Enantioselective indicator displacement assays (eIDAs), was transitioned to a high-throughput screening protocols, for the rapid determination of concentration and enantioselectivity (ee) of chiral diols and α-hydroxycarboxylic acid. To improve the design of our previously established receptor based on o-(N,N-dialkylaminomethyl)arylboronate scaffolds for eIDAs. The rigidity of the receptor, which pertinent from the formation of an intramolecular N-B dative bond was investigated. o-(Pyrrolidinylmethyl)phenylboronic acid its complexes with bifunctional substrates such as catechol, [alpha]-hydroxyisobutyric acid, and hydrobenzoin was studied in detail by x-ray crystallography and ¹¹B NMR. Our structural study predicts that the formation of an N-B dative bond, and/or solvolysis to afford a tetrahedral boronate anion, depends on the solvent and the complexing substrate present. To simplify the operation of eIDAs, we introduced an analytical method, which utilize a dual-chamber quartz cuvette, which reduces the number of spectroscopic measurements from two to one and introduced artificial neural networks (ANNs) which simplifies data analysis. In a second example a high-throughtput screening protocol for hydrobenzoin was developed. The method involves the sequential utilization of what we define herein as screening, training, and analysis plates. Several enantioselective boronic-acid based receptors were screened using 96-well plates, both for their ability to discriminate the enantiomers of hydrobenzoin and to find their optimal pairing with indicators resulting in the largest optical responses. The best receptor/indicator combination was then used to train an ANN to determine concentration and ee. To prove the practicality of the developed protocol, analysis plates were created containing true unknown samples of hydrobenzoin generated by established Sharpless asymmetric dihydroxylation reactions, and the best ligand was correctly identified. The system was extended to pattern recognition for the rapid determination of identity, concentration, and ee of chiral vicinal diols. A diverse enantioselective sensor array was generated with three chiral boronic acid receptors and pH indicators. The optical response produced by the sensor array, was analyzed by two pattern recognition algorithms: principal component analysis (PCA) and ANNs. The PCA plot demonstrated good chemoselective and enantioselective separation of the analytes, and ANNs was used to accurately determine the concentration and ee of five unknown samples. / text

Metastasis is one of the critical hallmarks of malignancy tumor and the principal cause of death in patients with cancer. Cell migration is the basic and essential step in cancer metastasis process. To systematically investigate functional genes regulating cell migration and cancer metastasis on large scale, we developed a novel on-chip method, SAMcell (self-assembled cell microarray). This method was demonstrated to be particularly suitable for loss-of-function high-throughput screening because of its unique advantages. The first application of SAMcell was to screen human genome miRNAs, considering that more and more miRNAs had been proved to govern cancer metastasis. We found that over 20 % of miRNAs have migratory regulation activity in diverse cell types, indicating a general involvement of miRNAs in migratory regulation. Through triple-round screenings, we discovered miR-23b, which is down-regulated in human colon cancer samples, potently mediates the multiple steps of metastasis, including cell motility, cell growth and cell survival. In parallel, the second application of SAMcell was to screen human genome kinase genes, considering that more and more kinase genes had become successful diagnostic marker or drug targets. We found over 11% migratory kinase genes, suggesting the important role of kinase group in metastasis regulation. Through both functional screening and bioinformatics analysis, we discovered and validated 6 prospective metastasis-related kinase genes, which can be new potential targets in cancer therapy. These findings allow the understanding of regulation mechanism in human cancer progression, especially metastasis and provide the new insight into the biological and therapeutical importance of miRNAs or kinases in cancer.

