Rozprawy doktorskie: „Biology - biotechnology”

1

Cupples, Gemma. "Fibre-laden flows in biology and biotechnology". Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8308/.

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Fibre-laden fluids are ubiquitous in biological and physical systems; the fibres alter the rheology of the fluid and hence the emergent behaviour of the system. This thesis investigates two physical situations associated with fibrous media. Firstly we optimise the shear-induced alignment of suspensions of elongated particles, motivated by collaboration with Linear Diagnostics Ltd who are developing handheld devices to detect disruptions in fibre alignment due to pathogen presence in biological samples. Incorporating the effects of fibre dispersion and the mechanical anisotropy induced by the particles, we model suspensions of elongated particles undergoing steady or oscillating ow using a Fokker-Planck framework, producing recommendations for designs which optimise the signal to noise ratio. Next, we investigate microscopic propulsion in perfectly aligned media; for example the evolving fibrous structure of cervical mucus and more generally the problem of propulsion and pumping of an active fluid with alignment. We model the swimming of spermatozoa by adapting Taylor's classical swimming sheet model using Ericksen's transversely isotropic constitutive law (a limit of the Fokker-Planck model), to account for an aligned fibrous network. We find that propulsion in fibre-laden fluids is drastically different from Newtonian fluids, supporting the requirement to investigate fibrous rheology.

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Willrodt, Christian. "Synthetic biology for synthetic chemistry - Microbial production and selective functionalization of limonene". Doctoral thesis, Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-201140.

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The progress in biotechnological disciplines such as metabolic engineering or synthetic biology increased the interest of chemical and pharmaceutical industries to implement microbial processes for chemical synthesis. However, most microorganisms, e.g., Escherichia coli or Saccharomyces cerevisiae, used in biotechnological applications are not evolved by nature for the production of industrially relevant compounds, which are often hydrophobic, non-charged, volatile, or toxic to the microbial organisms. Bioprocess design relies on an integrated approach addressing pathway, cellular, reaction, and process engineering to combine the results of natural evolution with the demands of industrial applicability. In this thesis, the microbial de novo production and selective oxyfunctionalization of the highly volatile isoprenoid limonene has been investigated as a model system featuring reactants with challenging physicochemical characteristics. Key constraints that limit limonene biosynthesis and its oxyfunctionalization in recombinant E. coli, related to genetics, physiology, and reaction engineering, were identified and relieved.

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Hudson, Cheryl A. "Impact of biotechnology labs on high school biology students". Montana State University, 2011. http://etd.lib.montana.edu/etd/2011/hudson/HudsonC0811.pdf.

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There is a growing interest and emphasis on teaching biotechnology methods and concepts to high school level students in order to help prepare them to be able to participate in highly technological careers. Numerous biotechnology professional development programs exist for science teachers to gain knowledge and skills that are necessary to teach biotechnology. While it is an easy transition to teach biotechnology methods in advanced and AP level courses, there is uncertainty about the limitations and accommodations that will be necessary to incorporate biotechnology labs into a regular high school biology classroom with 28 students or more of various levels and exceptionalities. The additional expense and time necessary to incorporate biotechnology are justified if students gain increased conceptual understanding and demonstrate improved attitude toward science as a result of the labs. The primary question I sought to answer with this project was what are the effects of incorporating biotechnology labs on high school students' understanding of molecular biology concepts? Secondary questions related to the project are: What were the effects of incorporating biotechnology labs on students' interest in science, students' confidence in their abilities to do science, and on my teaching practices? The sequence of biotechnology labs that occurred within the curriculum of compulsory high school biology were quantitative protein analysis of food, DNA fingerprinting, pGLO bacterial transformation, and GMO investigation of food. The labs utilized Vernier Probeware and Bio-Rad Explorer kits. Conceptual understanding of molecular biology concepts was assessed using student developed concept maps and free-response questions. Anonymous student surveys and one-on-one student interviews were used to assess attitude toward science, which is defined in this project as interest, confidence, and relevance. Results for improved attitude were inconclusive; however gains in conceptual understanding were substantial with the biotechnology labs.

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4

Madani, Fatemeh. "Biophysical studies of peptides with functions in biotechnology and biology". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-66948.

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My thesis concerns spectroscopic studies (NMR, CD and fluorescence) of peptides with functions in biotechnology and biology, and their interactions with a model membrane (large unilamellar phospholipid vesicles). The resorufin-based arsenical hairpin binder (ReAsH) bound to a short peptide is a useful fluorescent tag for genetic labeling of proteins in living cells. A hairpin structure with some resemblance to type II β-turn was determined by NMR structure calculations (Paper I). Cell-penetrating peptides (CPPs) are short (30-35 residues), often rich in basic amino acids such as Arg. They can pass through the cell membrane and deliver bioactive cargoes, making them useful for biotechnical and pharmacological applications. The mechanisms of cellular uptake and membrane translocation are under debate. Understanding the mechanistic aspects of CPPs is the major focus of Papers II, III, and IV. The effect of the pyrenebutyrate (PB) on the cellular uptake, membrane translocation and perturbation of several CPPs from different subgroups was investigated (Paper II). We concluded that both charge and hydrophobicity of the CPP affect the cellular uptake and membrane translocation efficiency. Endosomal escape is a crucial challenge for the CPP applications. We modeled the endosome and endosomal escape for different CPPs to investigate the corresponding molecular mechanisms (Papers III and IV). Hydrophobic CPPs were able to translocate across the model membrane in the presence of a pH gradient, produced by bacteriorhodopsin proton pumping, whereas a smaller effect was observed for hydrophilic CPPs. Dynorphin A (Dyn A) peptide mutations are associated with neurodegenerative disorders, without involvement of the opioid receptors. The non-opioid activities of Dyn A may involve membrane perturbations. Model membrane-perturbations by three Dyn A mutants were investigated (Paper V). The results showed effects to different degrees largely in accordance with their neurotoxic effects.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

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5

Funder, Joshua V. "Biology, information and property : the legal appropriation of plant biotechnology". Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365449.

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6

Soenksen, Martinez Luis Rubén. "Cell-free freeze-dried synthetic biology for wearable biotechnology applications". Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/127730.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2020
Cataloged from PDF of thesis. "February 2020."
Includes bibliographical references (pages 163-173).
Synthetic biology aims to develop modular genetic networks for computation, sensing, and control of biological systems, holding great promise for next-generation biosensing platforms. Similarly, advances in material sciences have allowed for the design of substrates and textiles engineered to exhibit novel mechanical, electrical, and optical properties for sensing and actuation. Wearable biosensors using synthetic biology principles and smart materials could expand on this potential, especially as solutions for continuous, fine-grained monitoring of physiological status, disease states, and pathogen/toxin exposure difficult to assess with other methods. Despite this, only few examples of synthetic biology sensors compatible with wearable use-cases have been described, all of which rely on the use of live engineered bacteria with sustainment limitations.
Thus, we report on the development of novel shelf-stable, genetically-programmable, and highly sensitive wearable sensing platforms based on cell-free synthetic biology components freeze-dried into flexible substrates and textiles; as well as on a new class of smart programmable synthetic biology materials capable of reacting to environmental queues. These systems were designed to exhibit colorimetric, fluorescent, luminescence, electrical, or mechanical outputs that can be passively or actively interrogated within isolated modules or in larger-scale garments with wireless networking capabilities. We functionally validated such platforms using a variety of synthetic biology circuits for detecting several relevant environmental exposure targets such as metabolites, chemicals, and pathogen-associated nucleic acids.
These findings suggest that cell-free synthetic biology tools have the potential to enable highly programmable wearable systems for rapid on-body detection or adaptation to external threats in first responders, warfighters or clinical personnel, as well as the assessment of athletic performance and monitoring to complex disease states.
by Luis Rubén Soenksen Martinez.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Mechanical Engineering

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7

Camsund, Daniel. "Engineering Transcriptional Systems for Cyanobacterial Biotechnology". Doctoral thesis, Uppsala universitet, Molekylär biomimetik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-223599.

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Cyanobacteria are solar-powered cell factories that can be engineered to supply us with renewable fuels and chemicals. To do so robust and well-working biological parts and tools are necessary. Parts for controlling gene expression are of special importance in living systems, and specifically promoters are needed for enabling and simplifying rational design. Synthetic biology is an engineering science that incorporates principles such as decoupling, standardization and modularity to enable the design and construction of more advanced systems from simpler parts and the re-use of parts in new contexts. For these principles to work, cross-talk must be avoided and therefore orthogonal parts and systems are important as they are decoupled by definition. This work concerns the design and development of biological parts and tools that can enable synthetic biology in cyanobacteria. This encompasses parts necessary for the development of other systems, such as vectors and translational elements, but with a focus on transcriptional regulation. First, to enable the development and characterization of promoters in different cyanobacterial chassis, a broad-host-range BioBrick plasmid, pPMQAK1, was constructed and confirmed to function in several cyanobacterial strains. Then, ribosome binding sites, protease degradation tags and constitutive, orthogonal promoters were characterized in the model strain Synechocystis PCC 6803. These tools were then used to design LacI-regulated promoter libraries for studying DNA-looping and the behaviour of LacI-mediated loops in Synechocystis. Ultimately, this lead to the design of completely repressed LacI-regulated promoters that could be used for e.g. cyanobacterial genetic switches, and was used to design a destabilized version of the repressed promoter that could be induced to higher levels. Further, this promoter was used to implement an orthogonal transcriptional system based on T7 RNAP that was shown to drive different levels of T7 promoter transcription depending on regulation. Also, Gal4-repressed promoters for bacteria were engineered and examined in Escherichia coli as an initial step towards transferring them to cyanobacteria. Attempts were also made to implement a light-regulated one-component transcription factor based on Gal4. This work provides a background for engineering transcription and provides suggestions for how to develop the parts further.

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8

Bigler, Amber L. "Student Content Knowledge Increases After Participation in a Hands-on Biotechnology Intervention". BYU ScholarsArchive, 2010. https://scholarsarchive.byu.edu/etd/2522.

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Hands-on learning is at the heart of science learning. This study examined increased changes of student content knowledge in biology, particularly biotechnology, after a hands-on biotechnology intervention was implemented into a secondary school. A traditional learning school was selected for a control. Both teachers had participated in a biotechnology professional development program called Project Crawfish. Students from both schools took the same assessment before and after their respective units (biotechnology intervention and genetics unit), and the classroom was the unit of analysis (n=5, n=6, respectively). The assessment was compared as a whole and then divided into five components, eight questions each: DNA extraction/gel electrophoresis, polymerase chain reaction (PCR), DNA sequencing, bioinformatics, and phylogenetics. The pre-tests were compared to establish a baseline between the two schools. The biotechnology intervention school began with a higher pre-test raw score than the traditional learning school. After adjusting for the pre-test scores, each school was analyzed for increases in student content knowledge and then compared to each other for any significant increases between the two schools. When the entire assessment was analyzed, each school had statistically significant increases in student content knowledge (<0.0001 for the biotechnology intervention school and 0.0481 for the traditional learning school). When the schools were compared to each other, a p-value of 0.0543 provided a suggestive relationship that the biotechnology intervention school had a larger increase in student content knowledge. When the assessment was divided into the five components, the traditional learning school had statistically significant increases in student content knowledge in the PCR and DNA sequencing components (0.0459, 0.0043, respectively). The biotechnology intervention school had statistically significant increases in student content knowledge in all five components. However, there were no significant differences in learning between the two schools. Implementing biotechnology through hands-on teaching methods should be considered by secondary science teachers. Further research would scale up this study to include more classrooms.

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9

Dias, Camila Arnaldo Olhê [UNESP]. "Análise estrutural e funcional de eIF5A selvagem e mutadas". Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/100727.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e é essencial para a viabilidade celular. Este fator tem sido associado com o início da tradução, proliferação celular, transporte nucleocitoplasmático e decaimento de mRNA. Estudos recentes associam eIF5A com a elongação, ao invés do inicio da tradução. eIF5A é a única proteína conhecida que contém o aminoácido essencial hipusina, gerado pelas enzimas desoxihipusina sintase e desoxihipusina hidroxilase. O objetivo deste estudo foi a caracterização estrutural e funcional de eIF5A de S. cerevisiae. Primeiramente, a estrutura terciária de eIF5A foi determinada por cristalografia e foi demonstrada a sua dimerização em solução, independentemente do resíduo hipusina. Foram obtidos e caracterizados 40 mutantes novos de eIF5A, dos quais 19 não complementaram o nocaute do gene selvagem, 13 apresentaram fenótipo de termossensibilidade e 8 não apresentaram nenhuma alteração nos fenótipos investigados. A maioria dos mutantes novos tem seus fenótipos resultantes da degradação da proteína eIF5A. Curiosamente, este é o primeiro estudo que sugere que a α-hélice presente no C-terminal de eIF5A é essencial para a manutenção da sua estrutura. Descrevemos também, que a extensão N-terminal de eIF5A, presente apenas em eucariotos, não é essencial para estrutura e função dessa proteína. Além disso, os mutantes contendo substituições na alça onde está localizado o aminoácido hipusina são inviáveis ou termossensíveis. Embora estes mutantes produzam eIF5A, inclusive na temperatura não permissiva, a proteína produzida não é hipusinada. Finalmente, dois mutantes termossensíveis (tif51AK56A e tif51AQ22H/L93F) produzem a proteína eIF5A estável na temperatura não permissiva, no entanto, apresentam...
The translation initiation factor 5A (eIF5A) is highly conserved from archae to mammals and is essential for cell viability. This factor has been associated with translation initiation, cell proliferation, nucleocytoplasmatic transport and mRNA decay. Recent studies show eIF5A involved in elongation, rather than translation initiation. eIF5A is the only protein known to contain the essential amino acid residue hypusine, generated by the enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase. The main goal of this study was the structural and functional characterization of S. cerevisiae eIF5A. First of all, the tertiary structure of eIF5A was determined by crystallography and this protein was defined as a dimer in solution, independently of the hipusine residue. We obtained and characterized 40 new mutants, which 19 cannot complement tif51A knockout cells, 13 are temperature-sensitive and 8 show no detectable phenotype. The phenotypes of most mutantes are caused by protein folding defects. Interestingly, this is the first study suggesting that the C-terminal -helix present in yeast eIF5A may be an essential structural element. Moreover, we describe that the eIF5A N-terminal extension present only in eukaryotic homologues is not essential in yeast. Furthermore, the mutants containing substitutions surrounding the hypusine modification site showed unviable or temperature-sensitive phenotypes. Although these mutant proteins were stable, they were defective in hypusine modification. Finally, two of the temperature-sensitive mutant strains (tif51AK56A and tif51AQ22H/L93F) produced stable eIF5A protein but showed defects in growth and protein synthesis and these mutants revealed polysome profile defect similar to that described for mutations in factors involved in translation... (Complete abstract click electronic access below)

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10

Kim, Daniel. "Characterization of the MATα pre-/pro- peptide by mutagenesis as a means to optimize secretion in pichia pistoris". Scholarly Commons, 2009. https://scholarlycommons.pacific.edu/uop_etds/738.

