Orientador: Yoon Kil Chang / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T06:53:45Z (GMT). No. of bitstreams: 1 Schmiele_Marcio_D.pdf: 9722936 bytes, checksum: 95d9146270f349c5f3e7ad761ac0d266 (MD5) Previous issue date: 2014 / Resumo: Os análogos de carne obtidos por extrusão termoplástica de proteínas vegetais são caracterizados pelo seu elevado teor proteico e estrutura semelhante às fibras da carne, envolvendo diversos tipos de ligações e/ou interações químicas entre as proteínas. O objetivo deste trabalho foi avaliar as características tecnológicas e físico-químicas de análogos de carne, à base de isolado proteico de soja, obtidos por processo de extrusão termoplástica a alta umidade (AU) e baixa umidade (BU). Para cada condição de umidade foi utilizado um Delineamento Composto Central Rotacional de três variáveis independentes (glúten vital, umidade de condicionamento e temperatura de extrusão). As variáveis dependentes avaliadas foram a textura instrumental, cor instrumental, capacidade de absorção de água, índice de solubilidade em água, capacidade de absorção de óleo, índice de dispersibilidade de proteína, energia mecânica específica e o tipo de interações proteicas. Estas interações foram avaliadas através de sete tipos de solventes específicos: (i) tampão fosfato para as proteínas no estado nativo; (ii) dodecil sulfato de sódio para as interações hidrofóbicas e iônicas; (iii) Triton 100X para as interações hidrofóbicas; (iv) ureia para as interações hidrofóbicas e pontes de hidrogênio; (v) ß-mercaptoetanol para as ligações dissulfeto; e (vi) ß-mercaptoetanol e ureia e (vii) dodecil sulfato de sódio e ureia, para avaliar o efeito sinérgico entre os sistemas. O ponto otimizado (caracterizado principalmente por promover maiores valores de L* e de capacidade de absorção de água, menores valores de índice de solubilidade em água, de capacidade de absorção de óleo, de desnaturação proteica e valores intermediários de textura instrumental e de energia mecânica específica) foi processado juntamente com uma amostra controle para ambos os processos com o intuito de validar os modelos matemáticos e avaliar as possíveis alterações na morfologia dos análogos de carne, na massa molecular das proteínas, na composição de aminoácidos totais e na desnaturação proteica. As melhores condições de processamento foram obtidos para os análogos de carne contendo de 12 e 5 % de glúten vital, 58 e 18 % de umidade de condicionamento e 135 e 100 °C para a temperatura de extrusão, para o processo AU e BU, respectivamente. As principais interações proteína-proteína encontradas nos análogos de carne foram as ligações dissulfeto e ligações de hidrogênio para o processo AU e as ligações dissulfeto e interações iônicas para o processo BU. A adição de glúten vital promoveu uma aparência mais lisa e melhor orientação na estrutura das fibras. Verificou-se que ocorreu aumento nas proteínas de baixa massa molecular e diminuição nas proteínas de alta massa molecular. No perfil de aminoácidos totais houve maior variação negativa para os aminoácidos essenciais (triptofano e treonina), semi essenciais (cisteína) e não essenciais (serina), indicando que houve redução no valor nutricional. As estruturas secundárias (a-hélice, ß-folha, ß-volta e a estrutura desordenada) mostraram alteração na sua conformação devido à desnaturação proteica e formação de novos agregados / Abstract: Meat analogue obtained by termoplastic extrusion of vegetable proteins are characterized by its high protein levels and structure similar to meat fibers, which comprises many types of chemical bonds and/or interactions between proteins. The aim of this work was to evaluate the technological and physico-chemical characteristics of meat analogue based on isolated soy protein obtained by thermoplastic extrusion process at high moisture (HM) and low moisture (LM) content. For each moisture condition was used a Central Rotational Composite Design with three independent variables (vital gluten, moisture content and extrusion temperature). The dependent variables evaluated were instrumental texture, instrumental color, water absorption capacity, water solubility index, oil absorption capacity, protein dispersibility index, specific mechanical energy, and the type of protein interactions. These interactions were evaluated using seven specific solvents types: (i) phosphate buffer for proteins in native state; (ii) sodium dodecil sulphate for hydrophobic and ionic interactions; (iii) Triton 100X for hydrophobic interactions; (iv) urea for hydrophobic interactions and hydrogen bonds; (v) ß-mercaptoethanol for dissulfide bonds; and (vi) ß-mercaptoethanol and urea and (vii) sodium dodecil sulphate and urea, for the synergistic effect between the systems. The optimized point (characterized mainly by promoting higher values for L* and water absorption capacity, lower values for water solubility index, oil absoption capacity and protein denaturation and intermediate values for instrumental texture and specific mechanical energy) was processed, together with a control sample for each processes, in order to validate the mathematical models and to evaluate possibles changes in the meat analogues morphology, in the protein molecular weight, in the total amino acid composition, and in the protein denaturation. The best processing conditions were obtained for the meat analogue containing 12 and 5 % of vital gluten, 58 and 18 % of moisture content and 135 and 100 °C of extrusion temperature, for the HM and LM processes, respectively. The main protein-protein interactions found in meat analogues were the dissulfide bonds and hydrogen bonds for the LM process and the dissulfide bonds and ionic interactions for the HM process. The addition of vital gluten promoted a smoother appearance and better orientation in the fiber structure. It was found that occured an increase in the protein with low molecular weight and a reduction in the protein with high molecular weight. There were a greater negative variation for the essential (tryptophan and threonine), semi-essential (cysteine) and nonessential (serine) amino acids in the total amino acid profile, indicating a reduction of the nutritional value. The secondary structure (a-helix, ß-sheet, ß-turn and disordered structure) showed alteration in its conformation due to the protein denaturation and formation of new aggregates / Doutorado / Tecnologia de Alimentos / Doutor em Tecnologia de Alimentos

