Global ETD Search

71	Advancing Cell-Free Protein Synthesis Systems for On-Demand Next-Generation Protein Therapeutics and Clinical Diagnostics Zhao, Emily Ann Long 16 December 2021 (has links) Recombinant proteins have many medical and industrial applications, but their use is complicated by commercial production and stability constraints. These issues are particularly challenging for recombinant proteins used in pharmaceutical therapeutics and clinical diagnostics. Expensive production and distribution limit the accessibility of therapeutics and diagnostics especially in the developing world. Additionally, clinical use of recombinant proteins face further challenges within biological systems including biological degradation and immunogenicity. To increase the accessibility of recombinant proteins, the cost and inefficiencies of protein manufacturing and distribution need to be significantly reduced. A powerful tool to aid in this endeavor is cell-free protein synthesis (CFPS) technology. CFPS is a versatile platform for recombinant protein production due to its open reaction environment, flexible reaction conditions, and rapid protein expression capabilities. These avoid the disadvantages of conventional manufacturing and present the capability of on-demand protein therapeutic production outside of centralized facilities. To improve the efficacy of recombinant proteins for medicinal use, protein engineering techniques such as PEGylation, or the conjugation of PEG polymers to protein surfaces, can be employed. PEGylation is widely used to enhance the pharmacokinetic properties of protein therapeutics. Deciphering optimal PEG conjugation sites is a continuing area of research that can be facilitated by CFPS systems that enable high-throughput, site-specific PEGylation. This dissertation presents advances in CFPS technology to promote increased accessibility and stability of life-saving therapeutics and diagnostics. The work presented here (1) improves on-demand therapeutic production capabilities by creating shelf-stable, endotoxin-free CFPS systems, (2) aids the rational design of next-generation PEGylated protein therapeutics through an in silico-in vitro CFPS screening platform, and (3) advances the development of portable clinical diagnostics for rapid and sustainable deployment at point-of-care through CFPS biosensor technology. The innovations of this dissertation are described in four publications. Specifically, an endotoxin-free CFPS system lyophilized with lyoprotectants is demonstrated that shows improved shelf-stability over standard lyophilized systems. A streamlined procedure for preparing endotoxin-free extract using auto-induction media is presented that significantly reduces CFPS preparation labor and time. A combinatorial screening approach is demonstrated in which coarse-grain molecular simulation informs PEGylation site selection as verified by CFPS experimental results. An inexpensive paper-based, saliva-activated CFPS biosensor platform is developed for the detection of SARS-CoV-2 sequences. Cell-free protein synthesis TXTL site-specific PEGylation protein therapeutic biologic unnatural amino acid cell-free biosensor RNA toehold switch Covid-19 on-demand therapeutic production point-of-care rapid diagnostic Engineering
72	Mechanism of N-Type Inactivation in Shaker Potassium Channels Pandey, Roshan 08 1900 (has links) Hyperexcitabilité est l'un des changements les plus importants observés dans de nombreuses maladies neuro-dégénératives telles que la sclérose latérale amyotrophique (SLA) et la maladie d'Alzheimer. De nombreuses recherches études se sont concentrées sur la réduction de l'hyperexcitabilité, soit en inactivant les canaux sodiques ce qui va réduire la génération de potentiels d'action, soit en prolongeant l'ouverture des canaux potassiques ce qui va qui ramener la membrane à son état de repos et réduire l’activité des neurones. Ainsi, pour cibler l'hyperexcitabilité, il faut tout d’abord comprendre les différents aspects de la fonction des canaux ioniques au niveau. Les objectifs des travaux présentés dans cette thèse consistent à déterminer le mécanisme d'inactivation dans les canaux potassiques Shaker. Les canaux Shaker Kv s'inactivent rapidement pour culminer le potentiel d'action et maintenir l'homéostasie des cellules excitables. L'inactivation de type N est causée par les 46 premiers acides aminés situés de l'extrémité N-terminale du canal, encore appelé, peptide d'inactivation (IP). De nombreuses études mutationnelles ont caractérisé l'inactivation de type N au niveau fonctionnel, cependant, la position de l'IP à l'état de repos et leur transition