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-18T09:33:31Z (GMT). No. of bitstreams: 1 Costa_BrunaZucolotoda_M.pdf: 3730198 bytes, checksum: aee2e21aa8a8998b409bff4636299519 (MD5) Previous issue date: 2011 / Resumo: A versatilidade enzimática foi abordada nesta dissertação desde a triagem de microorganismos para a seleção de biocatalisadores adequados, e estudos de interações intermoleculares presentes em sistemas complexos de promiscuidade enzimática, até avaliação da inibição de enzimas relacionadas a ocorrência de diversas enfermidades. As triagens enzimáticas de micro-organismos isolados de água de drenagem de mina de prospecção de cobre permitiram a identificação de hidrolases e monoxigenases, sendo que as cepas de bactérias que apresentaram as melhores atividades oxidativas mostraram-se promissoras para oxidação de diversas classes de sulfetos, em ensaios de biocatálise convencional. A atuação promíscua de lipases foi avaliada frente a oxidação de cetonas cíclicas em meio orgânico, e as interações intermoleculares deste sistema foram estudadas por RMN, onde foi observado características de um sistema organizado em micelas reversas. A partir destas evidências, foi proposto que a condição necessária para a reação enzimática é o confinamento da enzima num ambiente aquoso restrito limitado por uma barreira molecular de ácido graxo. Finalmente a inibição da atividade enzimática de proteína tirosina fosfatases foi avaliada com implementação de uma metodologia fluorimétrica rápida e eficiente. Logo, este trabalho apresentou uma visão ampla no campo da enzimologia com o emprego de biocatalisadores, sejam eles enzimas isoladas ou células íntegras, em estudos relacionados a aspectos químicos, bioquímicos e terapêuticos das enzimas / Abstract: In this thesis the enzymatic versatility was evaluated in microorganisms selecting specific biocatalysts, in studies of intermolecular interactions of complex systems focusing enzyme promiscuity, and last in enzyme inhibition related to the occurrence of various diseases. The enzymatic screening of microorganisms isolated from copper mine water drainage led to the identification of hydrolases and monooxygenases, and bacterial strains with relevant oxidative activities were further investigated in larger scale oxidation of several sulfides. The promiscuous activity of lipases was evaluated in organic medium catalyzing the oxidation of cyclic ketones. The supramolecular interactions involved in these reactions were studied by NMR, revealing the presence of reverse micelles in a highly organized system. Therefore confinement of the enzyme in a restricted aqueous environment limited by a narrow molecular barrier of fatty acids in an organic medium is the necessary condition for this enzymatic reaction to occur. Finally, inhibition of enzymatic activity of protein tyrosine phosphatases was assessed with the implementation of a quick and efficient fluorimetric method. The data presented herewith provide a broad vision of enzyme application, as isolated enzymes or whole cells, in studies addressing chemical, biochemical and therapeutic issues / Mestrado / Quimica Organica / Mestre em Química

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disease resulting in episodic but progressive extraskeletal bone formation. FOP is caused by missense mutations in the cytoplasmic domain of the type I bone morphogenetic protein (BMP) receptor ACVR1, leading to dysregulated activation. Currently there are no available drug treatments and the structural mechanism of mutant activation is still poorly characterised. To address this, a number of BMP and TGFβ receptors, including FOP mutants of ACVR1 were cloned, expressed and purified for both structural and biophysical experiments. The arginine at the site of most recurrent FOP mutation, R206H, is common across all type I receptors except BMPR1A and BMPR1B which have a lysine at this site. The novel structure of BMPR1B differed to wild-type ACVR1 showing some of the conformational changes expected of the active conformation. However, a variety of disease related ACVR1 mutant structures, including ACVR1 R206H, revealed a surprisingly persistent inactive conformation in the kinase domain. Some conformational changes suggestive of activation were observed in the mutant Q207D affecting the ATP pocket, the β4–β5 hairpin and the activation loop. Additionally, the structure of the Q207E mutant showed a slight release of the regulatory glycine-serine rich domain from its inhibitory position. These subtle changes suggest that the mutant inactive conformation is destabilised and potentially more dynamic. In agreement, all of the ACVR1 mutants showed reduced binding to the inhibitory protein FKBP12. However, mutant phosphorylation of the substrate Smad1 was not constitutive, but dependent on the co-expression of the partner ACVR2, consistent with recent evidence from transgenic knock-out mice. A novel 2-aminopyridine inhibitor scaffold with favourable specificity for ACVR1 was identified using a fluorescence-based thermal shift assay. Further derivatives were characterised with improved potency and selectivity. The crystal structures of ACVR1 bound to these inhibitors showed exquisite shape complementarity, contributing to their favourable specificity. This work has increased the understanding of FOP-associated mutant activation and provided a novel starting scaffold for potential drug development.

