Thesis (Ph.D.)--Baylor University, 2008. / Includes bibliographical references (p. 100-109)

Thesis (Ph.D.)--Baylor University, 2008. / Includes bibliographical references (p. 121-133)

The creation of enzymes displaying desired substrate specificity is an important objective of enzyme engineering. To help achieve this goal, computational protein design (CPD) can be used to identify sequences that can fulfill interactions required to productively bind a desired substrate. Standard CPD protocols find optimal sequences in the context of a single state, for example an enzyme structure with a single substrate bound at its active site. However, many enzymes catalyze reactions requiring them to bind multiple substrates during successive steps of the catalytic cycle. The design of multi-substrate enzyme specificity requires the ability to evaluate sequences in the context of multiple substrate-bound states because mutations designed to enhance activity for one substrate may be detrimental to the binding of a second substrate. Additionally, many enzymes undergo conformational changes throughout their catalytic cycle and the equilibrium between these conformations can have an impact on their substrate specificity. In this thesis, I present the development and implementation of two multistate computational protein design methodologies for the redesign of multi-substrate enzyme specificity and the modulation of enzyme conformational equilibrium. Overall, our approaches open the door to the design of multi-substrate enzymes displaying tailored specificity for any biocatalytic application.

Made available in DSpace on 2016-06-02T20:18:09Z (GMT). No. of bitstreams: 1 1788.pdf: 1563222 bytes, checksum: d55e992347a7bcc9f276f927b6f940f4 (MD5) Previous issue date: 2008-05-07 / Financiadora de Estudos e Projetos / Esta pesquisa é o resultado de quatro anos dedicados ao doutorado. Apresenta caráter pioneiro em vários aspectos. Primeiro, este trabalho utilizou a metodologia de Branched de DNA (bDNA) para análise de expressão gênica em tecidos biológicos. Inicialmente, a técnica que era destinada para cultura de células, foi adaptada para homogenatos de tecido. Primeiramente, validamos a metodologia mediante o uso do protocolo QuantiGene® Reagent Systems Evaluation Program (Genospetra). Mediante este protocolo, vários ensaios experimentais foram realizados, e resultou na adequação metodológica que a empresa utilizou para padronizar os procedimentos para análise com homogenatos de tecidos animais. Atualmente, nosso protocolo faz parte do manual do Sistema de Reagentes QuantiGene Panomics® que está sendo utilizado pela própria empresa para análise da expressão gênica com homogenatos de tecido muscular. Segundo, tendo em vista a existência de poucos trabalhos na literatura que investiga o efeito do laser λ=514 nm durante a regeneração do músculo esquelético, decidimos realizar esta investigação para fins de desenvolvimento do laser de argônio de baixa intensidade para fins terapêuticos. Terceiro, consiste no primeiro trabalho que investiga três comprimentos de onda de laser de baixa intensidade sobre a expressão de genes durante o processo de regeneração muscular in vivo. Ao final desse estudo obtivemos resultados relevantes que deram origem a dois manuscritos submetidos em revistas internacionais, em anexo. Para apresentação desta tese, os resultados obtidos foram organizados em capítulos que correspondem aos dois artigos apresentados a seguir.

The purpose of this thesis is to investigate and test the usage of HTTP/2 in dynamic adaptive video streaming as well as to take a look into how it can be used to benefit prefetching algorithms used with branched video. With a series of experiments the performance gains of using HTTP/2 rather than the older standard HTTP/1.1 has been investigated. The results has shown no significant change to player quality and buffer occupancy when using HTTP/2, though our tests has shown in a slight decrease in overall playback quality when using HTTP/2. When using a linear prefetch of two fragments an average quality improvement of 4.59% has been shown, however, the result is inconclusive due to variations in average quality between different values for how many fragments to prefetch. Average buffer occupancy has shown promise with a maximum increase of 12.58%, when using linear prefetch with three fragments. The values for buffer occupancy gains are conclusive. Two implementations for non-linear prefetching has been made. The first one uses HTTP/2 server push to deliver fragments for prefetching and the second one uses client-side invoked HTTP requests to pull fragments from the server. Using HTTP/2 server push has shown in a decrease of 2.5% in average total load time while using client-side pulling has shown in a decrease of 34% in average total load time.

