú grandi e ritardati. La seconda parte di questa tesi è dedicata alla risoluzione di un problema di ottimizzazione riguardante gli xilemi, che sono i condotti interni degli angiospermi e forniscono con acqua ed altri nutrienti le piante, dalle radici ai piccioli. Basandosi sui criteri di ottimizzazione per minimizzare l’energia dissipata in un flusso di fluido, nel capitolo 5 proponiamo un modello biofisico con l’obiettivo di spiegare il meccanismo fisico sottostante che influenza la struttura di condotti dello xilema nelle piante vascolari, che si traducono in profili di xilema affusolati. Affrontiamo questo problema di ottimizzazione formulando il modello nel contesto del calcolo delle variazioni. I risultati di queste indagini, oltre a fornire supporto quantitativo sulle precedenti teorie sulla selezione naturale, dimostra come i processi dell’ottimizzazione possono essere identificati in diversi sistemi biologici applicando metodi statistici come l’ottimalitá di Pareto e il variazionale uno, mostrando la rilevanza di impiegare questi approcci statistici a vari sistemi biologici.

Aryl hydrocarbon receptor (AHR) is a sensor protein, activated by aromatic chemical species for transcriptionally regulating xenobiotic metabolizing enzymes. AHR is also known to be involved in a variety of pathogenesis such as cancer, diabetes mellitus, cirrhosis, asthma, etc. The AHR signaling induced by xenobiotics has been intensively studied whereas its physiological role in the absence of xenobiotics is poorly understood. Despite a number of ligands of AHR have been reported thus far, further applications are still hampered by the lack of specificity and/or the partially agonistic activity. Thus, a pure AHR antagonist is needed for deciphering the AHR cryptic as well as potential therapeutic agent. The Proteolysis Targeting Chimera (PROTAC) is a bi-functional small molecule containing a ligand and proteolysis inducer. PROTAC recruits the target protein to proteolysis machinery and elicits proteolysis. Thus far, a number of PROTAC have been prepared and demonstrated to effectively induce the degradation of targeted protein in cultured cells, validating PROTAC as a useful research tool. In the present study, PROTACs based on apigenin was prepared and demonstrated to induce the degradation of AHR, providing the proof of concept. To improve activity, a synthetic structure, CH-223191, was optimized for antagonistic activity by positional scanning identifying several AHR antagonists. PROTACs based on the optimal structure were prepared and assessed their biological activity. The products and synthetic scheme described hereby will be helpful for the further understanding on AHR biology as well as for developing therapeutic agents targeting AHR.

Nowadays the number of hip joints arthroplasty operations continues to increase because the elderly population is growing. Moreover, the global life expectancy is increasing and people adopt a more active way of life. For this reasons, the demand of implant revision operations is becoming more frequent. The operation procedure includes the surgical removal of the old implant and its substitution with a new one. Every time a new implant is inserted, it generates an alteration in the internal femur strain distribution, jeopardizing the remodeling process with the possibility of bone tissue loss. This is of major concern, particularly in the proximal Gruen zones, which are considered critical for implant stability and longevity. Today, different implant designs exist in the market; however there is not a clear understanding of which are the best implant design parameters to achieve mechanical optimal conditions. The aim of the study is to investigate the stress shielding effect generated by different implant design parameters on proximal femur, evaluating which ranges of those parameters lead to the most physiological conditions.