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The unicellular yeast, Pichia pastoris has currently emerged as one of the most popular host systems for heterologous proteins due to its relatively cheap cost, easy genetic manipulability, ability to perform post-translational modifications on proteins, and respiratory growth capabilities which allow it to be cultured in very high concentrations. Over 700 foreign proteins have been recombinantly expressed using P. pastoris. Although P. pastoris appears to be an ideal host system, its main drawback is its inability to efficiently export some heterologous proteins into the extracellular medium. The incorporation of S. cerevisiae's MATα pre-pro signal leader (MATα) has led to increased protein secretion in most cases. MATα is thus used in the production of 90% of all proteins secreted in P. pastoris. However secretion efficiency still remains a problem. It has been suspected that low secretion may be attributed to improper extracellular targeting (a function of MATα). In order to address these issues there has been a precedent for performing limited mutagenesis of a signal leader peptide (like MATα) to increase protein secretion. In one study the insertion of a 10 amino-acid residue into MATα resulted in a 5-fold increase in secretion of bacterial phytase, an important industrial enzyme. Despite this success there have been no systematic mutagenesis processes which would help elucidate the reason behind this case of increased secretion. In our study, we performed a series of mutagenesis events, both random and site directed, with the intent of illuminating the mechanisms of MATα that contribute to secretion. As a result were able to create a novel secretion signal (pLL3) with enhanced secretion levels of our reporter protein HRP.

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Uys, Lafras. "Computational systems biology of sucrose accumulation in sugarcane". Thesis, Link to the online version, 2006. http://hdl.handle.net/10019/245.

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Bücking, Clemens [Verfasser], i J. [Akademischer Betreuer] Gescher. "Biology and biotechnology of dissimilatory metal reduction in Shewanella oneidensis / Clemens Bücking. Betreuer: J. Gescher". Karlsruhe : KIT-Bibliothek, 2012. http://d-nb.info/1037154258/34.

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Brusman, Anna-Lena. "Etiska aspekter av preimplantatorisk genetisk diagnostik och genterapi". Thesis, Linköping University, The Department of Physics, Chemistry and Biology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-10019.

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The research in the field of biotechnology is rapidly developing all over the world. Modern biotechnology offers unique opportunities, simultaneously as it gives rise to a number of ethical issues. Preimplantation genetic diagnosis (PGD), PGD/HLA (Human Leucocyte Antigen) and germline gene therapy (GLGT) are controversial techniques. PGD gives a possibility to identify a genetic disease prior to the embryo’s implantation in the uterus. PGD/HLA involves selecting an embryo with genes coding for a specific tissue type, so that the child to be born can act as a donor to an existing sibling who requires a stem cell transplant. GLGT seeks to eliminate or change “bad” genes.

The purpose of this study is to investigate student’s ethical attitude concerning PGD, PGD/HLA and GLGT.

The empirical study was based on focus group discussions. Four group interviews were made, with 15 participants in all. The students are taking courses in biology or religion.

The result from the interviews shows that the ethical issues are difficult to have a definite opinion in, because there are possibilities and risks involved in all these techniques, according to the students. A central part of the discussion was devoted to human dignity and the moral status of the embryo. They also see risks such as bioterrorism, designing the perfect humans, economic interests, medical risks, among many other risks.

Forskningen på det bioteknologiska området utvecklas snabbt över hela världen. Den moderna bioteknologin erbjuder unika möjligheter, samtidigt som den ger upphov till en rad frågor av etiskt slag. Preimplantatorisk genetisk diagnostik (PGD), PGD/HLA (Human Leucocyte Antigen) och zygotisk genterapi är kontroversiella tekniker. PGD ger en möjlighet att identifiera genetiska sjukdomar före embryots implantering i livmodern. Med hjälp av PGD/HLA väljs ett embryo ut vars gener kodar för en specifik vävnadstyp, vilket gör att barnet som föds kan fungera som donator till ett existerande syskon som är i behov av en stamcells transplantation. Med zygotisk genterapi kan man ta bort eller byta ut ”dåliga” gener.

Syftet med uppsatsen är att undersöka studenters etiska värderingar rörande PGD, PGD/HLA och zygotisk genterapi.

Den empiriska studien baserades på fokusgrupp diskussioner. Fyra gruppintervjuer gjordes, med sammanlagt 15 deltagare. Studenterna studerar på programutbildningar i biologi eller religion.

Resultatet från intervjuerna visar att de etiska frågeställningarna är svåra att ha en klar uppfattning om, eftersom det finns möjligheter och risker med alla dessa tekniker, enligt studenterna. En central del av diskussionen ägnades åt människovärdet och embryots moraliska status. De ser också risker som bioterrorism, designa perfekta människor, ekonomiska intressen, medicinska risker, bland många andra risker.

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14

Lang, Claus. "Magnetosome-specific expression of chimeric proteins in Magnetospirillum gryphiswaldense for applications in cell biology and biotechnology". Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-105376.

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Camargo, Ramírez Rosany del Carmen. "Function of microRNAs in plant innate immunity". Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/405716.

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Esta tesis aborda el estudio de miARNs en la inmunidad innata en plantas. El trabajo se ha desarrollado en arroz (Capítulo I y Capítulo II) y en Arabidopsis (Capítulo III), En el capítulo I se describe la identificación y caracterización funcional de nuevos miARNs de arroz en su interacción con el hongo Magnaporthe oryzae. Este hongo es responsable de la piriculariosis, una de las enfermedades más devastadoras para el cultivo del arroz a nivel mundial. A partir de la información generada mediante secuenciación masiva de bibliotecas de pequeños ARNs de arroz, se seleccionaron secuencias candidatas a representar nuevos miARNs de arroz, habiéndose estudiado 5 de estos candidatos (miR-64, miR-75, miR-96, miR-98 y miR-203). La obtención de líneas transgénicas de arroz ha permitido demostrar que la sobreexpresión de MIR-64 y MIR-75 confiere resistencia a M. oryzae, tratándose por lo tanto de miARNs que funcionan como reguladores positivos en la respuesta inmune de arroz. Por otra parte, la sobreexpresión de MIR-96, MIR-98 o MIR-203 aumenta la susceptibilidad a la infección por M. oryzae en plantas de arroz (reguladores negativos de la respuesta inmune). El análisis de mutantes de arroz afectados en la biogénesis de miARNs (mutantes dcl1, dcl3 y dcl4) indican que la producción del miARN maduro miR-64, miR-75 o miR-96 es dependiente de DCL3 y/o DCL4, lo cual apoya la idea de que se trata de nuevos miARNs de arroz. Además, mediante edición génica por CRISPR/Cas9, se ha comprobado que una delección de 22 nucleótidos en el precursor miR-75 resulta en un fenotipo de susceptibilidad a M. oryzae (Capítulo II), lo que concuerda con el fenotipo de resistencia que se observa en las plantas que sobreexpresan este miARN. En el capítulo III se ha estudiado la función de miR858 en la inmunidad innata de Arabidopsis thaliana frente a la infección por hongos patógenos. Este miARN reprime la expresión de factores de transcripción de tipo MYB que actúan como activadores de la expresión de genes que participan en la biosíntesis de flavonoides. Cuando la actividad del miR858 se encuentra bloqueada por la expresión de un gen de imitación de díana (plantas MIM858), las plantas son resistentes a la infección por hongos patógenos (Plectosphaerella cucumerina, Fusarium oxysporum f. sp. Conglutinans and Colletotrichum higginsianum), mientras que la sobreexpresión de este miARN confiere mayor susceptibilidad a la infección. Además, la interferencia con la actividad de miR858, y consiguiente aumento de la expresión de genes MYB, en las plantas MIM858 afecta de manera importante el metabolismo de fenilpropanoides, priorizándose la síntesis y acumulación de flavonoides, a expensas de la síntesis de precursores de lignina. La actividad antifúngica que se observa para kaempferol, naringenina (flavonoides) y ácido p-cumárico, explicaría el fenotipo de resistencia a la infección por hongos que se observa en las plantas MIM858. En su conjunto, los resultados obtenidos en este trabajo demuestran que los miARNs son componentes importantes en la resistencia/susceptibilidad a la infección por patógenos fúngicos en plantas de arroz y Arabidopsis. Un mayor conocimiento de función de miARNs en la inmunidad innata de las plantas, y de los procesos que son regulados por estos riboreguladores, puede ser de utilidad en el diseño de nuevas estrategias para el control de enfermedades en plantas.
This thesis comprises the study of miRNAs in innate immunity in plants. The work has been developed in rice (Chapter I and Chapter II) and in Arabidopsis (Chapter III), model systems used in studies of functional genomics in monocotyledonous and dicotyledonous species, respectively. Chapter I describes the functional identification and characterization of new rice miRNAs in their interaction with the fungus Magnaporthe oryzae. This fungus is responsible for blast disease, one of the most devastating diseases for rice cultivation worldwide. From the information generated by high-throughput sequencing of small rice RNA libraries, candidate sequences to represent novel rice miRNAs were selected. In this work 5 of these candidates have been studied (miR-64, miR-75, miR-96, miR-98 and miR-203). Obtaining transgenic rice lines has demonstrated that the overexpression of MIR-64 and MIR-75 confers resistance to M. oryzae, therefore these miRNAs function as positive regulators in the rice immune response. Moreover, overexpression of MIR-96, MIR-98 or MIR-203 increase susceptibility to M. oryzae in rice plants (negative regulators of immune response). Analysis of rice mutants affected in the miRNA biogenesis (dcl1, dcl3 and dcl4 mutants) indicate that the mature miRNA production of miR-64, miR-75 or miR-96 depends on DCL3 and/or DCL4, which supports the idea that they are novel rice miRNAs. Furthermore, by gene editing using CRISPR/Cas9, it has been found that a 22 nucleotides deletion in miR-75 precursor results in a susceptibility phenotype under M. oryzae infection (Chapter II), in agreement with a resistance phenotype that was observed in overexpressor plants for this miRNA. In chapter III, the miR858 function in Arabidopsis thaliana innate immunity to infection by pathogenic fungi was studied. This miRNA represses the expression of MYB transcription factors, which act as activators of the expression of genes involved in flavonoids biosynthesis. Plants are resistant to infection by pathogenic fungi (Plectosphaerella cucumerina, Fusarium oxysporum f. sp. Conglutinans and Colletotrichum higginsianum) when the activity of miR858 is blocked by the expression of target mimicry (MIM858 plants), while the overexpression of this miRNA confers greater susceptibility to infection. Additionally, interference with miR858 activity and consequent increase of MYB gene expression in MIM858 plants significantly affects phenylpropanoids metabolism, favoring the synthesis and accumulation of flavonoids, and disfavoring the synthesis of lignin precursors. The antifungal activity that was observed for Kaempferol, naringenin (flavonoids) and p-Coumaric acid, would explain the resistant phenotype by fungi infection which is observed in the MIM858 plants. Altogether, the results obtained in this work demonstrate that miRNAs are an important component in the resistance/susceptibility to infection by pathogenic fungi in Arabidopsis and rice plants. Greater knowledge of miRNA function in plant innate immunity and processes that are regulate by these riboregulators, can be useful in the design of new strategies for the control of diseases in plants.

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Arnosti, Lis Velosa [UNESP]. "Reação de trans-splicing in vitro utilizando extrato nuclear livre de células e sequencia parcial de alfa tubulina de Trypanosoma cruzi". Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/88008.