Nesse trabalho obtiveram-se membranas de poli(imida) ramifricada com tamanho de poro ajustável usando melamina como agente de ramificação e indutor da formação de poros. Estas poli(imidas) foram sulfonadas usando-se ácido sulfúrico concentrado eficazmente. Finalmente, obtivemos membranas de carbono molecular por pirólise sob atmosfera inerte das poli(imidas) ramificadas. Estas membranas apresentam canais micrométricos paralelos à superfície e uma estrutura assimétrica constituída de uma camada densa filtrante e uma camada com canais micrométricos paralelos à superfície altamente ordenados. A membrana apresentou boa estabilidade química frente ao ataque por radicais hidroxila gerados via reação de Fenton. / In this work was obtained branched poly(imides) with tuned pore size using melamine as branching and pore induction agent. The poly(imides) with were efficiently sulfonated with concentrated sulfuric acid. Finally, the molecular sieve carbon membranes were obtained by pyrolysis under inert atmosphere of the branched poly(imides). These membranes have an asymmetric structure with a dense filtering layer and a porous layer with highly ordered channels standing parallel to the surface. These membranes display good chemical stability toward hydroxyl radical attack produced by Fenton reaction.

INTRODUÇÃO: O destreinamento físico está relacionado com alterações moleculares associadas à perda de massa muscular, rápido acréscimo da massa adiposa, ganho de peso e resistência à insulina. Estudos apontam que a restrição calórica reduz a gordura corporal, contudo, associada com a inatividade física, altera o metabolismo proteico acelerando o catabolismo muscular. Nesse sentido, estudos com suplementação de aminoácidos essenciais, em especial a leucina, observam aumento na síntese proteica e redução da degradação proteica em situações de restrição ou recuperação nutricional. Dessa forma sugere-se que a restrição calórica associada à suplementação com leucina poderia atenuar os efeitos desencadeados pelo destreinamento físico. OBJETIVO: Investigar a influência da suplementação crônica de leucina na via de sinalização da síntese proteica e degradação proteica no tecido muscular a partir de parâmetros moleculares em ratos destreinados, submetidos à restrição calórica. MÉTODOS: Foram utilizados 64 ratos Sprague-Dawley machos e adultos, inicialmente distribuídos em 2 grupos: Controle (CON) (n = 16) representados pelos animais sedentários, e Treinamento (TREIN) (n = 48) que foram submetidos ao treinamento em esteira ergométrica durante oito semanas. Após esse período, os animais foram redistribuído em 6 grupos: Sedentário (SED), Treinamento (TREIN), Destreinamento (DT), Destreinamento + Leucina (LEU), Destreinamento + Restrição Calórica (DTRC) e Destreinamento + Restrição Calórica + Leucina (DTRC + LEU). Foram analisados massa corporal, consumo da ração, composição corporal, sensibilidade a insulina bem como os marcadores de inflamação (IL-6; IL-10; MCP-1; TNF-α; 1L-1α; PAI-1; leptina; adponectina) e parâmetros moleculares como genes e proteínas envolvidas na via de sinalização da síntese protéica (mTOR, P-4EBP1, P-s6K1 e eIF4E); degradação proteica (MAFBx e MURF) além de transportadores de aminoácidos (LAT-1 e SNAT 2 e CD98). ESTATÍSTICA: Os valores foram expressos em média e desvio padrão. As comparações entre os grupos após o período de destreinamento físico foram avaliadas por meio de análise de variância (ANOVA), seguida do teste de Tukey. Em todas as análises foi considerado nível de significância de 5%. A análise estatística foi realizada no software SPSS versão 17.0. RESULTADOS: Em relação à composição corporal, foi observada diferença estatisticamente significativa na gordura corporal e massa livre de gordura entre os grupos DTRC e DTRC+LEU, em relação aos demais grupos experimentais. Porém não houve diferença estatística entre o DTRC e DTRC+LEU. No entanto não foi observada diferença estatisticamente significativa quando avaliado a proteína da carcaça. Em relação aos parâmetros moleculares, não foi observada diferença estatisticamente significativa entre os grupos, quando avaliada a expressão de proteínas relacionadas com a via de sinalização de síntese proteica (mTOR, P-4EBP1, P-s6K1 e eIF4E) e transportadores de leucina (LAT- 1;SNAT-2;CD(98). Quanto avaliada a expressão gênica da via de degradação, foi observada uma menor expressão do gene MURF quando suplementado com leucina, porém sem diferença estatisticamente significativa. CONCLUSÃO: A restrição calórica associada com a suplementação com leucina foi efetiva na redução da gordura corporal, e aumento da massa livre de gordura, porém não houve diferença estatisticamente significante entre os dois grupos DTRC e DTRC+LEU, tampouco quando avaliada a proteína da carcaça desses animais. Dessa forma, pode-se concluir que a suplementação crônica com leucina não reverteu os efeitos desencadeados pelo destreinamento físico, e, além disso, não foi suficiente para alterar os parâmetros moleculares envolvidos na via de sinalização de síntese e degradação proteica desses animais. / INTRODUCTION: Physical detraining is related to molecular changes associated with loss of muscle mass, rapid increase in fat mass, weight gain and insulin resistance. Studies show that caloric restriction reduces body fat; however, associated with physical inactivity, it alters protein metabolism accelerating muscle catabolism. Accordingly, studies with supplementation of essential amino acids, particularly leucine, observed increase in protein synthesis and reduced protein degradation in situation of nutritional restriction or recovery. Thus, it is suggested that caloric restriction associated with leucine supplementation could attenuate the effects triggered by physical detraining. OBJECTIVE: To investigate the influence of chronic leucine supplementation in the signaling pathway of protein synthesis and degradation in muscle tissue from molecular parameters in detrained rats, subjected to caloric restriction. METHODS: Sixty-four adult male and female Sprague-Dawley rats were used, initially divided into 2 groups: Control (CON) (n = 16) represented by sedentary animals, and Trained (TRAIN) (n = 48) who underwent treadmill training for eight weeks. After this period, the animals were re-distributed into 6 groups: Sedentary (SED), Trained (TRAIN), Detrained (DT), Detrained + Leucine (LEU), Detrained + Caloric Restriction (DTRC) and Detrained + Caloric Restriction + Leucine (DTRC + LEU). Body mass, food consumption, body composition, insulin sensitivity were analyzed, as well as inflammation markers (IL-6; IL-10; MCP-1; TNF-α; 1L-1α; PAI-1; leptin; adiponectin) and molecular parameters, such as genes and proteins involved in signaling pathways of protein synthesis (mTOR, P-4EBP1, P-s6K1 and eIF4E); protein degradation (MAFBx and MURF) and also amino acid transporters (LAT-1, SNAT 2 and CD98). STATISTICAL ANALYSIS: Values were expressed as mean and standard deviation. Analysis of variance (ANOVA) was used for comparisons between groups after physical detraining, followed by Tukey\'s test. A 5% significance level was considered in all analyses. Statistical analysis was performed using SPSS software, version 17.0. RESULTS: In relation to body composition, a statistically significant difference was observed in body fat and fat free mass between groups DTRC and DTRC+LEU, compared with other experimental groups. However, there was no statistical difference between groups DTRC and DTRC+LEU. Nevertheless, no statistically significant difference was found when carcass protein was assessed. In relation to molecular parameters, no statistically significant difference was observed between groups, when protein expression related to the signaling pathway of protein synthesis (mTOR, P-4EBP1, P-s6K1 and eIF4E) and leucine transporters (LAT-1;SNAT-2;CD(98) was assessed. When gene expression of the degradation pathway was investigated, a lower expression of gene MURF was found with leucine supplementation; however, this was not statistically different. CONCLUSION: Caloric restriction associated with leucine supplementation was effective in reducing body fat, and increasing fat free mass; however, no statistically significant difference was found between groups DTRC and DTRC+LEU, nor when carcass protein of these animals was assessed. Therefore, it was concluded that chronic leucine supplementation did not reverse the effects triggered by physical detraining and, in addition, it was not sufficient to change the molecular parameters involved in the signaling pathway of protein synthesis and degradation of these animals.

