Orientador: Pedro de Oliva Neto / Banca: Iolanda Cristina Silveira Duarte / Banca: Maria das Graças de Almeida Felipe / Banca: Eleonora Cano Carmona / Banca: Crispin Humberto Garcia Cruz / Resumo: O presente trabalho teve por objetivo otimizar a autólise de levedura fresca de cervejaria (Saccharomyces cerevisiae), visando a extração máxima de ácido ribonucléico da biomassa na produção do extrato de levedura. As variáveis estudadas foram pH, temperatura, % de NaCl, % de NH3, tempo de processo e, métodos de recuperação de RNA do autolisado. Os experimentos foram realizados por meio de quatro ensaios delineados segundo Box & Benken (1989) e avaliado pela Metodologia da Superfície de Resposta, utilizando-se o Software Statística 5.1 e a análise estatística ANOVA. A otimização foi concluída por meio do quinto ensaio com a produção do extrato nas condições otimizadas (55,2ºC, 9,8% de NaCl em pH=5,1 por 24 horas e, 12,2% de NH3 a 60ºC sob agitação a 200 rpm/15minutos. Três métodos foram avaliados para recuperação do RNA e das frações de extrato e parede celular: 1) autólise/plasmólise; 2) choque térmico por 1 minuto a 68ºC seguido da autólise/plasmólise 3) hidrólise química alcalina. Pelo processo de autólise em combinação com 9,8% de NaCl, a taxa de extração de RNA em 24 horas foi de 89,7%, com um rendimento de 51,3% em massa de extrato com 57,9% de proteína e, 48,7% de parede celular desidratada com 21,7 % de proteína. A utilização de 12,2% de NH3 em base seca de levedura permitiu o aumento na taxa de extração de RNA de 89,7 para 93,6%, mas um forte escurecimento foi verificado no extrato obtido. Na recuperação do RNA após precipitação protéica em pH 4,3 com posterior uso de 2 volumes de etanol em pH=2, recuperou-se 15,47%, 13,80% e 7,42% de RNA respectivamente com purezas de 49,85%, 51,70% e 38,70%. As taxas de extração de RNA da biomassa foram de 87,45% para o método 1; 91,40% para o método 2 e 78,80% para o terceiro método, indicando uma boa alternativa para redução do teor de RNA da biomassa e produção do extrato rico... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present work had for objective to optimize the autolysis of fresh brewery's yeast (Saccharomyces cerevisiae), aiming the maximum extraction of ribonucleic acid of biomass in the yeast extract production. The studied variables were pH, temperature, % of NaCl, % NH3, processing time and RNA recovering methods from autolysed. The experiments were accomplished by mean of four delineated assays according to Box & Benken (1989) and evaluated by Surface Methodology of Answer, utilizing the software Statistica 5.1. and the analysis statistics "ANOVA". The optimization was concluded by mean of the fifth assay with an extract production in the optimized conditions (55.2ºC, 9.8% of NaCl in pH 5.1 for 24 hours and, 12.2% of NH3 at 60ºC under agitation at 200 rpm/15 minutes. Tree methods were evaluated for RNA recovering and of the extract fractions and cell wall: 1) autolysis/plasmolysis; 2) thermic shock during 1 minute at 68ºC followed of autolysis/plasmolysis; 3) alkaline chemical hydrolysis. The process of autolysis in combination with 9.8% of NaCl, the RNA extraction yield in 24 hours was of 89.7%, with a yield of 51.3% in extract mass with 57.9% of protein and, 48.7% of dehydrated cell wall with 21.7% of protein. The utilization of 12.2% of NH3 in dried base of yeast allowed the increase in the RNA yield extraction from 89.7 to 93.6%, but a strong darkness was observed in the obtained extract. The RNA recovering after 4.3 pH proteic precipitation with posterior use of 2 ethanol volumes in pH 2.0, it was recovered 15.47%, 13.8% and 7.42% of RNA respectively with purities of 49.85%, 51.70% and 38.70%. The RNA extraction yields of biomass were of 87.45% for the method 1; 91.40% for the method 2 and 78.80% for the third method, indicating a good alternative for RNA content reduction of biomass and rich extract production in nucleotides. The extract fractions were evaluated... (Complete abstract click electronic access below) / Doutor