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A família Trypanosomatidae compreende um grande número de protozoários parasitas, incluindo importantes agentes etiológicos de doenças humanas. Entre os causadores de doenças, destaca-se o Trypanosoma brucei (responsável pela Doença do Sono) e o Trypanosoma cruzi (agente causador da Doença de Chagas). Ambos possuem como mecanismo de processamento dos seus mRNAs o “trans-splicing”, que envolve a excisão de íntrons e a união dos éxons de dois transcritos independentes, o splice-leader, e o pré-mRNA aceptor. As reações de cis e trans-splicing in vitro com extrato nuclear de células HeLa já foram padronizadas e utilizada como modelo em diversos experimentos. Entretanto, em tripanosomas, as únicas referências sobre a reação de transsplicing in vitro são de Vianna et. al., (2001) e Skaked et. al., (2010), com extratos de parasitas preparados de modos diferentes. A reação com células permeáveis do Trypanosoma cruzi foi usada como modelo para análise de drogas tripanocidas na reação de trans-splicing (Barbosa et. al., 2007); porém, não é um sistema livre de células conforme mencionado acima. Diante disso, este trabalho propõe reproduzir a reação de “trans-splicing” in vitro com extratos nucleares livre de parasitas utilizando as formas epimastigotas de T. cruzi e/ou as formas prociclicas de T. brucei e uma sequência parcial de alfa tubulina de T. cruzi como pré-mRNA aceptor. A padronização desta reação in vitro possibilitará avanços no entendimento da maquinaria do trans-spliceossomo, tornando-se também um modelo interessante para avaliação de mecanismo de ação de drogas tripanocidas
Trypanosomatidae family comprises a large number of protozoan parasites, including important etiological agents of neglected human diseases. Among the diseases’ agents, Trypanosoma brucei (responsible for sleeping sickness) and Trypanosoma cruzi (causative agent of Chagas' disease) are the most important parasites. Both have trans-splicing as a mechanism for the processing of its mRNA, which involves the excision of introns and union of exons form two independent transcripts, the spliceleader (SLRNA), and the acceptor pre-mRNA. The reaction of cis-and transsplicing in vitro with nuclear extract of HeLa cells has already been standardized and used as a model in several experiments. However, in trypanosomes, the only references on the reaction of trans-splicing in vitro are from Vianna et. al., (2001) and Skaked et. al., (2010), using parasitefree extracts prepared on different methods. For trypanocidal drugs analyze, trans-splicing reaction using T. cruzi permeable cells was used as a model (Barbosa et. al., 2007), but this is not the same as free-cell nuclear extract as mentioned above. Thus, this work shows trans-splicing in vitro reaction using nuclear extracts either from T. cruzi epimastigote forms and/or T. brucei procyclic forms reacting with the T. cruzi alpHa-tubulin cloned sequence as pre-mRNA acceptor. The standardization of this in vitro reaction will be able to promote advances to understanding the transspliceosomo machinery, as well as occurred in mammalian cis- and/or trans-splicing, becoming also an interesting model for evaluating trypanocidal drugs interference

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Dias, Camila Arnaldo Olhê. "Análise estrutural e funcional de eIF5A selvagem e mutadas /". Araraquara : [s.n.], 2010. http://hdl.handle.net/11449/100727.

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Resumo: O fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e é essencial para a viabilidade celular. Este fator tem sido associado com o início da tradução, proliferação celular, transporte nucleocitoplasmático e decaimento de mRNA. Estudos recentes associam eIF5A com a elongação, ao invés do inicio da tradução. eIF5A é a única proteína conhecida que contém o aminoácido essencial hipusina, gerado pelas enzimas desoxihipusina sintase e desoxihipusina hidroxilase. O objetivo deste estudo foi a caracterização estrutural e funcional de eIF5A de S. cerevisiae. Primeiramente, a estrutura terciária de eIF5A foi determinada por cristalografia e foi demonstrada a sua dimerização em solução, independentemente do resíduo hipusina. Foram obtidos e caracterizados 40 mutantes novos de eIF5A, dos quais 19 não complementaram o nocaute do gene selvagem, 13 apresentaram fenótipo de termossensibilidade e 8 não apresentaram nenhuma alteração nos fenótipos investigados. A maioria dos mutantes novos tem seus fenótipos resultantes da degradação da proteína eIF5A. Curiosamente, este é o primeiro estudo que sugere que a α-hélice presente no C-terminal de eIF5A é essencial para a manutenção da sua estrutura. Descrevemos também, que a extensão N-terminal de eIF5A, presente apenas em eucariotos, não é essencial para estrutura e função dessa proteína. Além disso, os mutantes contendo substituições na alça onde está localizado o aminoácido hipusina são inviáveis ou termossensíveis. Embora estes mutantes produzam eIF5A, inclusive na temperatura não permissiva, a proteína produzida não é hipusinada. Finalmente, dois mutantes termossensíveis (tif51AK56A e tif51AQ22H/L93F) produzem a proteína eIF5A estável na temperatura não permissiva, no entanto, apresentam... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The translation initiation factor 5A (eIF5A) is highly conserved from archae to mammals and is essential for cell viability. This factor has been associated with translation initiation, cell proliferation, nucleocytoplasmatic transport and mRNA decay. Recent studies show eIF5A involved in elongation, rather than translation initiation. eIF5A is the only protein known to contain the essential amino acid residue hypusine, generated by the enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase. The main goal of this study was the structural and functional characterization of S. cerevisiae eIF5A. First of all, the tertiary structure of eIF5A was determined by crystallography and this protein was defined as a dimer in solution, independently of the hipusine residue. We obtained and characterized 40 new mutants, which 19 cannot complement tif51A knockout cells, 13 are temperature-sensitive and 8 show no detectable phenotype. The phenotypes of most mutantes are caused by protein folding defects. Interestingly, this is the first study suggesting that the C-terminal -helix present in yeast eIF5A may be an essential structural element. Moreover, we describe that the eIF5A N-terminal extension present only in eukaryotic homologues is not essential in yeast. Furthermore, the mutants containing substitutions surrounding the hypusine modification site showed unviable or temperature-sensitive phenotypes. Although these mutant proteins were stable, they were defective in hypusine modification. Finally, two of the temperature-sensitive mutant strains (tif51AK56A and tif51AQ22H/L93F) produced stable eIF5A protein but showed defects in growth and protein synthesis and these mutants revealed polysome profile defect similar to that described for mutations in factors involved in translation... (Complete abstract click electronic access below)
Orientador: Sandro Roberto Valentini
Coorientador: Cleslei Fernando Zanelli
Banca: Henrique Ferreira
Banca: Paulo Sergio Rodrigues Coelho
Banca: Nilson Ivo Tonin Zanchin
Banca: Beatriz Amaral de Castilho
Doutor

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Chakraborty, Ranjan. "Iron Uptake in Bacteria with Emphasis on E. coli and Pseudomonas". Digital Commons @ East Tennessee State University, 2013. http://amzn.com/9400760876.

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Contents: Ferric Siderophore Transport via Outer Membrane Receptors of Escherichia coli: Structural Advancement and A Tribute to Dr. Dick van der Helm -- An 'Ironman' of Siderophore Biology -- The Tricky Ways Bacteria Cope with Iron Limitation -- Iron Transport Systems and Iron Homeostasis in Pseudomonas. Abstract: Iron is essential for the growth of most bacteria because it serves as a cofactor for vital enzymes and for the components of the electron transport chain. Moreover, Iron plays an important role in bacterial pathogenicity; in fact, the iron transport systems in bacteria works as target for designing novel antibiotics. Because iron is not soluble under aerobic conditions, bacteria have had to find ways to overcome iron deficiency. One of them is producing an iron-chelating small organic molecule called siderophore. Indeed, most bacteria and fungi produce structurally and chemically diverse siderophores which are transported back to the cytoplasm using complex energy dependent transport systems.
https://dc.etsu.edu/etsu_books/1036/thumbnail.jpg

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SCALVENZI, Laura. "Amazonian plants from ethnomedicine to biotechnology through pharmaceutical biology approaches: a PhD experience in connecting forest with laboratory". Doctoral thesis, Università degli studi di Ferrara, 2011. http://hdl.handle.net/11392/2389376.

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The South american Natives, Shuar and Achuar people and their ethnomedical culture constitute the background subject of the Phd research, performed both in Ecuador (Salesian Politechnic University, Quito), and in Italy (Pharmaceutical biology labs, University of Ferrara). Based on ethnomedical responses, Piper aduncum, Maytenus macrocarpa, Schinus molle, Tecoma stans and Eugenia hallii were chosen as amazonian plant species subject of the research. AIMS The research has been focused on: − checking the presence of endophytic fungi in plants; − isolating and subculturing pure endophytic strains; − checking the biotransformation capacity of the isolated endophytes on pure compounds; the most performing endophytes were also tested on phytocomplexes and pure chemicals obtained by the plant from which the fungi were isolated; − phytochemical characterization and bioactivity assays of plant extracts: P. aduncum. − METHODS Biotransformations. Fresh aerial plant parts were properly washed in sanitizing solutions and in vitro cultured using adequate solid media to isolate endophytes. (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one, acetophenone, 1-indanone, 2-furyl methyl ketone, 2-methylcyclopentanone, 2-methylcyclohexanone, 2- methoxycyclohexanone were chosen as substrate model for biotransformations. The cultures were sampled after 1, 3, 7, 10 days of culturing, and ethyl acetate extracted to verify by GC-MS the presence of possible biotransformation products. Biotransformations were also checked on P. aduncum whole essential oil and on dillapiol, cis-ocimene, piperitone, (-)-terpinen-4-ol as most abundant chemicals. Chemical fingerprinting of P. aduncum essential oil. Steam distillation was adopted to obtain the essential oil, then characterized by GC-MS, NMR analyses. In vitro bioassays of P. aduncum essential oil. Antimicrobial activities were checked in vitro using proper agarized media to reach MIC. Antioxidant capacities were checked through DPPH test, ABTS and photochemiluminescence assays. Born's turbidimetric method and Writhing test were respectively adopted to check platelet-aggregation and anti-nociceptive properties. Mutagenic, antimutagenic properties and toxicity were assayed using classical and modified Ames test. MAIN RESULTS 364 fungal strains were in vitro isolated. Among all, 5 strains performed biotransformations on acetophenone to (S)-1-phenylethanol, with important yields (78-97%) and enantiomeric excess (78- 100%). Three strains gave also phenols probably by enzymatic reactions (Baeyer-Villiger oxidations). 15 fungal strains gave the lactones (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-3- oxabicyclo[3.3.0]oct-6-en-2-one from (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one, probably as result of monooxygenase activation. Phytochemical characterization of P. aduncum essential oil has evidenced dillapiol as the most abundant terpene, followed by cis-ocimene, piperitone and terpinen-4-ol. Only cisocimene and piperitone gave several biotransformation products through dehydrogenation and hydroxylation reactions. The essential oil has evidenced non-mutagenic properties and interesting antifungal and antioxidant activities. CONCLUSIONS Several endophytic fungal strains from Amazonian plants were isolated and checked for biotransformations on pure chemicals and on P. aduncum essential oil. Data obtained will be useful for possible following patents about micro-organisms able to transform pharmaceutically interesting chemicals. Taxonomical characterization of the most performing fungal strains is still in progress. P. aduncum essential oil can be considered genotoxically safe and provides interesting antifungal and antioxidant properties, supporting its ethnomedical use as cicatrising and disinfectant crude drug and suggesting an extension of its employ as preservative ingredient.

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Baktula, Avinash M. "A Method Based on Conserved Multiple Amino Acid Properties to Predict Amino Acid Substitutions Which Maintain the Protein Structure". TopSCHOLAR®, 2004. http://digitalcommons.wku.edu/theses/1107.

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A METHOD BASED ON CONSERVED MULTIPLE AMINO ACID PROPERTIES TO PREDICT AMINO ACID SUBSTITUTIONS WHICH MAINTAIN THE PROTEIN STRUCTURE Avinash M. Baktula September 16, 2004 1-117 Directed by: Claire A. Rinehart, Doug McElroy and Sigrid Jacobshagen Department of Biology Western Kentucky University Proteins often contain several domains, each with a distinct structure. Such domains have evolved as units that, when combined in various arrangements, produce proteins of unique structure. This study was conducted to identify amino acid substitutions that don’t change structure. Amino acid properties which were conserved in proteins with identical structures were used to predict a set of amino acids profiles at each sequence position that can serve as viable substitutions. To test this analysis ten different protein sets were taken from the Conserved Domain Database of National Center for Biotechnology Information (NCBI). An amino acid index database of numerical indices representing various physicochemical and biochemical properties of amino acids were mapped onto the amino acid sequences in each dataset and these were used to select properties common to the proteins with the same structure. Based on these conserved properties, a substitution index percentage (SI%) was calculated to represent the relative ability of an amino acid to substitute at a given position and still maintain a protein structure. Amino acid profiles from different SI% ranges were used to create a set of substitutions into the consensus sequence of each dataset (AASCS). The AASCS from each SI% range were submitted to two validation programs, RPS-BLAST and PSI-PRED. The number of matches between the AASCS and the primary data set sequences for each SI% range was used to select the substitution profile ranges that best maintained the structure. It was concluded that amino acid, substitutions with SI% greater than 90% consistently conserved the structure of the protein. This method may prove useful in predicting the structure of proteins with induced mutations (site-directed mutagenesis), and in studies pertaining to protein engineering.

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Guragain, Bhuwan. "Transcriptional co-repressor response of Arabidopsis thaliana to different abiotic stress". ScholarWorks@UNO, 2013. http://scholarworks.uno.edu/td/1738.

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Plants adapt to the complex environmental challenges by regulating their gene expression. Analyses of plant genomes have identified many genes that are either expressed or repressed during environmental stress. However we do not have much information on gene repression. Transcriptional repression in Arabidopsis thaliana is caused by co-repressors that lack the DNA binding domain and are recruited by transcription factors to regulate target gene expression. The Sridhar lab has identified co-repressors SLK1, SLK2, and LUH, which prevent the expression of stress response genes under non-stress conditions. Arabidopsis transgenic lines expressing the GUS under the control of co-repressor’s promoter were created, to determine the conditions during which the co-repressor are induced. In addition to that, transgenic plants expressing YFP fused with the co-repressor were created to study the sub-cellular localization of the co-repressor. I found that SLK1, SLK2, and LUH are expressed ubiquitously in most of the plants tissue evidenced by the promoter fusion to the GUS reporter. SLK1, SLK2, and LUH are induced by osmotic, cold and dehydration stress conditions. Furthermore, these proteins are localized in the nucleus of the cell. Key words: Arabidopsis thaliana, Co-repressor, SLK1, SLK2, LUH, Transcription factors, Trangenesis, Stress condition, GUS, GFP

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Maynard, Ronald J. "Risking the future : biomedicine and modernity in the lives of adults with cystic fibrosis /". Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/6402.