lors de l'inactivation est encore débattue. L'objectif de la première étude consiste à évaluer le mouvement des IP pendant leur inactivation à l'aide de la fluorométrie en voltage imposé. En insérant un acide aminé non naturel, la 3-[(6-acétyl-2-naphtalényl) amino]-L-alanine (Anap), qui est sensible aux changements d'environnement, nous avons identifié séparément les mouvements de la boule et de la chaîne. Nos données suggèrent que l'inactivation de type N se produit dans un mouvement biphasique en libérant d'abord le IP, ce qui va bloquer le pore du côté cytoplasmique. Pour affiner davantage la position de repos des IP, nous avons utilisé le transfert d'énergie de résonance à base de lanthanide et le métal de transition FRET. Nous proposons que le IP se situe dans la fenêtre formée par le canal et le domaine T1, interagissant avec les résidus acides-aminés du domaine T1. Dans notre deuxième étude, nous avons montré que le ralentissement de l'inactivation de type N observé dans la première étude est causée par une expression élevée des canaux Shaker. En effet, l'extrémité C-terminale du canal interagit avec les protéines d'échafaudage associées à la membrane pour la formation d'amas. Nous avons aussi montré qu'en tronquant les quatre derniers résidus C-terminaux impliqués dans la formation des amas, nous empêchons également le ralentissement de la cinétique d'inactivation dans les canaux Shaker. Nous avons également démontré que l'inactivation lente de type N n'est pas affectée par l'accumulation des cations potassiques [K+] externe ou toute diaphonie entre les sous-unités voisines. Cette étude élucide non seulement la cause du ralentissement de l'inactivation, mais montre également que les canaux modifient leur comportement en fonction des conditions d'expression. Les résultats trouvés au niveau moléculaire ne peuvent donc pas toujours être extrapolés au niveau cellulaire. / Hyperexcitability of neurons is a major symptom observed in many degenerative diseases such as ALS and Alzheimer’s disease. A lot of research is focused on reducing hyperexcitability, either by inactivating sodium channels that will reduce the generation of action potentials, or by prolonging the opening of potassium channels which will help to bring the membrane back to resting state and thus, reduce firing frequency of neurons. At the molecular level, it is important to understand different aspects of ion channel function to target hyperexcitability. The aim of this thesis was to investigate in two projects the inactivation mechanism in Shaker potassium channels. Shaker Kv channels inactivate rapidly to culminate the action potential and maintain the homeostasis of excitable cells. The so-called N-type inactivation is caused by the first 46 amino acids of the N-terminus of the channel, known as the inactivation peptide (IP). Numerous mutational studies have characterized N-type inactivation functionally, however, the position of the IP in the resting state and its transition during inactivation is still debated. The aim of the first project was to track the movement of IP during inactivation using voltage clamp fluorometry. By inserting an unnatural amino acid, 3-[(6-acetyl-2-naphthalenyl) amino]-L-alanine (Anap), which is sensitive to changes in environment, we identified the movements of ball and chain separately. Our data suggests that N-type inactivation occurs in a biphasic movement by first releasing the IP, which then blocks the pore from the cytoplasmic side. To further narrow down the resting position of the inactivation peptide, we used Lanthanide-based Resonance Energy transfer and transition metal FRET. We propose that the inactivation peptide is located in the window formed by the channel and the T1 domain, interacting with the acidic residues of the T1 domain. In a follow-up study, we explored the reason underlying slow inactivation kinetics observed during the study of N-type inactivation in the first project. High expression of Shaker channels results in slowing of the N-type inactivation. The C-terminus of the channel interacts with membrane associated scaffold proteins for cluster formation. In this study, we have shown that by truncating the last four C-terminal residues involved in cluster formation, and hence preventing channel clustering, we also prevent slowing of the inactivation kinetics in Shaker channels. We also showed that slow N-type inactivation is not affected by accumulation of external [K+] or any crosstalk between the neighboring subunits. The second project not only elucidates the cause of the inactivation slow-down but illustrates that the channels alter their behavior dependent on the expression conditions. Results found on the molecular level can thus not always be extrapolated to the cellular level. Canaux Kv acides aminés non-naturels Anap fluorimétrie en voltage impose tmFRET LRET Kv channels Unnatural amino acids Voltage clamp fluorometry Lanthanide resonance energy transfer