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-01-20T15:41:36Z No. of bitstreams: 2 Dissertação - Amanda Gabrielle da Silva - 2015.pdf: 1218291 bytes, checksum: 56dd4b22c77bb02a83c9d20a4cc4e0ce (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-21T07:54:16Z (GMT) No. of bitstreams: 2 Dissertação - Amanda Gabrielle da Silva - 2015.pdf: 1218291 bytes, checksum: 56dd4b22c77bb02a83c9d20a4cc4e0ce (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-21T07:54:16Z (GMT). No. of bitstreams: 2 Dissertação - Amanda Gabrielle da Silva - 2015.pdf: 1218291 bytes, checksum: 56dd4b22c77bb02a83c9d20a4cc4e0ce (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-09-09 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Considering potential molecular methods for diagnostic and/or therapeutic purpose of infectious human diseases, such as dengue, highlights the systematic evolution of ligands by exponential enrichment, known as SELEX. In this method, ligands to a target are exponentially enriched generating aptamers of DNA or RNA with high specificity, being considered as artificial antibodies. In this work, we aimed to realize the isolation of aptamers binding to 3'-UTR (untranslated region) of the RNA from dengue virus (DENV), because present important conformational structures that act as functional elements into RNA necessary in the infectious process. Total RNA of C6/36 infected cells was extracted, submitted to reverse transcription reaction to obtain viral cDNA (serotypes 2 and 3) and amplified by symmetric and/or asymmetric PCR technique to produce the 3’-UTR from DENV as target, generating RNA-like by containing deoxyuridine triphosphate (dUTP) and biotin in the 5’ region of the target. The random library of RNA ligands was obtained by sonication of a pool from human genomic DNA used in three successive PCR, and in the last reaction was introduced the promoter of T7 RNA polymerase, presenting fragments ranging from 80 to 600-bp. Eight rounds of selection were performed between the target and the library by using paramagnetic particles coated with streptavidin. For each round, after the incubation, the non-ligands were removed by using magnetic platform, and the ligands were eluted with NaOH. The eluted ligands were precipitated, submitted to RT-PCR and transcription in vitro, completing one round of selection. For analysis of variability of ligands, the product obtained from the eighth round was cloned and fourteen clones were randomly selected for amplification. The results demonstrated that the aptamers presented sizes with estimated molecular weights varying from 80 to 100-bp. These data indicated the viability of aptamers isolation against conformational elements present in the 3'-UTR of RNA from dengue virus, which may contribute to future research focusing on prevention and/or control of the disease. / Dentre os potenciais métodos moleculares para o diagnóstico e/ou terapêutica de doenças que acometem a saúde humana, tais como a dengue, destaca-se a seleção de ligantes pela utilização da química combinatória, conhecida como SELEX. Nesse método, os ligantes a um alvo são enriquecidos exponencialmente tendo como produto final a obtenção de aptâmeros de DNA ou RNA com elevada especificidade, sendo considerados como anticorpos artificiais. No presente trabalho foi realizado o isolamento de aptâmeros de RNA ligantes à extremidade 3’-UTR (untranslated region) do RNA do vírus da dengue (DENV), por apresentar elementos de RNA conformacionais e funcionais importantes no processo infeccioso. A partir do RNA extraído de células C6/36 infectadas foi feita a transcrição reversa (RT) para produção de cDNA viral (sorotipos 2 e 3) e amplificação por PCR simétrica e/ou assimétrica para a produção do alvo 3’-UTR do DENV, na forma de RNA-like por conter desoxiuridina trifosfatada (dUTP) e biotina na extremidade 5’. A biblioteca randômica de ligantes de RNA foi obtida por sonicação de um pool de DNA genômico humano utilizado como alvo em três PCRs sucessivas sendo que na última reação foi introduzido o promotor da T7 RNA polimerase e cujos fragmentos variaram de 80 a 600-pb. Foram realizados oito rounds de seleção entre alvo e biblioteca utilizando partículas paramagnéticas revestidas com estreptavidina. A cada round, após o período de incubação, os oligonucleotideos não ligantes foram removidos com o auxílio de plataforma magnética, e os ligantes foram eluídos com NaOH. Os ligantes eluídos foram precipitados e submetidos à RT-PCR e transcrição in vitro, finalizando um round de seleção. Para verificar a variabilidade de ligantes, o produto do oitavo round foi clonado e 14 clones foram selecionados aleatoriamente para amplificação. Os resultados demonstraram que os aptâmeros isolados possuem tamanhos distintos, com pesos moleculares estimados variando de 80 a 100-pb. Os dados aqui obtidos indicaram a viabilidade do processo de isolamento de aptâmeros para elementos conformacionais presentes na extremidade 3’-UTR do RNA do vírus da dengue os quais poderão contribuir para pesquisas futuras com foco na prevenção e/ou controle da doença.