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Sarrión, Perdigones Manuel Alejandro. "Design and development of modular DNA assembly tools for Multigene Engineering and Synthetic Biology in Plants". Doctoral thesis, Universitat Politècnica de València, 2014. http://hdl.handle.net/10251/35399.

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The post-genomics era has put at the disposal of modern plant breeders an endless list of genetic building blocks for the design of new biotechnological crops. After a first wave of single-gene transgenic with controversial public acceptance, genomic information and technology is paving the way for increasingly complex designs based in multiple gene engineering. Those designs aiming at the production of inexpensive health-promoting compounds are most likely to be welcomed by consumers. In this project we plan to develop new multigene assembling tools. During this PhD, a standardized collection of interchangeable genetic parts (including promoters, CDS, P-DNAs, etc) and vectors will be developed. The collection, inspired in Synthetic Biology standards, will be made easy-to-assemble in an interchangeable, semi-idempotent and seamless fashion by the addition of flanking recognition sites of type IIS Restriction endonucleases. The construction of the collection will facilitate multigene engineering and will constitute a first step towards enabling Synthetic Biology in plants.
Sarrión Perdigones, MA. (2014). Design and development of modular DNA assembly tools for Multigene Engineering and Synthetic Biology in Plants [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/35399
TESIS

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Avaca, Crusca Juliana Sposto [UNESP]. "Análise de interação funcional de elF5A com a tradução, repressão da tradução e degradação de mRNA em Saccharomyces cerevisiae". Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/100736.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O provável fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e sofre uma modificação pós-traducional única e essencial, em que uma lisina específica é convertida em um resíduo de hipusina. Este fator já foi relacionado ao transporte nucleocitoplasmático, à degradação de mRNA e à proliferação celular. Dados recentes restabelecem uma função para eIF5A na tradução e sugerem a sua atuação na etapa de elongação ao invés de início, como originalmente proposto. Uma vez que o envolvimento de eIF5A com o degradação de mRNA ainda não foi elucidado, tornou-se interessante estudar qual a natureza desta relação. O metabolismo de mRNA é um processo complexo que envolve as etapas da tradução (mRNAs nos polissomos), repressão da tradução (mRNAs acumulados em grânulos de estresse) e degradação de mRNA (mRNAs nos P bodies). Os componentes da maquinaria de degradação de mRNA acumulam-se nos P bodies e, neste trabalho, foi verificado que eIF5A não se localiza nestes corpúsculos. Ainda, foi avaliada a formação de P bodies no mutante tif51A-3, na temperatura não permissiva (38ºC), e foi verificada uma inibição na formação de P bodies. Outros mutantes com defeitos na elongação da tradução, como cca1-1 e eft2H699K, também apresentaram defeito na agregação dos P bodies. Foi avaliada a existência de interação genética sintética entre os mutantes tif51A-1 e tif51A-3 e mutantes de fatores envolvidos com a repressão da tradução e/ou degradação de mRNA. Foi revelada uma supressão parcial do fenótipo de termossensibilidade com nocautes dos genes SBP1, DHH1 e PAT1, que codificam fatores ativadores da remoção do capacete e repressores da tradução. Por outro lado, uma interação sintético doente entre os mutantes tif51A-1 e xrn1Δ foi...
The putative translation initiation factor 5A (eIF5A) is highly conserved from archaea to mammals and undergoes an unique and essential post-translational modification, in which a specific lysine residue is converted into a hypusine residue. This factor has been involved in several cellular processes such as nucleocytoplasmic transport, mRNA decay and cell proliferation. Recent data have re-established a function for eIF5A in translation and suggests a role in elongation rather than initiation as originally proposed. Since the involvement of eIF5A with mRNA decay has not been elucidated it has become interesting to study the aspects of this relationship. mRNA metabolism is a complex process that involves the steps of translation (mRNAs on the polysomes), translation repression (mRNAs accumulated in granules) and mRNA degradation (mRNAs in P bodies). The mRNA degradation machinery accumulates in P bodies and in this study we have verified that eIF5A is not localized inside them. P bodies assembly was evaluated in the tif51A-3 mutant at non-permissive temperature and this aggregation was inhibited. Other mutant strains with defects in translation elongation, such as cca1-1 e eft2H699K, also presented defective P body assembly. The existence of synthetic genetic interactions between the eIF5A mutants, tif51A-1 and tif51A-3, and translation repression and/or mRNA decay mutants was evaluated. A partial suppression of the temperature sensitive phenotype of the eIF5A mutants was revealed when SBP1, DHH1 and PAT1 genes were deleted. Since those proteins are decapping activators and translational repressors these interactions reveal a putative function for eIF5A as an inhibitor of translation repression. However, a synthetic sick phenotype between tif51A-1 and xrn1Δ mutants was observed. Finally, stress granules... (Complete abstract click electronic access below)

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Brohée, Sylvain. "Etude bioinformatique du réseau d'interactions entre protéines de transport ches les Fungi". Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210432.

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Les protéines associées aux membranes sont d'une importance cruciale pour la cellule. Cependant, en raison d'une plus grande difficulté de manipulation, les données biochimiques les concernant sont très lacunaires, notamment au point de vue de la formation de complexes entre ces protéines.

L'objectif global de notre travail consiste à combler ces lacunes et à préciser les interactions entre protéines membranaires chez la levure Saccharomyces cerevisiae et plus précisément, entre les transporteurs. Nous avons commencé notre travail par l'étude d'un jeu de données d'interactions à grande échelle entre toutes les perméases détectées par une méthode de double hybride spécialement adaptée aux protéines insolubles (split ubiquitin). Premièrement, la qualité des données a été estimée en étudiant le comportement global des données et des témoins négatifs et positifs. Les données ont ensuite été standardisées et filtrées de façon à ne conserver que les plus significatives. Ces interactions ont ensuite été étudiées en les modélisant dans un réseau d'interactions que nous avons étudié par des techniques issues de la théorie des graphes. Après une évaluation systématique de différentes méthodes de clustering, nous avons notamment recherché au sein du réseau des groupes de protéines densément interconnectées et de fonctions similaires qui correspondraient éventuellement à des complexes protéiques. Les résultats révélés par l'étude du réseau expérimental se sont révélés assez décevants. En effet, même si nous avons pu retrouver certaines interactions déjà décrites, un bon nombre des interactions filtrées semblait n'avoir aucune réalité biologique et nous n'avons pu retrouver que très peu de modules de protéines de fonction semblable hautement inter-connectées. Parmi ceux-ci, il est apparu que les transporteurs d'acides aminés semblaient interagir entre eux.

L'approche expérimentale n'ayant eu que peu de succès, nous l'avons contournée en utilisant des méthodes de génomique comparative d'inférence d'interactions fonctionnelles. Dans un premier temps, malgré une évaluation rigoureuse, l'étude des profils phylogénétiques (la prédiction d'interactions fonctionnelles en étudiant la corrééélation des profils de présence - absence des gènes dans un ensemble de génomes), n'a produit que des résultats mitigés car les perméases semblent très peu conservées dès lors que l'on considère d'autres organismes que les \
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

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Zhang, Xiaoli. "Using Silkworms as a Host to Spin Spider Silk-Like Fibers". DigitalCommons@USU, 2017. https://digitalcommons.usu.edu/etd/6368.

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Using silkworms as the potential host to spin spider silk-like fibers is an area of intense research world-wide. The conventional methods used to create transgenic silkworms hosting spider silk-like gene limits the incorporation of spider silk-like protein and do not improve the mechanical performance of the composite silkworm/spider silk fibers. In this dissertation, synthetic spider ampullate genes were incorporated into the precise site of the fibroin heavy chain or light chain using the latest genome editing technology CRISPR/cas9 guided non-homologous end joining as opposed to conventional random integration using transposon-based piggyBac system. These protocols, with extensive applicability to other silkworm researches, improved the content of spider silk-like protein in the transgenic silkworm/spider silk fibers, increases genetic stability in offspring, and improves the mechanical performance of the transgenic fibers compared to traditional methods. In addition, an enhanced green fluorescence protein (eGFP) was successfully incorporated into the fibroin light chain of silkworms using CRISPR/C as 9 initiated homologous recombination. The transgenic silkworm/spider fibers emitted strong green fluorescence under excitation. These results demonstrate that the we successfully developed a protocol to make silkworm as a host to spin spider silk-like fibers.

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Campos, Katherine Helen de Sa. "Identification and characterization of components that overcome secretion limitations of the yeast Pichia pastoris". Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/844.

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The methylotrophic yeast. Pichia pastoris, is a powerful, adaptable, and inexpensive recombinant expression system commonly used to secrete heterologous protein. Although P. pastoris is a popular host organism, secretion inefficiency continues to be a major hurdle in its ability to produce high levels of foreign protein. Optimization of cis- and trans-acting factors has greatly enhanced the secretory capabilities of P. pastoris, however protein-specific engineering of a host organism is costly and not always effective. P. pastoris' secretion inefficiency is commonly due to trans-acting factors. Strains of S. cerevisiae have been engineered, through random genomic mutation, that are capable of overcoming these /ram-acting factors to secrete high levels of foreign protein. The Lin-Cereghino laboratory at University of the Pacific has developed a screen to identify mutations in P. pastoris capable of circumventing secretion obstacles. The P. pastoris genome was randomly disrupted through restriction enzyme-mediated integration of an antibiotic resistance marker. Supersecretion mutants were identified by their ability to secrete β-galactosidase, a reporter enzyme not natively secreted by P. pastoris. Sixteen β-galactosidase secretion (bgs) mutants were initially isolated by the Lin-Cereghino lab. This research focused on characterizing one of the resultant bgs mutants, ///. Initial sequencing and alignment studies identified the predicted LI1p sequence to be homologous to S. cerevisiae protein kinase C (PKC). Considering the role of PKC in the Cell Wall Integrity pathway of S. cerevisiae. the cell wall and secretory organelles of III were closely examined using transmission electron microscopy. Additionally, a qualitative alkaline phosphatase assay was used to evaluate the cell wall integrity of ///. Finally, the secretory phenotype of 111 was examined using a group of structurally and functionally diverse reporter proteins. In characterizing the bgs mutant, III, this research contributes to an understanding of cellular components that limit protein secretion in the yeast, P. pastoris.

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Antonio, Tatiana [UNESP]. "Obtenção de mutantes de Streptomyces clavuligerus e avaliação de condições de cultivo para a melhoria de produção de cefamicina C". Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/100744.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Mutantes foram obtidos através de tratamento mutagênico dos esporos de S. clavuligerus ATCC 27064 com metil-metanosulfonato. Um total de 822 colônias, obtidas por repiques sucessivos, foram selecionadas em testes qualitativos e/ou quantitativos. O melhor mutante S. clavuligerus 45.41, mostrou-se de duas a quatro vezes mais produtivo que a linhagem selvagem, e manteve-se estável após 35 repiques sucessivos ao longo deste estudo. O comportamento das linhagens foi investigado pela adição de diferentes diaminas ao meio de cultivo, uma a uma, na ausência de lisina. Também se investigou duas fontes distintas de C, além de compostos como o ácido 2,6- diaminopimélico. Um planejamento de dois fatores (cadaverina e lisina), três níveis, baseado no ponto central, faces centradas, indicou que a adição de lisina aumenta a produção de CefC para ambas as linhagens. O efeito positivo da cadaverina foi observado principalmente na linhagem mutante 45.41. Resultados obtidos em processo de batelada, em biorreator de bancada confirmaram as diferenças observadas entre as linhagens nos cultivos em frascos agitados. Procedimento de mutagênese clássica foi utilizado em conjunto com critérios bem definidos para se estabelecer um meio de cultura apropriado, com o objetivo de alcançar um aumento significativo da produção de CefC. Este é o primeiro estudo utilizando um mutante de S. clavuligerus obtido por mutagênese clássica, cuja produção de CefC é de três vezes maior que a da linhagem selvagem, em meios contendo diaminas
Mutants were obtained by treating S. clavuligerus ATCC 27064 spores with methyl-methanesulfonate. A total of 822 colonies, obtained by successive sampling, were selected by qualitative and/or quantitative tests. The best mutant, S. clavuligerus 45.41, was two to four times more productive than the wild-type strain, and remained stable even after 35 successive samplings throughout the study. Strains behavior was investigated by adding different diamines in media, one by one, in the absence of lysine. Also investigated two different sources of C, and compounds such as 2,6-diaminopimelic acid. The two-factor (cadaverine and lysine), three-level, central composite-based, face-centered experimental design indicates that adding lysine increases CephC production for both strains alike. The positive effect of cadaverine was observed mainly in the Mutant 45.41 strain. Results obtained in batch-processes in a bench-scale bioreactor confirmed differences among strains observed in shaken flasks cultures. Classical mutagenesis procedures in conjunction with the adoption of well-defined criteria to establish an appropriate culture medium promoted a significant improvement in CephC production. It is the first study indicating an increase in CephC production in media containing diamines employing a mutant of S. clavuligerus obtained by classical mutagenesis

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Arnosti, Lis Velosa. "Reação de trans-splicing in vitro utilizando extrato nuclear livre de células e sequencia parcial de alfa tubulina de Trypanosoma cruzi /". Araraquara : [s.n.], 2011. http://hdl.handle.net/11449/88008.