The central nervous system (CNS) is susceptible to several disorders that can affect the structure or function of the brain or spinal cord, such as stroke and spinal cord injury (SCI). CNS disorders are complex, frequently causing failure of cognitive, motor and sensory functions. Unfortunately, there are only a few care alternatives for patients with CNS disorders, due to the limited capacity of the CNS to spontaneously regenerate; what expresses the need to develop innovative solutions, such as scaffolds that also could act as drug delivery systems to promote tissue and functional repairs in the CNS. To achieve this goal, three main projects were developed in this thesis. In the first project, a novel drug releasing duraplasty that can be applied as part of decompressive craniectomy (DC) was designed and tested. While DC can significantly reduce the risk of death, this procedure does not reverse the stroke damage. Thus, biosynthesized cellulose (BC) was used to produce a new duraplasty loaded with growth factors. The in vivo animal studies revealed that our duraplasty had excellent biocompatibility when implanted onto rodents’ brains. In the second project, BC tubes were prepared and nerve growth factor was incorporated into the tubes to be used as potential nerve guides to assist with the reconstitution of nerve tissues across SCI lesion. Physical and mechanical properties of the drug delivery systems produced were evaluated and compared to the neural native tissue. In addition, cell cultures demonstrated that growth factors released from both drug delivery systems were bioactive for over 7 days. In the third project, linear and 2-branched peptides were synthesized as potential bioactive molecules to improve tissue regeneration. These peptides, containing the RGDS sequence, were synthesized through Solid Phase Peptide Synthesis and characterized by mass spectrometry, high-performance liquid chromatography, and their conformational structures were analyzed by an energy minimized 3D model. In summary, this thesis explores the use of BC as drug releasing systems, which are promising and clinically relevant strategies to enhance nerve regeneration for many patients facing physical, mental and financial strains due to stroke, SCI or other difficult-to-cure injuries to the CNS.