Orientadores: Lucimara Gaziola de la Torre, Patrícia Cruz Bergami-Santos / O texto da introdução e conclusão estão na lingua portuguesa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-22T15:41:58Z (GMT). No. of bitstreams: 1 Vitor_MicaelaTamara_M.pdf: 6469643 bytes, checksum: c14b8646a57fec71cc1e0eac8a82cddb (MD5) Previous issue date: 2013 / Resumo: Esta pesquisa teve como objetivo o desenvolvimento tecnológico de uma vacina lipossomal contendo RNA tumoral destinado à imunoterapia do câncer. Nesta estratégia, RNA total codificando o antígeno tumoral Her-2/neu extraído de linhagem de células de adenocarcinoma de mama humano SK-BR-3 foram incorporados em lipossomas catiônicos introduzidos in vitro em células dendríticas (DCs). A vacina de DCs tem a função de auxiliar o sistema imunológico a identificar antígenos tumorais para que as células cancerígenas sejam eliminadas. Porém uma das etapas críticas é a introdução (transfecção) de RNA nas DCs. Lipossomas catiônicos é uma alternativa promissora, pois além de ativarem as DCs, é capaz mediar a transfecção de ácidos nucléicos para células. A experiência prévia do grupo de pesquisa na área de lipossomas catiônicos mostrou a possibilidade da obtenção de lipossomas em larga escala para o desenvolvimento de vacina de DNA contra a tuberculose. Neste contexto, este trabalho avaliou os lipossomas catiônicos com a composição lipídica de fosfatidilcolina natural de ovo (EPC), 1,2-dioleoil-sn-glicero-3-fosfatidiletanolamina (DOTAP) e 1,2-dioleoil-3-trimetilamônio-propano (DOPE), na respectiva proporção molar de 50/25/25%. Metodologicamente, o trabalho foi dividido em quatro partes: na primeira parte foi apresentada uma visão geral do trabalho desenvolvido, demonstrando o potencial dos lipossomas catiônicos complexados com RNA na imunoterapia do câncer. Na segunda parte do trabalho investigaram-se os efeitos dos lipossomas produzidos através do processo laboratorial na diferenciação/maturação das DCs in vitro e as DCs estimuladas por estes lipossomas, na indução da proliferação de linfócitos T, resultando em lipossomas catiônicos incorporados pelas DCs, com a capacidade de ativar as DCs in vitro e de induzir proliferação de linfócitos T. A terceira parte do trabalho teve como finalidade a otimização da produção dos lipossomas catiônicos obtidos através do método de injeção de etanol utilizando a ferramentas estatísticas, obtendo lipossomas com menor polidispersidade e tamanho, que demonstraram in vitro serem incorporadas e ativarem as DCs e induzirem a proliferação de linfócitos T. A última etapa refere-se ao estudo da incorporação do RNA nos lipossomas catiônicos produzidos através do processo escalonado otimizado e comparado com o laboratorial no intuito de serem internalizados pelas DCs, transfectar o RNA e induzir a proliferação de linfócitos T através das DCs. Os resultados demonstraram que os complexos foram internalizados pelas DCs e que estas são capazes de induzir a proliferação de linfócitos T, porém há necessidade de se obter a condição ótima de transfecção. Dessa forma, conclui-se que os lipossomas catiônicos em questão têm potencial para serem usados como ferramenta em futuras estratégias na imunoterapia do câncer / Abstract: This research aimed at the technological development of a liposomal vaccine containing tumor RNA for cancer immunotherapy. In this strategy, total RNA encoding the Her-2/neu tumor antigen extracted from cell line of human breast adenocarcinoma SK-BR-3 were incorporated into cationic liposomes, which were introduced in vitro into dendritic cells (DCs). DCs vaccine has the function of helping the immune system to identify tumor antigens in order to eliminate cancerous cells. However, one of the critical steps is the introduction (transfection) of RNA in DCs. Cationic liposomes are a promising alternative, because besides activating DCs, they are able to mediate transfection of nucleic acids into cells. Previous work of our research group in the cationic liposomes field developed a liposomal nanostructure obtained by a scale up process containing DNA vaccine against tuberculosis. In this context, this work evaluated the cationic liposomes composed by egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoylphosphatidylethanolamine (DOPE), at 50/25/25% molar proportion respectively. Methodologically, the present work was carried out in four mean steps: in the first step, it was carried out an overview of all work, showing the relevancy of cationic liposomes complexed with RNA for cancer immunotherapy. The second part of this work investigated the effects of liposomes produced via laboratory process upon DCs differentiation/maturation in vitro and induction of T lymphocytes proliferation by DCs stimulated with these liposomes, resulting in cationic liposomes incorporated by DCs, capable to activate DCs in vitro and to induce proliferation of T lymphocytes. The third part of the work aimed at optimizing the production of cationic liposomes obtained via the ethanol injection method using statistical tools, obtaining liposomes with smaller size and polydispersity, which demonstrated to be incorporated and activate DCs in vitro and to induce T lymphocytes proliferation. The last step refers to the study of RNA incorporation in the cationic liposomes produced via optimized scalable process compared to the laboratory process in order to be internalized by DCs, transfected RNA and to induce T lymphocytes proliferation by DCs. The results showed that the complexes were internalized by DCs and they are able to induce T lymphocytes proliferation, however we still have to obtain the optimal transfection condition. In sum, we conclude that the cited cationic liposomes can be used as a potential tool in further strategies in cancer immunotherapy / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestra em Engenharia Química