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Orientador: Regina Maria Barretto Cicarelli
Banca: Marcia Aparecida da Silva Graminha
Banca: Maurício Bacci Junior
Resumo: A família Trypanosomatidae compreende um grande número de protozoários parasitas, incluindo importantes agentes etiológicos de doenças humanas. Entre os causadores de doenças, destaca-se o Trypanosoma brucei (responsável pela Doença do Sono) e o Trypanosoma cruzi (agente causador da Doença de Chagas). Ambos possuem como mecanismo de processamento dos seus mRNAs o "trans-splicing", que envolve a excisão de íntrons e a união dos éxons de dois transcritos independentes, o splice-leader, e o pré-mRNA aceptor. As reações de cis e trans-splicing in vitro com extrato nuclear de células HeLa já foram padronizadas e utilizada como modelo em diversos experimentos. Entretanto, em tripanosomas, as únicas referências sobre a reação de transsplicing in vitro são de Vianna et. al., (2001) e Skaked et. al., (2010), com extratos de parasitas preparados de modos diferentes. A reação com células permeáveis do Trypanosoma cruzi foi usada como modelo para análise de drogas tripanocidas na reação de trans-splicing (Barbosa et. al., 2007); porém, não é um sistema livre de células conforme mencionado acima. Diante disso, este trabalho propõe reproduzir a reação de "trans-splicing" in vitro com extratos nucleares livre de parasitas utilizando as formas epimastigotas de T. cruzi e/ou as formas prociclicas de T. brucei e uma sequência parcial de alfa tubulina de T. cruzi como pré-mRNA aceptor. A padronização desta reação in vitro possibilitará avanços no entendimento da maquinaria do trans-spliceossomo, tornando-se também um modelo interessante para avaliação de mecanismo de ação de drogas tripanocidas
Abstract: Trypanosomatidae family comprises a large number of protozoan parasites, including important etiological agents of neglected human diseases. Among the diseases' agents, Trypanosoma brucei (responsible for sleeping sickness) and Trypanosoma cruzi (causative agent of Chagas' disease) are the most important parasites. Both have trans-splicing as a mechanism for the processing of its mRNA, which involves the excision of introns and union of exons form two independent transcripts, the spliceleader (SLRNA), and the acceptor pre-mRNA. The reaction of cis-and transsplicing in vitro with nuclear extract of HeLa cells has already been standardized and used as a model in several experiments. However, in trypanosomes, the only references on the reaction of trans-splicing in vitro are from Vianna et. al., (2001) and Skaked et. al., (2010), using parasitefree extracts prepared on different methods. For trypanocidal drugs analyze, trans-splicing reaction using T. cruzi permeable cells was used as a model (Barbosa et. al., 2007), but this is not the same as free-cell nuclear extract as mentioned above. Thus, this work shows trans-splicing in vitro reaction using nuclear extracts either from T. cruzi epimastigote forms and/or T. brucei procyclic forms reacting with the T. cruzi alpHa-tubulin cloned sequence as pre-mRNA acceptor. The standardization of this in vitro reaction will be able to promote advances to understanding the transspliceosomo machinery, as well as occurred in mammalian cis- and/or trans-splicing, becoming also an interesting model for evaluating trypanocidal drugs interference
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Campanale, Joseph Paul. "EXPOSURE TO ULTRAVIOLET RADIATION CAUSES PROTEOMIC CHANGES IN EMBRYOS OF THE PURPLE SEA URCHIN, STRONGYLOCENTROTUS PURPURATUS". DigitalCommons@CalPoly, 2009. https://digitalcommons.calpoly.edu/theses/142.

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The amount of solar ultraviolet radiation (UVR, 290-400 nm) reaching Earth’s surface is increasing due to ozone depletion and global climate change. Embryos of the purple sea urchin, Strongylocentrotus purpuratus, provide an ideal system for examining how UVR affects developing marine organisms and cells in general. To model the protein-mediated cell cycle response to UV-irradiation, six batches of S. purpuratus embryos were exposed to UVR, monitored for delays in the first mitotic division and examined for global proteomic changes. Embryos from each batch were exposed to or protected from artificial UVR for 25 or 60 min. Embryos treated with UVR for 60 min cleaved an average of 23.24 min (±1.92 s.e.m) later than the UV-protected embryos. Protein expression of UV-protected and UV-treated embryos was examined at 30 and 90 min post-fertilization using two dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) and mass spectrometry (MS). Proteins were isoelectrically focused (pH 4-7) and separated by molecular weight using SDS-PAGE. At least 1,306 protein spots were detected in all gels. A total of 171 protein spots (13% of the detected proteome) migrated differently in UV-treated embryos at 30 min post-fertilization and 187 spots (14%) at 90 min post-fertilization (2-way ANOVA, P= 0.03, n=6). Our results identify the differential migration of proteins from multiple cellular pathways and are the first to indicate that the mechanisms involved in the protein mediated UV-induced developmental delay are integrated among pathways for cellular stress, protein turnover and translation, signal transduction, general metabolism and involve the cytoskeleton.

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Abeille, Fabien. "Automatisation et intégration d'un réacteur de culture cellulaire pour un fonctionnement en continu". Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENS036/document.

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Au cours des six dernières décennies, la culture cellulaire est devenue une pratique courante. Elle est un outil majeur de la recherche biologique pour la compréhension du vivant, l'étude de maladies et la découverte de nouveaux médicaments. Elle représente un outil très répandu dans de nombreuses industries étant impliquées dans la production de produits alimentaires, cosmétiques et pharmaceutiques.Cependant, les cultures cellulaires en recherche et en industrie sont aujourd'hui confrontées à des limites et soulèvent des besoins à satisfaire. Elles sont toutes deux associées à des coûts élevés du fait des ressources nécessaires (cellules, réactifs, opérateurs qualifiés). Plus précisément, la culture en recherche est caractérisée par le faible débit des expériences, une variabilité importante et un risque de contamination due à la répétition d'opérations manuelles. De plus, les expériences de culture sont effectuées dans des conditions statiques et sur des modèles (cultures 2D, animaux...) relativement éloignés de la physiologie humaine. La culture cellulaire industrielle, quant à elle, a besoin de systèmes miniaturisés qui miment les procédés des bioréacteurs à grande échelle et qui offrent des possibilités de criblage plus élevés.Les systèmes de culture microfluidique représentent un outil prometteur pour résoudre ces problèmes et ces besoins. Le changement de comportement de la physique à petite échelle dans ces dispositifs permet de contrôler temporellement et spatialement le microenvironnement des cellules. Ce qui n'est pas possible avec des méthodes de culture classiques. Le degré d'automatisation et d'intégration permet une nette augmentation du nombre d'expériences par système et la réduction conséquente de la consommation de ressources. Ainsi, de nombreuses petites architectures 3D cellulaires cultivées dans des conditions dynamiques et à haut débit ont été réalisées et ont démontré leur capacité à recréer rapidement des environnements plus physiologiques. En ce qui concerne la culture industrielle, des cultures miniaturisées ont déjà montré leur capacité à reproduire les caractéristiques observées dans les macrobioreactors avec des possibilités de criblages élevées.Dans ce contexte, un bioréacteur microfluidique de paillasse, se conformant aux formats standards utilisés dans le laboratoire d'accueil, a été fabriqué avec succès au cours de cette thèse pour effectuer des cultures cellulaires en continu. Des solutions intégrées ont été mises au point pour fournir de façon continue les conditions adéquates pour la prolifération cellulaire (perfusion, régulation de température…). Des études ont également été menées afin d'automatiser la récolte des cellules avec pour but final de cultiver ces cellules sur du long terme dans le bioréacteur.Le système fabriqué garantit ainsi des conditions stériles pour les cultures sur un simple banc de laboratoire. En outre, ces cultures ont été réalisées de façon autonome sans utiliser un incubateur encombrant. Dans ces conditions, le bioréacteur permet de réaliser des cultures en continu de divers types cellulaires sur plusieurs jours: des cellules d'insectes ont été cultivées pendant 5 jours et des cellules de mammifère pendant 3 jours. En ce qui concerne les cultures de cellules de mammifère, une avancée majeure a été effectuée par rapport aux cultures réalisées dans les systèmes microfluidiques en utilisant comme support de culture des microporteurs (diam. : 175 µm).Bien que la culture de cellules sur microporteurs soit réalisée en routine dans l'industrie, aucun système de culture microfluidique autonome n'a encore intégré ce type de culture. Ce genre de miniaturisation est une avancée majeure pour des applications en bioprocédés où il devrait permettre de raccourcir et réduire les coûts associés au développement de bioproduits
Over the past six decades, cell culture has become a common practice. It is a major tool in biological research for the understanding of life science, such as the study of disease and the discovery of new drugs. It plays an important role in many industries since it is involved in the production of many food, cosmetic, and pharmaceutical products.However, Research and the industry are now facing some limits and are expressing needs to be addressed. They are both associated with high costs due to a large consumption of resources (cells, reagents, qualified operators). More specifically, cell culture in research is characterized by low throughput of experiments, important variability and risk of contamination due to the recurrent manual operations performed by operators. Additionally, experiments are performed in static conditions and on models (2D cultures, animals…) which poorly resemble the human physiology. Industrial cell culture needs miniaturized systems that mimic the large scale bioreactors and offer higher screening possibilities.Microfluidic cell culture systems represent a promising tool to address the aforementioned issues and needs. The change of physical behaviors at the small-scale in microfluidic devices allow controlling temporally and spatially the cell microenvironment, unattainable with conventional cell culture methods. The level of automation and integration allows the substantial increase of the number of experience per system and considerable reduction of resource consumption. Thus, many small cellular 3D architectures grown under dynamic conditions and in high-throughput have been performed and have demonstrated their ability to quickly re-create more physiological environments. Regarding the industrial culture, miniaturized cultures have already shown their ability to reproduce the characteristics of the culture observed in macrobioreactors with higher screening capabilities.In this framework, a benchtop microfluidic bioreactor, complying with the standard microfluidic platform and format used in the host laboratory, has been successfully fabricated to perform continuous cell cultures. Integrated solutions were developed to provide continuously the adequate conditions for cell proliferation (perfusion, thermal regulation…). Integrated cell harvest was also performed with the final goal to achieve long-term cell culture in the bioreactor.The fabricated system proved to guarantee sterile conditions for cell cultures on a regular lab bench. Moreover, these cultures were achieved autonomously without requiring a cumbersome incubator. In these conditions, the bioreactor demonstrated the possibility to perform continuous cell cultures of various cell types during several days: insects cells were cultured during 5 days and mammalian cells during 3 days. Regarding the mammalian cell cultures performed, a breakthrough has been achieved compared to the cultures performed in microfluidic systems since microcarriers (diam.:175 µm) were used as growth support.Although microcarrier cell culture is routinely performed in the industry, no autonomous microfluidic culture system has addressed this type of culture yet. Such a miniaturization is a major step forward for bioprocess applications where the need to develop scale-down bioreactors that mimic large scale operation has been clearly identified to shorten and reduce the costs associated to bioproduct development

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Serrat, Gurrera Xavier. "Applied biotechnology to improve Mediterranean rice varieties = Biotecnologia aplicada a la millora de varietats d’arròs mediterrànies". Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/396188.

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The current world population is over 7.4 billion and expected to exceed 9 billion in 2040, causing a 70% increase in food demand. Global environmental degradation, in the form of salinization, pollution and global warming, has also reduced the availability of suitable arable land and water sources, contributing to promote crop improvement in order to increase the potential yields. Rice (Oryza sativa) is the most widely consumed staple food for a large proportion of the world population. Classical rice breeding programs use its natural variability to create new allelic combinations which are screened for selecting those presenting superior agronomic traits such as improved yield. Those improved lines are stabilized through inbreeding to maintain the phenotype in their progeny. Certified seed producers systematically select and propagate registered varieties year by year in order to maintain their uniformity and the original registered cultivar traits, since natural mutations, spontaneous breeding between varieties and alien grain contamination can introduce undesirable variability at this stage. Nowadays biotechnology is used to drive the improvement of rice traits such as increased yield and grain quality. Moreover it helps to rapidly bestow tolerance to biotic (diseases and insects) and abiotic (drought, salinity, cold temperatures, nutrients deficiency) factors. Some of the available biotechnological techniques applied for crop improvement are i) the genetic engineering, which allows the addition of foreign genes in the rice genome although being controversial due to the social and environmental concerns, ii) the anther culture, which fasten and improves the selection of new breeding lines, and iii) the Targeting Induced Local Lesions IN Genomes (TILLING) which combines the production of large mutant populations with the detection of mutants in genes of interest through molecular screening. The main aim of the thesis is to study different biotechnological tools and their applications to the improvement of Mediterranean rice varieties. To achieve this biotechnology is used to study the pollen dispersion of a genetically engineered rice line, to accelerate the stabilization process through anther culture technique and to introduce new variability using a mutagenesis protocol followed by molecular detection of mutants. In this thesis we first studied the pollen-mediated gene flow between wild rice, conventional rice and an herbicide resistant transgenic rice line in order to determine gene flow rates in relation to the distance and the prevailing wind speed and direction. Results showed that pollen dispersal is dramatically effected by the distances between rice plants and the speed and direction of the prevailing wind. Furthermore, the enhanced pollen dispersal capability of weedy rice can also play an important role in transgenic pollen dispersal, which unfortunately had been underestimated. Then, we adapted an anther culture protocol in order to efficiently obtain commercial dihaploid lines from a Mediterranean japonica variety. Furthermore, we described the greenhouse and field trials used to select the best lines for registration which are now being successfully commercialized. Finally, we developed a fast protocol for obtaining mutants with agronomic interest. This protocol is based on ethyl methanesulfonate mutagenesis of seed-derived calli. The in vitro regenerated mutant population plants were directly screened for senescence-related genes, allowing to shorten in more than eight months the common seed mutagenesis protocol. The molecular screening protocol was also optimized and several potential delayed senescence mutants were identified and tested.