73	Terapeutiese begeleiding van die jong kind in kindersorg wie se ouers op 'n onnatuurlike wyse gesterf het Engelbrecht, Stephanie 30 November 2002 (has links) Text in Afrikaans / In today's society our children are being incredibly exposed to trauma and death. Parents want to protect their children against hardship and loss and it is evident that todays society is characterized by social pathology. In view of the above the researcher studied literature extensively to investigate the affect of the phenomenon of death and trauma in the life of the young child. In order to establish the availability of therapy programs for the young child in childcare whose parents died in an unnatural way. It was evident that the present South African society is indeed characterized by conflict, homicide and violence. The death of a parent is a traumatic experience for the young child (aged two to seven years). VVhen death occurs suddenly it was found that the grief process is more complex. Through the process of literature study and empirical investigation it seems that many young children, who lost their parents through unnatural death, are placed in childcare institutions. In this study it was established that the fact that children were placed in childcare did not always imply that therapeutic intervention took place. Limited financial support and social services contributed to insufficient therapeutical intervention. Furthermore it was found that the investigated childcare institutions couldn't provide the researcher with therapeutic programmes. This study therefore indicates that there is a need for the development of therapeutic programmes for the young child in childcare. whose parents died in an unnatural way. / In die hedendaagse samelewing blyk dit dat kinders al hoe meer aan dood en trauma blootgestel word. Hoewel ouers graag hulle kinders wil beskerm teen hartseer en verlies, blyk dit dat vandag se samelewing gekenmerk word deur 'n vorm van sosiale patologie. In die lig van voorafgaande, het die navorser literatuurstudie onderneem om die teenwoordigheid en die fenomeen van dood en trauma in die lewe van die jong kind te ondersoek. 'n Verdere literatuurstudie is ook onderneem om die rou ervaring van die jong kind te ondersoek. Die navorser wou ook in hierdie studie vasstel of daar terapeutiese begeleiding aan die jong kind in kindersorg, wie se ouers op 'n onnatuurlike wyse gesterf het, beskikbaar is. Daar is in die studie gevind dat die huidige Suid-Afrikaanse samelewing inderdaad gekenmerk word deur konflik, moord en misdaad. Die afsterwe van 'n ouer is 'n traumatiese ondervinding vir die jong kind (twee tot sewe jaar). Wanneer die dood skielik en onverwags intree, is bevind dat die rouproses gekompliseerd is. Deur die proses van literatuurstudie en empiriese ondersoek blyk dit dat verskeie jong kinders wie se ouers op 'n onnatuurlike wyse gesterf het, in kindersorginstansies geplaas word. Daar is in hierdie studie bewys dat hoewel die kind in kindersorginstansies geplaas word, terapeutiese begeleiding nie altyd plaasvind nie. Hierdie gebrekkige terapeutiese begeleiding word toegeskryf aan die feit dat nie voldoende finansiele ondersteuning en maatskaplike dienste is nie. Daar is verder gevind dat kindersorginstansies, wat aan die studie deelgeneem het, nie oor traumaterapie programme vir terapeutiese begeleiding beskik nie. Hierdie studie toon dus dat daar 'n behoefte is vir die ontwikkeling van terapeutiese programme vir die jong kind in kindersorg wie se ouers op 'n onnatuurlike wyse gesterf het. / Educational studies / M. Ed. (Voorligting) Trauma Unnatural death Parent Young child Grief process Childcare Trauma Onnatuurlike afsterwe Ouer Jong kind Rouproses Kindersorg Terapeutiese begeleiding 155.937 Psychic trauma in children Bereavement -- Psychological aspects Grief in children Grief -- Psychological aspects Loss (Psychology) Death -- Psychological aspects Grief therapy Sudden death -- Psychological aspects Violent deaths -- Psychological aspects