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A critical event during the life cycle of Dictyostelium discoideum is glycogen turnover. This process is catalyzed by glycogen phosphorylase-2 (gp-2). Since gp-2 expression is first induced during the transition from growth to differentiation, understanding how this gene is controlled may provide some insight into the process of differentiation. In order to identify the trans-acting factors responsible for activating gp-2 expression, cDNA plasmid libraries of amoebae, and cells at 8 h and 12 h of development were generated. The long-term goals of this project involve screening expression libraries with identified cis-acting elements from the gp-2 promoter to yield the DNA binding proteins responsible for gene regulation. For this approach to succeed, a high-quality cDNA library is essential. The library must contain full-length cDNA that represents the complexity of mRNA present during the developmental stage of interest. Hence, all three libraries were subjected to extensive testing prior to and following cloning. RNA quality and the fidelity of the time points were determined by Northern blot analysis and by RTPCR for several marker genes. Following cDNA synthesis, the cDNA was assessed for complexity and full-length synthesis by PCR and radioactive primer extension, respectively. Ligation of the cDNA into a vector was performed using several ratios of vector:insert in order to ensure that long cDNA species were included in the plasmid library. Finally, the presence of the marker genes was confirmed by PCR amplification of plasmid extracted from bacteria transformed with the plasmid library. / Master of Science

In recent years, several interesting biological roles played by RNA have come to light. Apart from their known role in translation of genetic information from DNA to protein, they have been shown to act as enzymes as well as regulators of gene expression. Protein-RNA complexes are involved in regulating cellular processes like cell division, differentiation, growth, cell aging and death. A number of clinically important viruses have RNA as their genetic material. Defective RNA molecules have been linked to a number of human diseases. The ability of RNA to adopt stunningly complex three-dimensional structures aids in diverse functions like catalysis, metabolite sensing and transcriptional control. Several secondary structure motifs are observed in RNA, of which the double-helical RNA motif is ubiquitous and well characterized. Though DNA duplexes have been shown to be present in many polymorphic states, RNA duplexes are believed to exhibit conservatism. Early fibre diffraction analysis on molecular structures of natural and synthetically available oligo- and polynucleotides suggested that the double-helical structures of RNA might exist in two forms: A-form and A′-form. New improved crystallographic methods have contributed to the increased availability of atomic resolution structures of many biologically significant RNA molecules. With the available structural information, it is feasible to try and understand the contribution of the variations at the base pair, base-pair step and backbone torsion angle level to the overall structure of the RNA duplex. Further, the effect of protein binding on RNA structure has not been extensively analysed. These studies have not been investigated in greater detail due to the focus of the research community on understanding conformational changes in proteins when bound to RNA, and due to the lack of a significant number of solved RNA structures in both free and protein-bound state. While studies on the conformation of the DNA double-helical stem have moved beyond the dinucleotide step into tri-, tetra-, hexa- and octanucleotide levels, similar knowledge for RNA even at the dinucleotide step level is lacking. In this thesis, the results of detailed analyses to understand the contribution of the base sequence towards RNA conformational variability as well as the structural changes incurred upon protein binding are reported. Objectives The primary objective of this thesis is to understand the following through detailed analyses of all available high-resolution crystal structures of RNA. 1 Exploring sequence-dependent variations exhibited by dinucleotide steps formed by Watson-Crick (WC) base pairs in RNA duplexes. 2 Identifying sequence-dependent variations exhibited by dinucleotide steps containing non-Watson-Crick (NWC) base pairs in RNA duplexes. 3 Developing a web application for the generation of sequence-dependent non-uniform nucleic acid structures. 