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Xu, Ying. "Antifouling compounds from deep-sea bacteria and their potential mode of action /". View abstract or full-text, 2009. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202009%20XU.

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Loggan, Briley. "Evaluating the Success of Female Selected Sex-Sorted Semen at Western Kentucky University's Dairy Farm". TopSCHOLAR®, 2019. https://digitalcommons.wku.edu/theses/3109.

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The purpose of this study was to evaluate the use of female selected sex-sorted semen and to determine the association of variables on the success of Western Kentucky University’s Dairy Farm. Official breeding and calving records (n=144) were used to determine the relation of lactation number, breeding season, breeding number, breeding year and semen type on pregnancy results, sex of offspring, and the mortality of the offspring. Previous research has shown pregnancy results can be influenced by lactation number, breeding season, number of breedings and semen type. Results from this study show that pregnancy results were not associated with lactation number (P=0.21), breeding year (P=0.22), breeding number (P=0.52) or semen type (P=0.99). Breeding season was associated with pregnancy results (P=0.04). Lactation number (P=0.40), breeding season (P=0.20) or breeding number (P=0.12) did not influence the sex of the offspring. The year of breeding and semen type (conventional or sexed) had a significant or close to significant effect on the sex of the offspring (P=0.01) and (P=0.06). The mortality of offspring was not associated with lactation number (P=0.46), breeding season (P=0.94), breeding year (P=0.76), breeding number (P=0.40) or semen type (P=0.49).

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Avaca, Crusca Juliana Sposto. "Análise de interação funcional de elF5A com a tradução, repressão da tradução e degradação de mRNA em Saccharomyces cerevisiae /". Araraquara : [s.n.], 2011. http://hdl.handle.net/11449/100736.

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Orientador: Sandro Roberto Valentini
Coorientador: Cleslei Fernando Zanelli
Banca: Paulo Sérgio Rodrigues Coelho
Banca: Flávio Henrique da Silva
Banca: Iran Malavazi
Banca: Carla Columbano de Oliveira
Resumo: O provável fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e sofre uma modificação pós-traducional única e essencial, em que uma lisina específica é convertida em um resíduo de hipusina. Este fator já foi relacionado ao transporte nucleocitoplasmático, à degradação de mRNA e à proliferação celular. Dados recentes restabelecem uma função para eIF5A na tradução e sugerem a sua atuação na etapa de elongação ao invés de início, como originalmente proposto. Uma vez que o envolvimento de eIF5A com o degradação de mRNA ainda não foi elucidado, tornou-se interessante estudar qual a natureza desta relação. O metabolismo de mRNA é um processo complexo que envolve as etapas da tradução (mRNAs nos polissomos), repressão da tradução (mRNAs acumulados em grânulos de estresse) e degradação de mRNA (mRNAs nos P bodies). Os componentes da maquinaria de degradação de mRNA acumulam-se nos P bodies e, neste trabalho, foi verificado que eIF5A não se localiza nestes corpúsculos. Ainda, foi avaliada a formação de P bodies no mutante tif51A-3, na temperatura não permissiva (38ºC), e foi verificada uma inibição na formação de P bodies. Outros mutantes com defeitos na elongação da tradução, como cca1-1 e eft2H699K, também apresentaram defeito na agregação dos P bodies. Foi avaliada a existência de interação genética sintética entre os mutantes tif51A-1 e tif51A-3 e mutantes de fatores envolvidos com a repressão da tradução e/ou degradação de mRNA. Foi revelada uma supressão parcial do fenótipo de termossensibilidade com nocautes dos genes SBP1, DHH1 e PAT1, que codificam fatores ativadores da remoção do capacete e repressores da tradução. Por outro lado, uma interação sintético doente entre os mutantes tif51A-1 e xrn1Δ foi... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The putative translation initiation factor 5A (eIF5A) is highly conserved from archaea to mammals and undergoes an unique and essential post-translational modification, in which a specific lysine residue is converted into a hypusine residue. This factor has been involved in several cellular processes such as nucleocytoplasmic transport, mRNA decay and cell proliferation. Recent data have re-established a function for eIF5A in translation and suggests a role in elongation rather than initiation as originally proposed. Since the involvement of eIF5A with mRNA decay has not been elucidated it has become interesting to study the aspects of this relationship. mRNA metabolism is a complex process that involves the steps of translation (mRNAs on the polysomes), translation repression (mRNAs accumulated in granules) and mRNA degradation (mRNAs in P bodies). The mRNA degradation machinery accumulates in P bodies and in this study we have verified that eIF5A is not localized inside them. P bodies assembly was evaluated in the tif51A-3 mutant at non-permissive temperature and this aggregation was inhibited. Other mutant strains with defects in translation elongation, such as cca1-1 e eft2H699K, also presented defective P body assembly. The existence of synthetic genetic interactions between the eIF5A mutants, tif51A-1 and tif51A-3, and translation repression and/or mRNA decay mutants was evaluated. A partial suppression of the temperature sensitive phenotype of the eIF5A mutants was revealed when SBP1, DHH1 and PAT1 genes were deleted. Since those proteins are decapping activators and translational repressors these interactions reveal a putative function for eIF5A as an inhibitor of translation repression. However, a synthetic sick phenotype between tif51A-1 and xrn1Δ mutants was observed. Finally, stress granules... (Complete abstract click electronic access below)
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Stimple, Samuel Douglas. "Recent Advances in Developing Molecular Biotechnology Tools for Metabolic Engineering and Recombinant Protein Purification". The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1514494485801145.

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Stefanutti, Erin. "ANGIOSTATIN LIKE PEPTIDES IN MILK: POTENTIAL DEVELOPMENT FOR DAIRY PRODUCTS CAPABLE OF CANCER PREVENTION". DigitalCommons@CalPoly, 2011. https://digitalcommons.calpoly.edu/theses/479.

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For the past 40 years, antiangiogenic approaches have been of major interest in the development of methods to cure and prevent cancer. Angiogenesis, the development of blood vessels from pre-existing vascularization, is essential for cancer growth and spread of metastasis through the delivery of nutrients and oxygen essential to sustain the metabolic activity of these malignant cells. Blocking access to blood will cause cancerous cells to assume a dormant state creating inactive micro-tumors innocuous to the host. Angiostatin, the internal fragment of the fibrinolytic zymogen plasminogen, has shown great potential in reducing cancer size and number of metastatic colonies in animal models. Owing to the success of these preliminary results angiostatin is currently on clinical trials. Plasminogen is known to be transferred from blood to milk during lactation. The objectives of this research were to: 1) investigate the ability of various proteases in cleaving plasminogen, both from human and bovine sources, and consequently release the angiostatin like fragment; 2) determine the anticancer activity of bovine angiostatin; 3) examine ability of the antiangiogenic fragment to survive digestion; 4) purify the fragment of interest through column chromatography. Production of angiostatin was tested through hydrolysis of plasminogen via Bacillus Polymyxa protease (or dispase I), elastase, lactic acid bacteria and Bacilli originated enzymes. Once proteases capable of angiostatin like peptide production were identified, and sequence analysis of the fragments obtained conducted to confirm that bovine angiostatin was indeed produced, ability of angiostatin, both human and bovine, in inhibiting malignant melanoma as well as colon cancer cells was evaluated in vitro. From the results obtained we can confirm that bovine angiostatin inhibitory activity on cancerous cells is similar to that observed for human angiostatin. Analysis of bovine angiostatin survival through in vitro human digestion model was also examined. Results show good possibility of angiostatin surviving digestion, even if confirmation of these results is required through further in vivo studies. Additionally, digestive enzymes such as trypsin and α-chymotrypsin showed ability in cleaving plasminogen directly to release a 25kDa fragment. Knowing that each kringle has some degree of anticancer activity it would be of interest to further study the possibility of angiostatin related fragments to be produced during milk digestion. Finally, affinity chromatography through L-lysine used to purify human angiostatin resulted to be an adequate method for bovine angiostatin purification. Preliminary results obtained from this study open a new area worth investigating to uncover the potential of using bovine angiostatin in the development of novel food products capable of cancer prevention.

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KUMAR, ANIL. "Optimization of Transgene Expression in Chlamydomonas reinhardtii and its Biotechnological Applications". The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1293558274.

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Hewabostanthirige, Dhanushka. "Loss of Id4 Promotes Stemness In Prostate Cancer Cells". DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2019. http://digitalcommons.auctr.edu/cauetds/182.

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Inhibitor of differentiation 4 (ID4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer (PCa). Recent studies have shown that Id4 is highly expressed in the normal prostate and decreases in prostate cancer due to epigenetic silencing. Id4 knockdown in androgen sensitive LNCaP cells has been shown to lead to castration resistant prostate cancer (CRPC) in vitro and in vivo. Id4-/- mice leads to underdeveloped prostate with PIN like lesions without the loss of Androgen Receptor (AR) expression. In this study we demonstrate that the loss of ID4 expression in PCa cell line LNCaP and DU145 may promote tumorigenesis by promoting stemness. LNCaP cells with stably silenced ID4 ((-)ID4) using retroviral based shRNA and LNCaP transfected with non-specific shRNA were used to perform colony forming assay and prostatosphere formation using matrigel. Expression of cancer stem cell markers was determined using western blotting and immunocytochemistry (ICC). FACS analysis was used to sort stem cells and determine the ID4 expression. Xenograft study was performed on SCID mice using CD133 positive LNCaP cells. LNCaP(-)ID4 and DU145 cells lacking ID4 showed increased holoclone as well as decreased paraclone formation, which are believed to be derived from stem cells and differentiated cells respectively, as compared to non-silencing control in the colony forming assay. There was also an increase in prostatosphere development in the LNCaP (-) ID4 cells indicating that the loss of ID4 is responsible for promoting the LNCaP cells towards cancer stem cells. The results were further validated via western blotting and ICC using known cancer stem cell markers on the holoclones and paraclones formed by these cells. Xenograft study showed that 10,000 cells from CD133 positive LNCaP cells developed tumor on SCID mice. This study reports for the first time that loss of ID4 increases holoclone and prostatosphere formation indicating that Id4 may contribute to promoting stemness in prostate cancer cells.

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Choi, Adam. "Illuminating Biology with Membrane Penetrating Sulfonate Delivery Scaffolds and Near-Infrared Azasiline Fluorophores". eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/997.

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Near-infrared (NIR) light, with wavelengths of 650 to 900 nanometers, effectively penetrates tissues. The high signal to noise ratio and low phototoxicity of NIR light makes this wavelength range ideal for deep tissue imaging. However, current NIR fluorophores are generally large hydrophobic molecules that are prone to aggregation. Sulfonation can enhance aqueous solubility, but their anionic nature prevents membrane diffusion, and thus, restricts the applications of sulfonated molecules to in vitro or fixed cells. The repertoire of commercially available sulfonated NIR probes is mostly limited cyanines, which have low photostability. Moreover, larger cyanines require multiple sulfonates to maintain aqueous solubility. For example, Indocyanine Green is only sparingly soluble in PBS, despite having two sulfonates. My work has focused on the delivery of sulfonated dyes into live cells and the development of a new, ultra-compact NIR dye scaffold. First, to expand the in-cell applications of sulfonated fluorophores, I designed reductively-labile sulfonate protecting groups. Using these scaffolds, I have successfully delivered the fluorophore dansyl sulfonate into live cells, where the cytosolic reducing environment unmasks the anionic sulfonate. Secondly, to create a compact, photostable NIR fluorophore, I pioneered the discovery of azasilines dyes. The two azasiline derivatives, ASiFluor710 and ASiFluor730, fluoresce over 700 nanometers and are among the most compact NIR fluorophores currently known. ASiFluor730 also retains the high photostability of oxazine dyes, highlighting their potential in long exposure applications. Beyond the immediate applications in fluorescence microscopy and in vivo imaging, I envision that my work will serve as a framework for the future design of soluble, membrane permeable, NIR fluorescent probes.

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41

Pracheil, Tammy. "Regulation of the Target of Rapamycin Signaling Pathway in Saccharomyces cerevisiae". ScholarWorks@UNO, 2013. http://scholarworks.uno.edu/td/1662.

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An integrative, biochemical, genetic, and molecular biology approach utilizing gene manipulation, gene knock outs, plasmid based protein expression, and in vivo protein localization of fluorescence tagged proteins was employed to determine the function of an essential protein, Lst8, in TORC1 and TORC2 signaling and a previously uncharacterized complex, the Far3-7-8-9-10-11 complex (Far complex) in the budding yeast, Saccharomyces cerevisiae. Mutations in SAC7 and FAR11 suppressed lethality of both lst8 and tor2-21 mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2. Far11, a component of a six-member complex, was found to interact with Tpd3 and Pph21, conserved components of Protein Phosphatase 2A (PP2A) via co-immunoprecipitation. Mutations in FAR11 and RTS1, which encodes a PP2A regulatory B subunit, restore phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. These data suggest that TORC2 signaling is antagonized by Far11-dependent PP2A activity. To characterize the assembly of the Far complex in vivo, intracellular localization of the Far complex was examined by fluorescence microscopy. It was found that the Far complex localizes to the endoplasmic reticulum (ER). The data show that Far9 and Far10 are tail-anchored proteins that localize to the ER first and recruit a Far8-Far7-Far3 pre-complex. Far11 is found at the ER only when all other Far proteins are assembled at the ER. Surprisingly, ER localization is required for the Far Complex’s role TORC2 signaling because deletion of the tail-anchor domain of Far9 results in partial bypass of the tor2-21 mutant growth defect at 37 ˚C.

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42

Dumaine, Jennifer. "Acute Sleep Fragmentation Induces Tissue-Specific Changes in Cytokine Gene Expression and Increases Serum Corticosterone Concentration". TopSCHOLAR®, 2015. http://digitalcommons.wku.edu/theses/1480.

