abstract: The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either synthetic aptamers or natural components, conferring direct biological functions to the nucleic acid devices. The applications of nucleic acids greatly relies on the bio-reactivity and specificity when applied to highly complexed biological systems. This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo for constructing multi-input gene circuits with NOT logic. With the aid of a DNA nanoscaffold, pairs of hetero-bivalent aptamers for human alpha thrombin were identified with ultra-high binding affinity in femtomolar range with displaying potent biological modulations for the enzyme activity. The newly identified bivalent aptamers enriched the aptamer tool box for future therapeutic applications in hemostasis, and also the strategy can be potentially developed for other target molecules. Secondly, by employing a three-way junction structure in the riboregulator structure through de-novo design, we identified a family of high-performance RNA-sensing translational repressors that down-regulates gene translation in response to cognate RNAs with remarkable dynamic range and orthogonality. Harnessing the 3WJ repressors as modular parts, we integrate them into biological circuits that execute universal NAND and NOR logic with up to four independent RNA inputs in Escherichia coli. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

Three samples of the biopolymer hyaluronic acid (HA) were characterized in the following manner: the molecular weights were obtained via multi-angle laser light scattering; the intrinsic viscosities were calculated through dilute solution viscometry, and the rheology of HA solutions was determined with constant rate rotational viscometry and dynamical mechanical testing. In addition, the highly debated role of hyaluronic acid in wound healing was examined by studying the effect that HA has upon thrombin-catalyzed fibrin clotting. Fibrin, in phosphate-buffered saline, was clotted both alone and after being incubated with HA. It was determined that the presence of hyaluronic acid resulted in a slower clotting process; in effect, HA acts as an anti-coagulant. Based upon the experimental evidence, it is proposed that this anti-coagulant phenomenon arises through a combination of two mechanisms: 1) specific binding between HA and fibrin, which acts to retard fibrin clotting through steric hindrance, and 2) the formation of an HA network which slows fibrin clotting by hindering free diffusion of fibrin and thrombin. Finally, creation of a synthetic replacement for synovial fluid was attempted using xanthan gum and locust bean gum in phosphate-buffered saline. The phenomenon of gum synergism was utilized in an effort to exert some degree of fine-tuning over the final rheological properties of the solution. This also would provide the side benefit of reducing the weight of gum required per unit volume. By mixing the solutions at different temperatures, it was possible to exploit the tendency of xanthan gum to uncoil at higher temperatures and therefore bind more strongly to locust bean gum. However, it was determined that no combination of gum concentrations and processing conditions resulted in a gum solution that adequately mimicked the rheology of a hyaluronan solution. / Graduation date: 2003

The thesis aimed to develop and characterize a novel detection approach, which we termed aptaPCR exploiting nucleic acid aptamers as combined recognition and reporter biocomponents for the ultrasensitive detection of analytes. Nucleic acid aptamers are synthetic ligands selected from vast combinatorial libraries through a process referred to as SELEX – Systematic Evolution of Ligand By Exponential Enrichment. As compared to other natural and synthetic receptor, aptamers possess unique chemical and biochemical characteristics, such as: a well known chemistry, remarkable stability, an ability to be selected against virtually any target even in non-physiological conditions

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that acts as a molecular link between the coagulation and fibrinolytic cascades. TAFI can be activated by thrombin and plasmin but the reaction is enhanced significantly when thrombin is in a complex with the endothelial cofactor thrombomodulin (TM). The in vitro properties of TAFI have been extensively characterized. Activated TAFI (TAFIa) is a thermally unstable enzyme that attenuates fibrinolysis by catalyzing the removal of basic residues from partially degraded fibrin. The in vivo role of the TAFI pathway, however, is poorly defined and very little is known about the role of different activators in regulating the TAFI pathway. In the present study, we have constructed and characterized various TAFI mutants that are resistant to activation by specific activators. Based on peptide sequence studies, these mutants were constructed by altering key amino acid residues surrounding the scissile R92-A93 bond. We measured the thermal stabilities of all our mutants and found them to be similar to wild type TAFI. We have identified that the TAFI mutants P91S, R92K, and S90P are impaired in activation by thrombin or thrombin-TM, thrombin alone, and thrombin alone or plasmin, respectively. The TAFI mutants A93V and S94V were predicted to be resistant to activation by plasmin but this was not observed. The triple mutant, DVV was not activated by any of the aforementioned activators. Finally, we have used in vitro fibrin clot lysis assays to evaluate the antifibrinolytic potential of our variants and were able to correlate their effectiveness with their respective activation kinetics. In summary, we have developed activation resistant TAFI variants that can potentially be used to explore the role of the TAFI pathway in vivo. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-30 11:44:37.191

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Zugl.: Giessen, Universiẗat, Diss., 2008.

Thesis (Ph. D.)--University of Wisconsin, 1944. / Typescript. Includes bibliographical references.