>Magister Scientiae - MSc / Recent advances in the field of plant genetics and application of molecular technologies has lead to greater understanding of various crop genomes and their organization.The applications of these techniques include molecular markers which have been used to examine DNA variation within crop species. This allows for the creation of further genetic variation for new and favourable traits.Molecular markers or DNA markers are short fragments of DNA that can be used to locate desirable genetic traits in the genome or show specific genetic differences. The Maloideae subfamily includes fruit species such as pear. Pears (Pyrus communis L.) are large edible fruit that are grown in cool climates, native to coastal regions in Africa, Asia and Europe. The external appearance of this fruit plays a vital role on its rate of sale potential. Thus it is important for the appearances of the pear to meet the expectations of the consumer.External factors affecting the appearance of fruit, such as shape and colour, can have a large influence on the consumer’s first impression and opinion of what the fruit may taste like(Jaeger and MacFie, et al., 2001). The South African pear industry is the fourth largest in the fruit industry after apple, citrus and grape, exporting 3.8% to Europe (Ferrandi, et al., 2005).Increase in production and export of the pear is dependant on the variety of cultivars with desired traits. New cultivars, especially ranges of new cultivars, with harvest dates from early to late in the season, can fill gaps in the marketing strategy of exporters and in the local markets (Human, et al., 2005) Therefore, development of molecular markers allows for their possible use in maker-assisted selection and for the construction of a genetic linkage map thus leading to the location of favourable traits and ultimately the improvement of the quality of the pear.In this study high throughput genomic DNA extractions were performed. The Cetyltrimethyl ammonium bromide (CTAB) method was employed as the results proved to be most promising. Furthermore the screenings of molecular markers were conducted in order to obtain DNA variation. Molecular markers were used to locate specific genetic differences.Multiplexing PCR was conducted using fluorescent primers for further screening and results proved to be useful as many variations could be observed.

Le microbiote du rumen bovin est un écosystème très diversifié et efficace pour la dégradation de substrats complexes, notamment issus de la biomasse végétale. Composé majoritairement de microorganismes non cultivés, il constitue un réservoir très riche de nouvelles enzymes d’intérêt potentiel pour les biotechnologies industrielles, en particulier les bioraffineries et la bioremédiation. Dans le cadre de cette thèse, nous avons mis en œuvre une approche de criblage fonctionnel du métagenome ruminal pour accélérer la découverte d’enzymes de dégradation des lignocelluloses, mais aussi de divers polluants synthétiques. En particulier, de nouvelles estérases capables de dégrader un insecticide de la famille des carbamates, le fenobucarb, ainsi qu’un polyuréthane commercial, l’Impranil DLN, ont pu être identifiées. De plus, le développement d’une nouvelle stratégie de criblage d’oxydoréductases nous a permis d’isoler trois enzymes bactériennes originales, très polyspécifiques, ne requérant ni cuivre ni manganèse pour dégrader différents substrats polycycliques tels que des polluants majeurs de l’industrie textile, mais aussi des dérivés de lignine. Enfin, le criblage de deux banques issues d’enrichissements in vivo et in vitro du microbiome du rumen sur paille de blé a permis d’isoler des cocktails d’enzymes lignocellulolytiques au profil fonctionnel et d’origine taxonomique différents, constitués de glycoside-hydrolases, estérases et oxydoréductases. Quinze nouveaux modules CAZy, correspondant à des familles enzymatiques jamais caractérisées, ont été identifiés. L’ensemble de ces résultats met en lumière l’immense potentiel d’innovation biotechnologique contenu dans les écosystèmes microbiens, en particulier dans le microbiote du rumen bovin / Bovine rumen microbiota is a highly diverse and efficient ecosystem for the degradation of complex substrates, especially those issued from plant biomass. Predominantly composed of uncultivated microorganisms, it constitutes a rich reservoir of new