Malaria is prevalent in over 100 countries which is populated by half of the world’s population and culminates in approximately one million deaths per annum, 85% of which occurs in sub-Saharan Africa. The combined resistance of the mosquitoes and parasites to the currently available pesticides and antimalarial chemotherapeutic agents requires the concerted effort of scientists in the malaria field to identify and develop novel mechanisms to curb this deadly disease. In this study, a thorough understanding of the role players in the polyamine pathway of the parasite was obtained, which could aid future studies in the development of novel inhibitory compounds against these validated drug targets. The uniquely bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) of Plasmodium falciparum forms an important controlling node between the polyamine and methionine metabolic pathways. It has been speculated that the unique bifunctional association of the rate-limiting enzymes allows for the concerted regulation of the respective enzyme activities resulting in polyamine synthesis as per requirement for the rapidly proliferating parasite while the methionine levels are strictly controlled for their role in the methylation status. The results of this study showed that the enzyme activities of the bifunctional complex are indeed coordinated and subtle conformational changes induced by complex formation is suggested to result in these altered kinetics of the individual AdoMetDC and ODC domains. Studies also showed that the identification of the interaction sites between the domains, which allows for communication across the complex, may be targeted for specific interference with the enzyme activities. Furthermore, these studies showed that the current knowledge on the different subclasses of the AdoMetDC family should be re-evaluated since P. falciparum AdoMetDC shows diverse properties from orthologues and therefore points towards a novel grouping of the plasmodial protein. The extensive biochemical and biophysical studies on AdoMetDC has also provided important avenues for the crystallisation and solving of this protein’s 3D structure for subsequent structure-based identification of drug-like lead compounds against AdoMetDC activity. The application of structure-based drug design on malarial proteins was additionally investigated and consequently proved that the rational design of lead inhibitory compounds can provide important scaffold structures for the identification of the key aspects that are required for the successful inhibition of a specific drug target. Spermidine synthase, with its intricate catalytic mechanism involving two substrate binding sites for the products of the reactions catalysed by AdoMetDC/ODC, was used to computationally identify compounds that could bind within its active site. Subsequent testing of the compounds identified with a dynamic receptor-based pharmacophore model showed promising inhibitory results on both recombinant protein and in vitro parasite levels. The confirmation of the predicted interaction sites and identification of aspects to improve inhibitor interaction was subsequently investigated at atomic resolution with X-ray protein crystallography. The outcome of this doctoral study shows the benefit in applying a multidisciplinary and multinational approach for studying drug targets within the malaria parasite, which has led to a thorough understanding of the targets on both biochemical and structural levels for future drug design studies. / Thesis (PhD)--University of Pretoria, 2011. / Biochemistry / unrestricted

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions.

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Information fusion aims to develop intelligent approaches of integrating information from complementary sources, such that a more comprehensive basis is obtained for data analysis and knowledge discovery. Our Protein Biological Unit (ProtBuD) database is the first database that integrated the biological unit information from the Protein Data Bank (PDB), Protein Quaternary Server (PQS) and Protein Interfaces, Surfaces and Assemblies (PISA) server, and compared the three biological units side-by-side. The statistical analyses show that the inconsistency within these databases and between them is significant. In order to improve the inconsistency, we studied interfaces across different PDB entries in a protein family using an assumption that interfaces shared by different crystal forms are likely to be biologically relevant. A novel computational method is proposed to achieve this goal. First, redundant data were removed by clustering similar crystal structures, and a representative entry was used for each cluster. Then a modified k-d tree algorithm was applied to facilitate the computation of identifying interfaces from crystals. The interface similarity functions were derived from Gaussian distributions fit to the data. Hierarchical clustering was used to cluster interfaces to define the likely biological interfaces by the number of crystal forms in a cluster. Benchmark data sets were used to determine whether the existence or lack of existence of interfaces across multiple crystal forms can be used to predict whether a protein is an oligomer or not. The probability that a common interface is biological is given. An interface shared in two different crystal forms by divergent proteins is very likely to be biologically important. The interface data not only provide new interaction templates for computational modeling, but also provide more accurate data for training sets and testing sets in data-mining research to predict protein-protein interactions. In summary, we developed a framework which is based on databases where different biological unit information is integrated and new interface data are stored. In order for users from the biology community to use the data, a stand-alone software program, a web site with a user-friendly graphical interface, and a web service are provided. / Computer and Information Science

It remains a major challenge in biophysics to understand the conformational dynamics of biomolecules. As powerful tools, molecular dynamics (MD) simulations have become increasingly important in studying the full atomic details of conformational dynamics of biomolecules. In addition, many statistical models have been developed to give insight into the big datasets from MD simulations. In this work, I first describe three statistical models used to analyze MD simulation data: Lifson-Roig Helix-Coil theory, Bayesian inference models, and Markov state models. Then I present the applications of each model in analyzing MD simulations and revealing insight into the conformational dynamics of biomolecules. These statistical models allow us to bridge microscopic and macroscopic mechanisms of biological processes and connect simulations with experiments. / Chemistry