Made available in DSpace on 2014-10-09T12:49:28Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:02:37Z (GMT). No. of bitstreams: 1 10386.pdf: 2709613 bytes, checksum: e5193eaf23e32490bcf76fc5ad0d8789 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares, IPEN/CNEN-SP

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-03-20T19:23:41Z No. of bitstreams: 1 JeffersonRomarioDuarteDaLuz_DISSERT.pdf: 1201751 bytes, checksum: 75f1b32406c3dfccabe706f4f9d4a66e (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-03-21T22:23:37Z (GMT) No. of bitstreams: 1 JeffersonRomarioDuarteDaLuz_DISSERT.pdf: 1201751 bytes, checksum: 75f1b32406c3dfccabe706f4f9d4a66e (MD5) / Made available in DSpace on 2017-03-21T22:23:37Z (GMT). No. of bitstreams: 1 JeffersonRomarioDuarteDaLuz_DISSERT.pdf: 1201751 bytes, checksum: 75f1b32406c3dfccabe706f4f9d4a66e (MD5) Previous issue date: 2016-11-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O emprego de plantas medicinais para o tratamento, cura e preven??o de patologias ? um h?bito que acompanha a humanidade desde seus prim?rdios. Considerando que o Brasil apresenta uma grande biodiversidade para a produ??o de fitoter?picos, ? de suma import?ncia o estudo de plantas como fontes alternativas de tratamento, visando a busca de susbt?ncias que possam auxiliar os f?rmacos atualmente prescritos na terap?utica cl?nica. Durante muitos anos, doen?as cardiovasculares e doen?as tromboemb?licas tem sido as principais causas de morte por doen?as no mundo, sendo respons?veis pela morte de cerca de 17,5 milh?es de pessoas at? o ano de 2012 (31% das mortes em todo o mundo). O sistema de coagula??o est? centralmente envolvido na forma??o do trombo venoso. Indica??es atualmente definidas para anticoagulantes incluem a profilaxia e tratamento do tromboembolismo. Heparinas n?o fracionadas e heparinas de baixa massa molar s?o utilizadas como medicamentos anticoagulantes, no entanto, estes compostos s?o acompanhados de alguns efeitos secund?rios, tais como, trombocitopenia e um elevado risco de hemorragia. A efeito deste problema tem se gerado uma procura de novas subst?ncias, a fim de auxiliar a terap?utica anticoagulante. Neste contexto, este estudo teve como objetivo avaliar o potencial anticoagulante, efeitos t?xicos e hemorr?gicos dos extratos foliares de Licania rigida Benth e Turnera subulata, esp?cies vegetais amplamente encontradas no semi?rido nordestino. Os extratos foram obtidos a partir de etanol (50%), com posterior parti??o com solventes de polaridades crescentes, incluindo hexano e acetato de etila. A avalia??o dos extratos frente ao sistema de coagula??o mostrou uma atividade anticoagulante satisfat?ria pelo Tempo de Tromboplastina parcial Ativada e Tempo de Protrombina (100% de atividade), atividade Anti-Xa (~ 40% de inibi??o) e uma grande capacidade de inibibir diretamente da trombina (~ 80 a 100% de inibi??o) como principal mecanismo de a??o. Al?m disso, observou-se que os extratos apresentam um baixo efeito hemorr?gico, bem como, a aus?ncia de toxicidade em modelos in vitro (Citotoxicidade por MTT) e in vivo (Toxicidade oral aguda). Este trabalho relata pela primeira vez o potencial anticoagulante de Licania rigida Benth e Turnera subulata. / The use of medicinal plants for the treatment, cure and prevention of pathologies is a habit that accompanies humanity since its beginnings. Considering that Brazil presents a great biodiversity for the production of phytotherapics, it is of paramount importance the study of plants as alternative sources of treatment, aiming at the search for substances that may help the drugs currently prescribed in clinical therapeutics. Over many years, cardiovascular disease and thromboembolic disorders have been the leading cause of death by disease in the world, being responsible for the death of approximately 17.5 million people by the year 2012 (31% of deaths worldwide). The coagulation system is centrally involved in the formation of venous thrombus. Currently defined indications for anticoagulants include prophylaxis and treatment of thromboembolism. Unfractionated heparins and low molecular heparins are used as anticoagulant drugs. However, these compounds are accompanied by several side effects such as thrombocytopenia and a high risk of systemic bleeding. The effect of this problem demanded the search for new substances in order to assist prolonged anticoagulant therapy. In this context, this study aimed to evaluate the anticoagulant potential, toxic and hemorrhagic effects from Licania rigida Benth and Turnera subulata leaves, species widely found in Northeast semiarid. The extracts were obtained from ethanol (50%) with subsequent partition with solvents of increasing polarities, including hexane and ethyl acetate.The crude extracts were obtained from ethanol (50%) and subsequent partition with increasingly polar solvents including hexane and ethyl acetate. The evaluation of the extracts against the coagulation system showed a satisfactory anticoagulant activity by Activated Partial Thromboplastin Time and Prothrombin Time (100% activity), Anti-Xa activity (~ 40% inhibition) and a large capacity to inhibit directly from Thrombin (~ 80 to 100% inhibition) as the main mechanism of action. In addition, the extracts were found to have a low hemorrhagic effect, as well as the absence of toxicity in in vitro models (MTT cytotoxicity) and in vivo (acute oral toxicity). This paper reports for the first time the anticoagulant potential of Licania rigida Benth and Turnera subulata.

Made available in DSpace on 2014-10-09T12:49:28Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:02:37Z (GMT). No. of bitstreams: 1 10386.pdf: 2709613 bytes, checksum: e5193eaf23e32490bcf76fc5ad0d8789 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares, IPEN/CNEN-SP