enzymes of potential interest for industrial biotechnologies, especially biorefineries and bioremediation. As part of this thesis, we used the functional screening of the ruminal metagenome to increase the discovery of enzymes able to degrade lignocelluloses, as well as different synthetic pollutants. In particular, new esterases able to degrade a carbamate insecticide, fenoucarb, and a commercial polyurethane, Impranil DLN, have been identified. Moreover, the development of a new screening strategy for oxidoreductases allowed the isolation of three original bacterial enzymes that are very polyspecific, and do not need copper nor manganese to degrade different polycyclic substrates, like major pollutants of the textile industry, as well as lignin derivatives. Finally, the screening of two libraries from in vivo and in vitro enrichments of the ruminal microbiome on wheat straw allowed the isolation of lignocellulolytic enzymatic cocktails, with different functional profiles and taxonomical origins, comprising glycoside-hydrolases, esterases and oxidoreductases. Fifteen novel CAZy modules, related to enzymatic families never characterized, were identified. All these results highlight the vast potential of microbial ecosystems, in particular the bovine rumen microbiota, for biotechnological innovation

L’innovation thérapeutique progresse traditionnellement par la combinaison du criblage expérimental et de la modélisation moléculaire. En pratique, cette dernière approche est souvent limitée par la pénurie de données expérimentales, particulièrement les informations structurales et biologiques. Aujourd'hui, la situation a complètement changé avec le séquençage à haut débit du génome humain et les avancées réalisées dans la détermination des structures tridimensionnelles des protéines. Cette détermination permet d’avoir accès à une grande quantité de données pouvant servir à la recherche de nouveaux traitements pour un grand nombre de maladies. À cet égard, les approches informatiques permettant de développer des programmes de criblage virtuel à haut débit offrent une alternative ou un complément aux méthodes expérimentales qui font gagner du temps et de l’argent dans la découverte de nouveaux traitements.Cependant, la plupart de ces approches souffrent des mêmes limitations. Le coût et la durée des temps de calcul pour évaluer la fixation d'une collection de molécules à une cible, qui est considérable dans le contexte du haut débit, ainsi que la précision des résultats obtenus sont les défis les plus évidents dans le domaine. Le besoin de gérer une grande quantité de données hétérogènes est aussi particulièrement crucial.Pour surmonter les limitations actuelles du criblage virtuel à haut débit et ainsi optimiser les premières étapes du processus de découverte de nouveaux médicaments, j’ai mis en place une méthodologie innovante permettant, d’une part, de gérer une masse importante de données hétérogènes et d’en extraire des connaissances et, d’autre part, de distribuer les calculs nécessaires sur les grilles de calcul comportant plusieurs milliers de processeurs, le tout intégré à un protocole de criblage virtuel en plusieurs étapes. L’objectif est la prise en compte, sous forme de contraintes, des connaissances sur le problème posé afin d’optimiser la précision des résultats et les coûts en termes de temps et d’argent du criblage virtuel / Therapeutic innovation has traditionally benefited from the combination of experimental screening and molecular modelling. In practice, however, the latter is often limited by the shortage of structural and biological information. Today, the situation has completely changed with the high-throughput sequencing of the human genome, and the advances realized in the three-dimensional determination of the structures of proteins. This gives access to an enormous amount of data which can be used to search for new treatments for a large number of diseases. In this respect, computational approaches have been used for high-throughput virtual screening (HTVS) and offer an alternative or a complement to the experimental methods, which allow more time for the discovery of new treatments.However, most of these approaches suffer the same limitations. One of these is the cost and the computing time required for estimating the binding of all the molecules from a large data bank to a target, which can be considerable in the