Le nombre de proteines membranaires dont la structure est connue est tres faible en regard du nombre de structures resolues, ceci en raison des difficultes a obtenir des cristaux de qualite suffisante pour les etudes par diffraction des rayons x. La finalite de ce travail est de comprendre l'implication du detergent, utilise pour solubiliser puis cristalliser les proteines membranaires, dans le processus de cristallisation et dans la qualite des cristaux obtenus. En particulier, on s'est interesse a l'organisation prise par le detergent dans les cristaux, et aux differentes interactions auxquelles il participe. Les structures du detergent dans les cristaux ont ete resolues par cristallographie des neutrons a basse resolution. Les micelles de detergent, pures ou complexees avec les proteines, ont elles aussi ete etudiees, en solution, par diffusion neutronique aux petits angles. Ces deux techniques sont utilisees en combinaison avec la methode de variation de contraste. Les structures formees par le detergent dans deux cristaux de porine (de rhodobacter capsulatus et de escherichia coli) de groupes d'espace differents ont ete modelisees, ainsi que les micelles de detergent presentes dans la solution de cristallisation de l'une de ces porines. On a essaye d'en tirer une analyse generale du role du detergent, dans l'optique d'optimiser la formation de cristaux de proteines membranaires de bonne qualite. En outre les interactions entre la membrane et la proteine, mises en lumiere par la localisation du detergent au niveau de la zone transmembranaire de la proteine, ont ete abordees.

Islet transplantation is an attractive method to achieve blood glucose homeostasis. However, β-cell function declines over time. Therefore, it is necessary to explore strategies to enhance the β-cell mass and function. Also, because there is a severe shortage of human cadaver tissue, alternative sources of insulin secreting tissue need to be examined. Neonatal porcine islet (NPI) tissue has emerged as an attractive alternative source of β-cells. The aim of this thesis was to optimize the culturing conditions of NPIs pre-transplantation so that the available tissue can be used as efficiently and economically as possible. The results from this study indicate that the treatment of NPI cultures with z-VAD-FMK, a pan caspase inhibitor and general protease inhibitor significantly enhances β-cell survival. Additionally, the optimum length of culturing NPIs pre-transplantation appears to be 3-5 days. Since widespread cell death stimulates immunogenic response, this treatment also has the potential benefit of reducing immunosuppression needs in the recipient.<br>Experimental Surgery

碩士<br>國立高雄海洋科技大學<br>水產食品科學研究所<br>97<br>To produce high protease and chitinase activities, extra addition of chitins and CMC to the medium was conducted on the cultivation of Bacillus subtilis YJ-1. When this strain was incubated in a complex medium containing 0.5% (w/v) commercial chitin at 37oC, 150 rpm for 3 days, the highest neutral (655.2 U/mL•min) and alkaline (638.2 U/mL•min) proteases activities were obtained. The chitinases activities were 438.04 U/mL•min and 4.22 U/mL•min measured by neocuproine and DMBA methods, respectively. The shrimp shell solution was subjected to the hydrolysis by the above crude selective enzymes at 50oC for 3-4 hr, the soluble proteins, peptides, total free amino acid and reducing sugar of shrimp hydrolysates increased significantly (p<0.05), compared with those before hydrolysis. This phenomenon suggested the degradation of proteins contained in shrimp shell solution occurred. This study also investigated the effects of glucose content and pH on the fermerntation by Lactobacillus acidophilus BCRC 17010, Bifidobacterium adolescentis BCRC 14608 or Lactobacillus casei BCRC 12272. It was found that addition of 2% glucose at neutra pH was benefit for the growth of Bifidobacterium adolescentis BCRC 14608 (10.68 log CFU/mL after 36 hr fermentation). This result indicated that the shrimp shell hydrolysates were a good medium for Bifidobacterium adolescentis BCRC 14608. The molecular mass of the degraded products by electrophoresis weas approximately 2.5 kDa and the degraded chitooligosaccharides were pentaacetylchitopentaose and N-acetylchitobiose to pentaacetylchitopentaose measured by TLC and HPLC, respectively. The antioxidation ability including trolox equivalent antioxidant capacity, reducing power and Fe2+ ion chelating ability of the hydrolysate were 4 mM, 1.5 nm and 80.42% respectively. These results suggested that the combination use of selective enzymes produced by B. subtilis YJ1 with B. adolescentis BCRC 14608 fermentation could process the shrimp shell into a fermented functional chitooligosaccharide beverages.