4 Investigating the relationship between base sequence and backbone torsion-angle preferences in RNA double helices followed by molecular dynamics simulation using various force fields, to check their ability to reproduce the above experimental findings. Chapter 1 gives an overview of the structural features and polymorphic states of RNA duplexes and the present understanding of the structural architecture of RNA, thereby laying the background to the studies carried out subsequently. The chapter also gives a brief description on the methodologies applied. Relevant methodologies and protocols are dealt with in detail in the respective chapters. Sequence-dependent base-pair step geometries in RNA duplexes A complete understanding of the conformational variability seen in duplex RNA molecules at the dinucleotide step level can aid in the understanding of their function. This work was carried out to derive geometric information using a non-redundant RNA crystal structure dataset and to understand the conformational features (base pair and base-pair step parameters) involving all Watson-Crick (WC) (Chapter 2) and non-Watson-Crick (NWC) base pairs (Chapter 3). The sequence-dependent variations exhibited by the base-pair steps in RNA duplexes are elaborated. Further, potential non-canonical hydrogen bond interactions in the steps are identified and their relationship with dinucleotide step geometry is discussed. Comparison of the features of dinucleotide steps between free and protein-bound RNA datasets suggest variations at the base-pair step level on protein binding, which are more pronounced in non-Watson-Crick base pair containing steps. Chapter 4 describes a web-server NUCGEN-Plus, developed for building and regeneration of curved and non-uniform DNA and RNA duplexes. The main algorithm is a modification of our earlier program NUCGEN that worked mainly for DNA. The WC step parameters and intra-base parameters for RNA were obtained from the work detailed in Chapter 2. The FORTRAN code and input sequence file format was modified. The program has two modules: a) Using the model-building module, the program can build duplex structures for a given input DNA/RNA sequence. Options are available for selecting various derived or user specified base-pair step parameters, and fibre diffraction parameters that can be used in the building process. The program can generate double-helical structures up to 2000 nucleotides in length. In addition, the program can calculate the curvature of the generated duplex at defined length scale. b) Using the regeneration module, double-helical structures of nucleic acids can be rebuilt from the existing solved structures. Further, variants of an existing structure can be generated by varying the input geometric parameters. The web-server has a user-friendly interface and is freely available in the public domain at: http://nucleix.mbu.iisc.ernet.in/nucgenplus/index.html Sequence dependence of backbone torsion angle conformers in RNA duplexes RNA molecules consist of covalently linked nucleotide units. Each of these units has six rigid torsional degrees of freedom (α, β, γ, δ, ε, and ζ) for the backbone and one (χ) around the glycosidic bond connecting the base to the ribose, thereby providing conformational flexibility. An understanding of the relationship between base sequence and structural variations along the backbone can help deduce the rationale for sequence conservation and also their functional importance. Chapter 5 describes in detail the torsion angle-dependent variations seen in dinucleotide steps of RNA duplex. A non-redundant, high resolution (≤2.5Å) crystal structure dataset was created. Base-specific preferences for the backbone and glycosidic torsion angles were observed. Non-A-form torsion angle conformers were found to have a greater prevalence in protein-bound duplexes. Further validation of the above observation was performed by analysing the RNA backbone conformers and the effect of protein binding, in the crystal structure of E. coli 70S ribosome. Chapter 5 further describes the molecular dynamics simulation studies carried out to understand the effect of force fields on the RNA backbone conformer preferences. A 33mer long duplex was simulated using seven different force fields available in AMBER and CHARMM program, each for 100 ns. Trajectory analyses suggest the presence of sequence-dependent torsion angle preferences. Torsion angle conformer distribution closer to that of crystal structures was observed in the system simulated using parmbsc0 force field. Molecular dynamics simulation studies of AU/AU base-pair step A unique geometric feature, unlike that in other purine-pyrimidine (RY) steps in the crystal dataset analysis, was reported for AU/AU step (see Chapter 2). Appendix 1 describes the work carried out to validate these features observed in the crystal structures using simulation studies. Additionally, the effect of nearest-neighbor base pairs on the AU/AU step geometry were examined. General Conclusion Overall, the findings of this thesis work suggest that RNA duplexes exhibit sequence-dependent structural variations and sample a large volume of the double-helical conformational space. Further, protein binding affects the local base-pair step geometry and backbone conformation.

Thesis (Ph.D. in Biophysics & Genetics, Human Medical Genetics Program) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 123-147). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

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