context of the high-throughput. Also, the accuracy of the results obtained is another very evident challenge in the domain. The need to manage a large amount of heterogeneous data is also particularly crucial.To try to surmount the current limitations of HTVS and to optimize the first stages of the drug discovery process, I set up an innovative methodology presenting two advantages. Firstly, it allows to manage an important mass of heterogeneous data and to extract knowledge from it. Secondly, it allows distributing the necessary calculations on a grid computing platform that contains several thousand of processors. The whole methodology is integrated into a multiple-step virtual screening funnel. The purpose is the consideration, in the form of constraints, of the knowledge available about the problem posed in order to optimize the accuracy of the results and the costs in terms of time and money at various stages of high-throughput virtual screening

Orientador: Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T08:42:43Z (GMT). No. of bitstreams: 1 Tizei_PedroAugustoGalvao_M.pdf: 2670317 bytes, checksum: edbc1214fe54dca40dc54a503a4d4c85 (MD5) Previous issue date: 2013 / Resumo: A produção fermentativa de etanol a partir de substratos chamados de "primeira geração", como cana-de-açúcar e amido de milho, já atingiu níveis muito elevados de eficiência. As linhagens industriais utilizadas nestes processos já são adaptadas ao ambiente industrial e possuem características que dificultam o melhoramento por engenharia genética tradicional. Duas abordagens inovadoras foram utilizadas para buscar processos fermentativos mais eficientes: o uso de reatores bioelétricos para alterar os produtos da fermentação e um ensaio de engenharia evolutiva para otimizar fenótipos heterólogos. Foram feitas fermentações bioelétricas com Saccharomyces cerevisiae, obtendo aumentos de produtividade de etanol, sem alterar o rendimento final, e também mudanças nas proporções dos subprodutos glicerol e acetato. Uma resposta distinta foi observada para uma linhagem industrial cultivada nas mesmas condições. Foram realizadas análises de expressão gênica global de linhagens de laboratório e industrial fermentando sob estímulo bioelétrico. Não foram observadas alterações na via fermentativa, mas houve variações grandes na expressão de genes relacionados a outros aspectos da fisiologia da levedura, como genes para síntese de lipídios de membrana e genes desconhecidos ou com funções aparentemente não-relacionadas ao processo fermentativo. Também foi evidente uma resposta global diferente entre as duas linhagens. Foi estabelecido um método automatizado para ensaios de engenharia evolutiva, que permitiu a seleção de linhagens por crescimento em celobiose. Utilizando apenas a variabilidade presente no genoma de uma linhagem industrial diplóide, foi possível obter linhagens haplóides com desempenho superior à linhagem parental. Portanto, esta estratégia pode ser viável para se obter fenótipos superiores utilizando linhagens distantes de S. cerevisiae / Abstract: Fermentative ethanol production from substrates known as "first generation", such as sugar cane and corn starch, has reached very high levels of efficiency. The industrial strains used in these processes are already adapted to the industrial environment and possess characteristics that hinder further improvement by traditional genetic engineering. Two innovative approaches were used to seek more efficient fermentation processes: the use of bioelectric reactors to alter fermentation products and an evolutionary engineering assay to optimize heterologous phenotypes. Bioelectric fermentations were carried out with Saccharomyces cerevisiae, obtaining increases in ethanol productivity, without changing the final yield, and also changes in the proportions of byproducts glycerol and acetate. A distinct response was observed for an industrial strain cultivated under the same conditions. Global gene expression analyses were carried out for a laboratory and industrial strain under bioelectric stimulus. No changes were observed for the fermentative pathway, but there were large variations in expression for genes related to other aspects of yeast physiology, such as membrane lipid synthesis and unknown genes or genes with functions that are apparently unrelated to the fermentation process. A difference in the global response for the two strains was also evident. An automated method for evolutionary engineering assays was established, which allowed the selection of strains by growth on cellobiose. Using only the genetic variability present within the genome of a diploid industrial strain, it was possible to obtain haploid strains with superior growth rate when compared to the parental strain. Therefore, this strategy may be viable for obtaining superior phenotypes using distant strains of S. cerevisiae / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