When watching interactive branched video, the viewer is given the opportunity to tailor the storyline of the video playback. This type of video puts the users in control of their viewing experiences and provides content creators with great flexibility how to personalize the viewing experience for individual viewers. When it comes to regular linear videos, the concept of having a playback bar visually presenting the playback is a well established implementation used for most (if not all) video players, but for interactive branched videos, that is not the case. Instead, most branched video implementations are typically custom-made on a per-video basis (e.g., see custom-made Netflix and BBC movies) and do not use a playback bar. With the goal to fill this void, a branched video player with a generalized playback bar that visualizes the tree-like video structure was developed in 2018 by students of the Information Technology program at Linköping University and will be made public with a publication in the near future. Within the preparatory work prior to making this branched video player public, this project included a three-phase user study, where we evaluated the playback bar and its implemented features and compared the video player with alternative designs. With this thesis, we highlight the value of a branched video playback bar and provide interesting insights into how it, and other design customization features, may best be integrated into a branched video player. Furthermore, we present an improved version of the video player in which the new implementations are based on, and motivated by, the results from the user study. Finally, we describe how further investigations may be done to evaluate the improved version of the video player.

Par ce travail expérimental, nous essayons d’établir un lien entre les propriétés mécaniques de gels d’actine branchés de levure et la composition biochimique des réseaux. L’actine est un polymère semi-flexible qui fait partie du cytosquelette. De nombreux partenaires protéiques de l’actine (notés ABPs par la suite) se lient aux filaments d’actine et les agencent en différents types de réseaux. Arp2/3 est un complexe protéique qui génère la croissance de réseaux d’actine branchés. Les réseaux d’actine branchés en croissance intéressent tout particulièrement physiciens comme biologistes car ils sont capables de développer des forces nécessaires à de nombreux processus vitaux pour la cellule, comme l’endocytose. Nous avons ici étudié les propriétés mécaniques de gels d’actine branchés reconstitués in vitro, en nous focalisant sur le rôle d’un type d’ABPs en particulier, les protéines de réticulation. Il nous a été possible de quantifier et de comparer l’effet de trois protéines de réticulation différentes sur la mécanique des réseaux d’actine branchés de levure.Afin de mener à bien cette étude, nous avons combiné deux puissantes techniques expérimentales.Nous avons utilisé une technique de mesure des propriétés mécaniques basée sur l’utilisation de colloïdes superparamagnétiques développée au laboratoire. Cette technique permet de réaliser des mesures quantitatives et à haut débit sur des gels polymères très fins (quelques centaines de nanomètres d’épaisseur). Les réseaux ont été reconstitués in vitro grâce à la fonctionnalisation des billes superparamagnétiques avec Las17, une protéine que notre collaborateur biologiste a identifiée comme suffisant à activer Arp2/3 chez la levure. Nous avons de plus combiné deux approches complémentaires en travaillant à la fois sur des extraits cellulaires de levure contenant toutes les ABPs des réseaux Arp2/3 et à la fois sur des mélanges de quelques protéines purifiées.L’approche « top-down » est basée sur l’utilisation d’extraits cellulaires de mutants de la levure n’exprimant pas une ou des protéine(s) d’intérêt(s), et l’approche « bottom-up » sur l’addition de la protéine étudiée dans le système simplifié de quelques protéines purifiées. / In this experimental work we tried to quantify the mechanical properties of yeast branchedactin networks with regard to their biochemical composition. Actin is a semi-flexible biopolymerthat is assembled as part of the cytoskeleton. Proteins partners of actin (ABPs) shape itsfilaments into different type of networks. Arp2/3 is a protein complex that has the propertyto generate branched actin gels. Growing branched actin networks are of particular interest forboth biologists and physicists because of their ability to generate forces necessary to many vitalprocesses such as endocytosis. Here we study in vitro the mechanical properties of such networks,and we focus on the role of one type of actin binding proteins, the crosslinkers. This family ofproteins appears to play a role in both the elastic, viscous and plastic properties of the gels. Weare able to quantify and to compare the impact of three different crosslinkers on branched actinnetworks in yeast.In order to conduct said study, we combined two powerful experimental methods. We used asuperparamagnetic particle-based mechanical measurement technique that was developed in thelab and allows quantitative, high-throughput measurements on very thin gels. And the networkswere reconstituted in vitro by functionalization of the magnetic particles with Las17, which hasbeen showed to activate Arp2/3 for the yeast by our biologist collaborator. We furthermoreworked on both yeast extracts containing all the ABPs of the Arp2/3 networks, and with setsof a few purified proteins, in order to combine a « top-down » (use of mutations in yeast toprevent the expression of protein(s) of interest) and a « bottom-up » (addition of a protein ofinterest in a simplified system) approaches.