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Sleep fragmentation induces acute inflammation and increases glucocorticosteroids in vertebrates. Obesity and sleep fragmentation are often concurrent pro-inflammatory conditions in patients with obstructive sleep apnea. Despite the association between the two, their simultaneous effects on immune and endocrine profiles have not been explored. In the first experiment, we investigated changes in proinflammatory (IL-1β, TNF-α) and anti-inflammatory (TGF-β1) cytokine gene expression in the periphery (liver, spleen, fat, and heart) and brain (hypothalamus, prefrontal cortex, and hippocampus) in mice exposed to various intervals of sleep fragmentation. Serum corticosterone concentration was also assessed. Sleep was disrupted in male C57BL/6J mice using an automated sleep fragmentation chamber (Lafayette Industries), which involved movement of a sweeping bar at specified intervals. Mice were exposed to bar sweeps every 20 sec (high sleep fragmentation; HSF), 120 sec (low sleep fragmentation; LSF), or the bar remained stationary (control). Trunk blood and tissue samples were collected after 24 h of SF. It was found that HSF is a potent inducer of inflammation in the periphery (IL-1β: adipose, heart, and hypothalamus), but leads to upregulation of antiinflammatory cytokines in the brain (TGF-β1: hypothalamus and hippocampus), despite elevated serum corticosterone. Due to the association between obesity and SF, this experiment was replicated in male C57BL/6J mice (lean) and ob/ob KO mice (obese) using the previously described methods. We predicted the acute inflammatory response resulting from HSF would be different for the lean and the obese mice, with the greatest cytokine gene expression levels in the OB HSF group, due to a summative effect of the pro-inflammatory conditions. Obesity was the factor that most affected cytokine gene expression profiles. Additionally, the pro- vs anti-inflammatory gene expression profile varied with tissue type. While obesity resulted in neuroinflammation (hypothalamus, prefrontal cortex, hippocampus), it led to decreases in pro-inflammatory cytokine gene expression in the periphery (spleen, fat, heart). Serum corticosterone concentration was significantly elevated due to SF, but was not affected by obesity. As a result, the obese mice likely had neuroendocrine adaptations to combat the pre-existing pro-inflammatory condition of obesity, which impacted the acute inflammatory response to sleep loss.

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Grosse-Holz, Friederike. "Proteases and inhibitors in the interaction between Nicotiana benthamiana and Agrobacterium tumefaciens : systematic analysis and emerging solutions for molecular farming". Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:6146918c-3749-4604-88fa-01d426e4a817.

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Nicotiana benthamiana is now an established platform for molecular farming, the production of biopharmaceuticals in plants. Infiltration with Agrobacterium tumefaciens (agroinfiltration) is commonly used to transiently express one or multiple transgenes in N. benthamiana leaves. Agroinfiltrated N. benthamiana is a flexible and scalable recombinant protein (RP) production platform, but is impeded by low RP yields. Plant proteases can degrade RPs and thus limit RP accumulation. To inform, design and implement strategies for enhancing RP accumulation, I present four papers about proteases and protease inhibitors in agroinfiltrated N. benthamiana. First, I investigated the transcriptome, extracellular proteome and active secretome to understand the plant response to agroinfiltration and investigate the expressed proteases. I show that an extracellular immune response is mounted at the expense of photosynthesis. Comprehensive annotation and monitoring uncover a large, diverse repertoire of proteases in agroinfiltrated leaves, indicating that broad-range depletion of protease activity may be required to enhance RP accumulation. Second, I reviewed the literature on multifunctional plant protease inhibitors (PIs) and grouped them into three types of multifunctional PIs that evolved independently. Third, I screened candidate PIs and discovered that three new, unrelated PIs enhance RP accumulation. I present universal elements of the RP degradation machinery, uncovering new questions on our understanding of the protease network that degrades RPs. Fourth, I identified targets of SlCYS8, a PI that enhances RP accumulation. The target proteases of SlCYS8 are implicated in RP degradation and the high specificity of SlCYS8 can be used to study their role in other processes. By elucidating the immune response to agroinfiltration, by uncovering the N. benthamiana protease repertoire and by providing new tools to deplete the activity of specific proteases, this thesis makes a relevant contribution to both basic plant research and molecular farming.

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Antonio, Tatiana. "Obtenção de mutantes de Streptomyces clavuligerus e avaliação de condições de cultivo para a melhoria de produção de cefamicina C /". Araraquara [s.n.], 2012. http://hdl.handle.net/11449/100744.

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Resumo: Mutantes foram obtidos através de tratamento mutagênico dos esporos de S. clavuligerus ATCC 27064 com metil-metanosulfonato. Um total de 822 colônias, obtidas por repiques sucessivos, foram selecionadas em testes qualitativos e/ou quantitativos. O melhor mutante S. clavuligerus 45.41, mostrou-se de duas a quatro vezes mais produtivo que a linhagem selvagem, e manteve-se estável após 35 repiques sucessivos ao longo deste estudo. O comportamento das linhagens foi investigado pela adição de diferentes diaminas ao meio de cultivo, uma a uma, na ausência de lisina. Também se investigou duas fontes distintas de C, além de compostos como o ácido 2,6- diaminopimélico. Um planejamento de dois fatores (cadaverina e lisina), três níveis, baseado no ponto central, faces centradas, indicou que a adição de lisina aumenta a produção de CefC para ambas as linhagens. O efeito positivo da cadaverina foi observado principalmente na linhagem mutante 45.41. Resultados obtidos em processo de batelada, em biorreator de bancada confirmaram as diferenças observadas entre as linhagens nos cultivos em frascos agitados. Procedimento de mutagênese clássica foi utilizado em conjunto com critérios bem definidos para se estabelecer um meio de cultura apropriado, com o objetivo de alcançar um aumento significativo da produção de CefC. Este é o primeiro estudo utilizando um mutante de S. clavuligerus obtido por mutagênese clássica, cuja produção de CefC é de três vezes maior que a da linhagem selvagem, em meios contendo diaminas
Abstract: Mutants were obtained by treating S. clavuligerus ATCC 27064 spores with methyl-methanesulfonate. A total of 822 colonies, obtained by successive sampling, were selected by qualitative and/or quantitative tests. The best mutant, S. clavuligerus 45.41, was two to four times more productive than the wild-type strain, and remained stable even after 35 successive samplings throughout the study. Strains behavior was investigated by adding different diamines in media, one by one, in the absence of lysine. Also investigated two different sources of C, and compounds such as 2,6-diaminopimelic acid. The two-factor (cadaverine and lysine), three-level, central composite-based, face-centered experimental design indicates that adding lysine increases CephC production for both strains alike. The positive effect of cadaverine was observed mainly in the Mutant 45.41 strain. Results obtained in batch-processes in a bench-scale bioreactor confirmed differences among strains observed in shaken flasks cultures. Classical mutagenesis procedures in conjunction with the adoption of well-defined criteria to establish an appropriate culture medium promoted a significant improvement in CephC production. It is the first study indicating an increase in CephC production in media containing diamines employing a mutant of S. clavuligerus obtained by classical mutagenesis
Orientador: Maria Lucia Gonsales da Costa Araujo
Banca: Pedro de Oliva Neto
Banca: Cecilia Laluce
Banca: José Gregório Cabrera Gomez
Banca: Cristina Paiva de Sousa
Doutor

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Venter, Mauritz. "Isolation of grapevine promoters with special emphasis on the vacuolar pyrophosphatase". Thesis, Stellenbosch : University of Stellenbosch, 2004. http://hdl.handle.net/10019.1/16078.

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Dissertation (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Understanding the complex nature of grapevine molecular biology is of great importance for viticulturists. Progress in the elucidation of key events on a genetic level could provide further insight into the underlying cues responsible for the precise control of physiological and metabolic changes during a specific condition such as fruit development. The use and analysis of molecular ‘tools’, such as promoters controlling the site and level of gene activity, could assist in the understanding of grapevine biology and serve as a platform for the future design and development of recombinant DNA protocols and strategies for Vitis vinifera L. A high-throughput gene expression system, cDNA-AFLPs, was successfully used to analyse large-scale transcriptional activity during berry ripening. Candidate cDNA fragments were selected on the basis of desired expression patterns and/or known gene function for subsequent promoter isolation. From three candidate cDNAs selected, the promoter of a gene encoding vacuolar pyrophosphatase (V-PPase) was isolated for computational and comparative analyses. Promoter activity was evaluated on a transient level using the green fluorescent protein (GFP) reporter gene. Comparative integration has allowed for putative correlation of cis-elements, acting as receptors within promoter regions, to regulate V-PPase gene expression in response to development, environmental stress and tissue-specificity. In this study, integration of genetic data have advanced the understanding and transcriptional role of a key enzyme (V-PPase) during grape ripening. Although never a replacement for experimental verification, this integrative strategy of combining gene expression profiles with bioinformatics and regulatory data will greatly assist in further elucidation of various other key components and regulatory cues associated with grapevine molecular biology. This study has allowed us to use molecular tools that could assist in gaining further insight into genetic complexities and could serve as a platform for a more refined genetic manipulation strategy in Vitis vinifera L.
AFRIKAANSE OPSOMMING: Begrip van die komplekse aard van wingerd molekulêre biologie is van groot belang vir wingerdkundiges. Vooruitgang in die begrip van belangrike gebeurtenisse op ń genetiese vlak behoort verdere insig in die onderliggende instruksies vir die noukeurige beheer van fisiologiese en metaboliese veranderinge tydens ń spesifieke kondisie soos vrug rypwording te bevorder. Die gebruik en analise van molekulêre ‘instrumente’ soos promoters, wat die posisie en vlak van geen aktiwiteit beheer, kan bydra tot n beter begrip van wingerd biologie en sodoende dien as ń platform vir die toekomstige ontwerp en ontwikkeling van rekombinante DNS (deoksiribonukleiensuur) protokolle en strategieë vir Vitis vinifera L. ń Hoë-kapasiteit geen uitdrukkings sisteem, nl. kDNS-AFLPs (komplementêre deoksiribonukleiensuur- geamplifiseerde fragment lengte polimorfisme), is suksesvol gebruik vir die analise van grootskaalse transkripsionele aktiwiteit tydens druif rypwording. Kandidaat kDNS fragmente is geselekteer, gebaseer op verlangde uitdrukkings-patrone en/of bekende geen funksie vir daaropvolgende promoter isolering. Van drie geselekteerde kandidaat kDNS fragmente, is die promoter van ń geen wat vakuolêre pirofosfatase (V-PPase) kodeer geïsoleer vir rekenaar- en vergelykende analise. Promoter aktiwiteit is op ń nie-stabiele vlak deur die gebruik van ń groen-fluoresserende proteien (GFP) verklikker geen geëvalueer. Vergelykende integrering het dit moontlik gemaak om veronderstelde korrelasies van cis-elemente, wat as reseptore binne ń promoter area dien, en die regulering van V-PPase geen uitdrukking, in reaksie tot ontwikkeling, omgewings stres en weefsel-spesifisiteit, te maak. Tydens hierdie studie, het die integrering van genetiese data gehelp om die transkripsionele rol van ń belangrike ensiem (V-PPase) tydens druif rypwording beter te verstaan. Alhoewel dit nooit ń plaasvervanger vir eksperimentele bewyse sal wees nie, kan hierdie gëintegreerde strategie, wat die kombinasie van geen-uitdrukkingsprofiele met bioinformatika en regulatoriese data behels, grootliks bydra om verskeie ander belangrike komponente en regulatorieseaanwysings geassosieërd met wingerd molekulêre biologie te ontrafel. Hierdie studie het verdere insig in genetiese kompleksiteite verleen, en kan nou dien as ń platform vir ń meer presiese genetiese manipulering strategie in Vitis vinifera L.

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Agarwal, Stuti. "Pemetrexed, A Modulator of AMP-activated Kinase Signaling and an Inhibitor of Wild type and Mutant p53". VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/4003.