While protein therapeutics are indispensable in the treatment of a variety of diseases, including cancer, rheumatoid arthritis, and diabetes, key limitations including short half-lives, high immunogenicity, protein instability, and centralized production complicate long-term use and on-demand production. Site-specific polymer conjugation provides a method for mitigating these challenges while minimizing negative impacts on protein activity. However, the location-dependent effects of polymer conjugation are not well understood. Cell-free protein synthesis provides direct access to the synthesis environment and rapid synthesis times, enabling rapid evaluation of multiple conjugation sites on a target protein. Here, work is presented towards developing cell-free protein synthesis as a platform for both design and on-demand production of next generation polymer-protein therapeutics, including (1) eliminating endotoxin contamination in cell-free reagents for simplified therapeutic preparation, (2) improving shelf-stability of cell-free reagents via lyophilization for on-demand production, (3) coupling coarse-grain simulation with high-throughput cell-free protein synthesis to enable rapid identification of optimal polymer conjugation sites, and (4) optimizing cell-free protein synthesis for production of therapeutic proteins

<div>Protein N-terminal methyltransferases (NTMTs) are a family of enzymes that methylate the α-N-terminus of a variety of protein substrates. Both NTMT1 and NTMT2 recognize a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) to install 1-3 methyl group(s) on the substrates. NTMT1 plays important roles in mitosis regulation, chromatin interactions, and DNA damage repair. Another member NTMT2 shares ~50% sequence similarity and the same substrate recognition motif although NTMT2 was initially characterized as a mono-methyltransferase. To understand the molecular mechanism of NTMT2, we obtained the first co-crystal structure of NTMT2 in complex with its peptide substrate. After an extensive investigation of substrate recognition and methylated products of NTMT1/2, we found out that NTMT2 can fully methylate G/P-PKRIA peptides despite a predominant mono-methyltransferase. Moreover, we identified a gatekeeper N89 in NTMT2 that controls the substrate entry and the product specificity of NTMT2.</div><div>To elucidate the biological functions of NTMT1/2-catalyzed N-terminal methylation, we applied two different strategies to discover cell-potent inhibitors. Guided by the co-crystal structures of NTMT1 in complex with previously reported inhibitors, we designed and synthesized a series of new peptidomimetic inhibitors. By introducing more hydrophobic groups, the most cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM) exhibited over 2-fold increased inhibition on cellular N-terminal methylation levels with an IC50 value of ~50 µM compared to previously reported peptidomimetic inhibitor DC541. Meanwhile, we also discovered the first potent small molecule inhibitor Genz-682452 (IC50 = 0.5 ± 0.04 µM) after screening ~58,000 compounds. Subsequent structural modifications led to the discovery of GD433 (IC50 = 27 ± 0.5 nM) with a 20-fold increased potency compared to the initial hit Genz-682452. Inhibition mechanism indicated both inhibitors bind to peptide-binding pocket and co-crystal structures of both Genz-682452 and GD433 with NTMT1 confirmed their binding modes. Furthermore, GD433 shows over 7-fold selectivity over other major 40 protein methyltransferases and DNA methyltransferase and exhibits improved selectivity for NTMT1 over glucosylceramide synthase (GCS). GD433 significantly decreases the cellular N-terminal methylation level of NTMT1 substrates RCC1 and SET at 10 nM in both HEK293 and HCT116 cells, providing a valuable probe for cell-based studies in the future.<br></div><p><br></p>