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New drug discoveries and new approaches towards diagnosis and treatment have improved cancer therapeutics remarkably. One of the most influential and effective discoveries in the field of cancer therapeutics was antimetabolites, such as the antifolates. The interest in antifolates increased as some of the antifolates showed responses in cancers, such as mesothelioma, leukemia, and breast cancers. When pemetrexed (PTX) was discovered, our laboratory had established that the primary mechanism of action of pemetrexed is to inhibit thymidylate 22 synthase (TS) (E. Taylor et al., 1992). Preclinical studies have shown that PTX has a broad range of antitumor activity in human and murine models of cancer (Adjei, 2000; Adjei, 2004; S. Chattopadhyay, Moran, & Goldman, 2007; Miller et al., 2000). Accordingly, in February 2004, the FDA issued first-line treatment approval for pemetrexed in malignant pleural mesothelioma and in 2008 for first line treatment for locally advanced or metastatic NSCLC (reviewed in (Rollins & Lindley, 2005). As an antifolate this level of therapeutic activity of PTX against lung cancers was surprising and atypical (Hazarika, White, Johnson, & Pazdur, 2004). This led us to the question whether the effects of pemetrexed on other folate-dependent targets could explain the clinical activity of the drug. Our lab showed that, in addition to inhibiting thymidylate synthase, PTX also inhibits aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of de novo purine synthesis. Inhibition of AICART leads to massive accumulation of its substrate 5-amino-4-imidazolecarboxamide ribonucleotide (ZMP), causing activation of AMP-dependent kinase (AMPK), which ultimately leads to suppression of mTORC1 signaling, a central regulator of cell growth and proliferation. This secondary mechanism could explain the unusual activity of PTX against mesothelioma and lung cancers. The large proportion of lung cancers are either null or mutant for p53 function. Therefore, this thesis focused on defining what the role of p53 is in the PTX-mediated AMPK activation and mTORC1 inhibition and how the loss of p53 affects mTORC1 signaling. These two questions proved to be interlinked. Chapter 2 investigates this relationship in detail. We found that, upon loss of p53, mTORC1 signaling is enhanced to a significant degree in colon carcinoma and lung cancer cell lines. Clearly, this observation required explanation. We found that the major factors responsible for these differences in mTORC1 activity upon loss of p53 23 were lower levels of two p53 target genes Tuberin (TSC2) and sestrin2. Immunoprecipitation studies of mTORC1 complexes from p53 wt and p53 null cells revealed quite interesting differences in the components of the mTORC1 complex. Immunoprecipitates from p53 null cells had higher levels of mTOR and lower levels of TSC2 and PRAS40 bound to raptor. This suggested that, in comparision to p53 competent cells, p53 null cells have more mTORC1 complex with enhanced activity due to decreased interaction of TSC2 and PRAS40, both of which are inhibitors of mTORC1. These observations explained the higher mTORC1 in p53 null cells and laid the foundation for determining the role of p53 in PTX-activated AMPK and mTORC1 inhibition. In the experiments described in Chapter 3, we found that PTX-mediated AMPK activation inhibited mTORC1 regardless of the p53 status in colon carcinoma cells. This suggested that mTORC1 inhibition by PTX was either independent of p53 mediated negative regulation of mTORC1 or was somewhere bypassing it. Therefore, we compared the effects of PTX with the classic AMPK activator aminoimidazolecarboxamide ribonucleoside (AICAR). In spite of a common mechanism of AMPK activation, namely, expansion of cellular ZMP levels, signaling from AMPK activated by PTX or AICAR were quite different. PTX-activated AMPK phosphorylated the mTORC1 component Raptor but not tuberin (TSC2), whereas AICARactivated AMPK phosphorylated both the targets. This differential behavior of two AMPK activators was due to differential behavior of p53 under these two treatments. Both, AICAR and PTX treatment led to increase in p53 levels but the p53 that accumulated after AICAR treatment was transcriptionally active while the p53 that accumulated after PTX treatment was not. Transcription of p53 targets, including TSC2 and sestrin2, was activated in AICAR- but not in PTX-treated cells. In the absence of p53 function, TSC2 was deficient and mTORC1 activity 24 enhanced, but Raptor phosphorylation by AMPK following PTX was robust and independent of both p53 and TSC2. Therefore we concluded that p53 deficiency suppresses TSC2 and upregulates mTORC1, but AMPK-phosphorylation of Raptor after pemetrexed treatment was sufficient to suppress mTORC1, even in TSC2 deficiency. This suggested pemetrexed as a drug for treatment of Tuberous Sclerosis, a genetic disease caused by functional inactivity of TSC1 or TSC2 due to point mutations in these genes. Mutation of p53 is one of the most common genetic alterations in human cancers and tumors. Cancers that express mutant p53 tend to be more aggressive, resistant to chemotherapy and show worse prognosis then p53-null tumors (Elledge et al., 1993; Olivier et al., 2006). This tumor-promoting activity of mutant p53 has been correlated with acquired and novel transcriptional activities of mutant p53. It has been shown that mutp53 can activate the transcription of cell growth promoting genes, such as, NFκB2, PCNA, MDR1, Axl, EGFR, hTERT, and HSP70, which are not usually transcriptional targets of wt p53. Interestingly, we found that whereas DNA damaging drugs enhance the acquired oncogenic transcriptional activities of mutp53, PTX interferes with this transcription activation. We also found in Chapter 4 that PTX can limit or block the DNA damaging drug-mediated increment of transcriptional activation of mutp53. This suggests that blockade of transcriptional activation of mutp53 by pemetrexed may provide an additional therapeutic benefit in mutp53 bearing cancers. As discussed in Chapter Three, although pemetrexed (with TdR) increases the levels of p53 and its binding to the promoter of its target gene, p21, this p53 is transcriptionally inactive. In order to understand the mechanism of the pemetrexed-mediated transcriptional defect of wt p53, we studied the PTX-mediated signaling towards ATM and ATR and their effects on their substrates Chk2 and Chk1, respectively. These studies suggested that the difference between 25 signaling under AICAR treatment and PTX treatment was that, unlike PTX, AICAR treatment was leading to DNA damage, followed by Chk2 phosphorylation at Thr68. We found there were three major differences between AICAR and pemetrexed (+ TdR) mediated signaling: AICAR caused DNA damage, followed by ATM mediated phosphorylation of Chk2 at Thr68 and phosphorylation of p53 at Ser15 all of which lead to activation of p53 transcriptional activity, events which do not take place under PTX treatment. Studies aimed at understanding the effects of PTX on wt and mutp53 transcriptional activities are discussed in detail in Chapters Three and Four of this dissertation. Overall, we concluded that PTX interferes with the transcription activity of wild type as well as gain-of-function mutant p53. The blockade of DNA damaging agent-mediated enhancement of mutp53 transcription activity by PTX, suggests the clinical relevance of PTX in carcinomas with mutp53. We suggest that this could be one of the contributing factors in the effects of PTX against human lung cancers.

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Silva, Natan Versati da. "Produção e estudo de atividade antiangiogênica de proteínas de fusão endostatina-domínio BH3 das proteínas pró-apoptóticas PUMA e BIM". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-23032016-155417/.

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A endostatina (ES) é uma proteína inibidora da angiogênese, com ação específica sobre células endoteliais em proliferação, utilizada para tratamento de tumores sólidos. No entanto, o elevado efeito antitumoral da ES observado em animais não é reproduzido em humanos. Com o intuito de potencializar a eficácia terapêutica da ES, produzimos duas proteínas híbridas com dois domínios funcionais. O primeiro domínio é a ES, que apresenta especificidade por células endoteliais ativadas, dirigindo estas proteínas de fusão às células endoteliais em proliferação, promovendo sua internalização e seu efeito inibitório. Como segundo domínio funcional utilizamos os domínios BH3 próapoptóticos de duas proteínas BH3-only com o objetivo de promover a liberação de citocromo C e desencadear o processo de apoptose, aumentando a ação antiangiogênica da ES. Neste trabalho, foram desenhadas duas proteínas de fusão que contêm o domínio BH3 das potentes proteínas pró apoptóticas PUMA e BIM (ES-PUMA e ES-BIM), que deveriam apresentar efeito antiangiogênico potencializado em relação à ES selvagem. A inserção dos fragmentos de DNA codificantes para os domínios BH3 de PUMA e BIM no vetor contendo o gene da ES (pET-ES) foram realizadas por mutagênese sítiodirigida. Estas proteínas de fusão recombinantes foram expressas como corpos de inclusão em E.coli, renaturadas utilizando processo que utiliza alta pressão e purificadas em resina de afinidade por heparina. O tratamento de células endoteliais com as proteínas ES-PUMA e ES-BIM não levou à queda de viabilidade em ensaio de MTS ou de apoptose avaliado por citometria de fluxo, em comparação com os resultados obtidos pelo tratamento com ES.
Endostatin (ES) is an angiogenesis inhibitor protein, with specific effect on proliferating endothelial cells, used to treat solid tumors. However, the high antitumor effect observed in animals is not reproduced in humans. In order to enhance the therapeutic efficacy of ES, we produced two hybrid proteins with two functional domains. The first domain is the ES that is specific for activated endothelial cells, directing the fusion proteins to endothelial cells in proliferation, promoting the internalization and the inhibitory effect. As a second functional domain we used the pro-apoptotic BH3 domains of two BH3-only proteins in order to promote the release of cytochrome C and trigger the apoptosis process, increasing the ES antiangiogenic action. In this work, we produced two fusion proteins containing the BH3 domain of the potent pro-apoptotic proteins BIM and PUMA (PUMA-ES and ES-BIM), which should provide enhanced antiangiogenic effect in relation to ES. The insertion of DNA fragments coding for the BH3 domain and PUMA and BIM in a vector containing ES gene (pETES) was accomplished by site-directed mutagenesis. These recombinant fusion proteins were expressed as inclusion bodies in E. coli, refolded using process at high pressure and purified on heparin affinity resin. Treatment of endothelial cells with ES-PUMA and ES-BIM did not lead to loss in viability in MTS assay or increase of apoptosis evaluated by flow cytometry, in comparison with the results obtained by treatment with ES.

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Halle, Briana. "Production of a Cost-Effective, TMV-Based Rabies Vaccine through Recombinant DNA Technology". Scholarship @ Claremont, 2018. http://scholarship.claremont.edu/cmc_theses/1816.

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Infectious diseases remain a significant cause of human deaths, as approximately 15 million deaths were attributed to infectious diseases in 2010 (Dye, 2014). One such disease is rabies, which causes around 59,000 human deaths worldwide annually according to some estimates (Kessels et al., 2017). However, 95% of human deaths attributed to rabies occur in Asia and Africa (Singh et al., 2017). Rabies is preventable, yet it is still a major concern in developing, low-income countries that lack access to the medical care necessary to combat it (Hampson et al., 2015). Alternative techniques for low-cost vaccine production have the potential to resolve this issue. This research investigates the use of recombinant DNA techniques and plant biotechnology to produce a more cost-effective vaccine for rabies. Gene sequences from the rabies glycoprotein were inserted at the end of the coat protein portion of the Tobacco Mosaic Virus (TMV) genome. Plants were then infected with this recombinant virus, with hopes that TMV particles would assemble with proteins produced from the inserted glycoprotein sequence fused to the TMV coat protein. Results thus far suggest some of the sequences could be producing recombinant TMV particles, although issues involving successful extraction and reversion to wild type are still a challenge. Additionally, other research suggests that this is an effective method for vaccine development in general and for rabies.

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Jackson, Constanza. "Synthesis of 2’ Modified Primers to Characterize Extension Events by Mutant Taq DNA Polymerases". Scholarship @ Claremont, 2015. http://scholarship.claremont.edu/scripps_theses/592.

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Oligonucleotides enable many biotechnological applications; however they are easily degraded by nucleases. Many nucleotides modified at the 2’ position are degraded at decreased rates which improves oligonucleotide utility. Most applications of oligonucleotides rely on enzymatic synthesis. Unfortunately, native DNA polymerases do not recognize most useful modified nucleotide substrates. Directed evolution has been used to identify mutants of Taq DNA polymerase I (Taq) that recognize substrates with 2’ modifications. While mutant enzymes capable of modified nucleotide addition have been identified, to date, all of these enzymes are limited by their inability to synthesize full length modified DNA. Despite considerable efforts to evolve new activity there has been little work done to quantitatively characterize these evolved enzymes. This thesis work presents efforts to synthesize modified primers that will help comparatively and quantitatively characterize three enzymes previously evolved to recognize 2’ modified substrates. Using the methods developed in this thesis project, our lab will be able to characterize the relationship between the number of modified nucleotides in the primer terminus and the rate of modified and unmodified nucleotide addition. Future work will identify key enzymatic steps that limit extension in these enzymes with implications for the future design of Taq mutants capable of synthesizing long 2’ modified oligonucleotides.

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Hussain, Shahed. "The development of novel biocatalytic routes for the synthesis of enantiomerically-pure chiral amines". Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/the-development-of-novel-biocatalytic-routes-for-the-synthesis-of-enantiomericallypure-chiral-amines(eba89b88-6801-40cc-a3c3-9c5f1c518d0f).html.

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Chiral amines represent a pervasive structural motif found in various natural products, pharmaceuticals, agrochemicals and fine chemicals. Their preparation in single-enantiomer form continues to attract significant research attention and although many advances have been made in the area of synthetic organic chemistry to increase the scope of the routes to these moieties, there remains an ever-growing need of general strategies for the assembly of structurally-diverse amines which also conform to the efficiency and environmental requirements of modern manufacturing processes. This report investigates biocatalytic routes as a means for constructing chiral amine scaffolds, which offer a more environmentally benign approach when compared with traditional chemocatalysed processes. Probing the catalysts available in the biocatalytic toolbox of enzymes, several routes were examined in more detail. Imine reductases (IREDs) represent a recent addition to the toolbox, enzymes which by definition are able to reduce pre-formed imines to their corresponding amines with high selectivity. This report analyses the (R)-imine reductase [(R)-IRED] from Streptomyces sp. GF3587, one of the first imine reductases identified for its biocatalytic potential, in greater depth. The enzyme was found to catalyse the reduction of a broad range of cyclic imines while displaying high levels of activity and selectivity, thereby offering a direct route of access to chiral secondary and tertiary amines. Substrate kinetic parameters were established for the enzyme in order to understand its substrate preferences and the enzymeâs catalytic mechanism was probed through the generation of mutant (R)-IREDs. Owing to their operation under physiological conditions as well as the orthogonal nature of their reactions, it is possible to combine multiple enzyme reactions to enable cascades. This report examines a multi-enzyme reaction combining Ï-transaminases (ATAs) with imine reductases, for the synthesis of chiral disubstituted piperidines from simple diketone substrates. The cascade was then taken a step further by the inclusion of the carboxylic acid reductase (CAR) enzyme, for the synthesis of the nitrogen-containing heterocycles morpholine and thiomorpholine from ketoacid compounds. Finally, the well-established deracemisation technique, employing a selective amine oxidase (AO) with either a non-selective chemical reducing agent or a biocatalytic reductant (IRED), was explored in more detail by encompassing new substrate motifs. As biocatalysis becomes more readily accepted as a general technique in the synthetic chemistâs repertoire, the concept of carrying out enzymatic reactions in constant flow was explored as a means for applying this methodology with increased production and decreased processing rates.

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Rozprawy doktorskie na temat „Biology - biotechnology”

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