74	Terapeutiese begeleiding van die jong kind in kindersorg wie se ouers op 'n onnatuurlike wyse gesterf het Engelbrecht, Stephanie 30 November 2002 (has links) Text in Afrikaans / In today's society our children are being incredibly exposed to trauma and death. Parents want to protect their children against hardship and loss and it is evident that todays society is characterized by social pathology. In view of the above the researcher studied literature extensively to investigate the affect of the phenomenon of death and trauma in the life of the young child. In order to establish the availability of therapy programs for the young child in childcare whose parents died in an unnatural way. It was evident that the present South African society is indeed characterized by conflict, homicide and violence. The death of a parent is a traumatic experience for the young child (aged two to seven years). VVhen death occurs suddenly it was found that the grief process is more complex. Through the process of literature study and empirical investigation it seems that many young children, who lost their parents through unnatural death, are placed in childcare institutions. In this study it was established that the fact that children were placed in childcare did not always imply that therapeutic intervention took place. Limited financial support and social services contributed to insufficient therapeutical intervention. Furthermore it was found that the investigated childcare institutions couldn't provide the researcher with therapeutic programmes. This study therefore indicates that there is a need for the development of therapeutic programmes for the young child in childcare. whose parents died in an unnatural way. / In die hedendaagse samelewing blyk dit dat kinders al hoe meer aan dood en trauma blootgestel word. Hoewel ouers graag hulle kinders wil beskerm teen hartseer en verlies, blyk dit dat vandag se samelewing gekenmerk word deur 'n vorm van sosiale patologie. In die lig van voorafgaande, het die navorser literatuurstudie onderneem om die teenwoordigheid en die fenomeen van dood en trauma in die lewe van die jong kind te ondersoek. 'n Verdere literatuurstudie is ook onderneem om die rou ervaring van die jong kind te ondersoek. Die navorser wou ook in hierdie studie vasstel of daar terapeutiese begeleiding aan die jong kind in kindersorg, wie se ouers op 'n onnatuurlike wyse gesterf het, beskikbaar is. Daar is in die studie gevind dat die huidige Suid-Afrikaanse samelewing inderdaad gekenmerk word deur konflik, moord en misdaad. Die afsterwe van 'n ouer is 'n traumatiese ondervinding vir die jong kind (twee tot sewe jaar). Wanneer die dood skielik en onverwags intree, is bevind dat die rouproses gekompliseerd is. Deur die proses van literatuurstudie en empiriese ondersoek blyk dit dat verskeie jong kinders wie se ouers op 'n onnatuurlike wyse gesterf het, in kindersorginstansies geplaas word. Daar is in hierdie studie bewys dat hoewel die kind in kindersorginstansies geplaas word, terapeutiese begeleiding nie altyd plaasvind nie. Hierdie gebrekkige terapeutiese begeleiding word toegeskryf aan die feit dat nie voldoende finansiele ondersteuning en maatskaplike dienste is nie. Daar is verder gevind dat kindersorginstansies, wat aan die studie deelgeneem het, nie oor traumaterapie programme vir terapeutiese begeleiding beskik nie. Hierdie studie toon dus dat daar 'n behoefte is vir die ontwikkeling van terapeutiese programme vir die jong kind in kindersorg wie se ouers op 'n onnatuurlike wyse gesterf het. / Educational studies / M. Ed. (Voorligting) Trauma Unnatural death Parent Young child Grief process Childcare Trauma Onnatuurlike afsterwe Ouer Jong kind Rouproses Kindersorg Terapeutiese begeleiding 155.937 Psychic trauma in children Bereavement -- Psychological aspects Grief in children Grief -- Psychological aspects Loss (Psychology) Death -- Psychological aspects Grief therapy Sudden death -- Psychological aspects Violent deaths -- Psychological aspects
75	Ne-lidští vypravěči v literární fikci / Non-Human Narrators in Literary Fiction Hocková, Eva January 2018 (has links) This master thesis deals with the phenomenon of non-human narrator in the literary fiction. The theoretical part of the thesis provides a conceptual framework. The framework is based on the so-called unnatural narratology. Firstly, the thesis discusses non-human narrators that are conventionally accepted. Secondly, the thesis provides a case study of non-human narrators that are perceived as unnatural. The case study includes examples from both canonical works and contemporary works. The research focuses on two main levels related to the usage of the phenomenon of non-human narration as used in the narratives: the level of "meaning" and the level of "form and effect".
76	Designing Cell-Free Protein Synthesis Systems for Improved Biocatalysis and On-Demand, Cost-Effective Biosensors Soltani Najafabadi, Mehran 06 August 2021 (has links) The open nature of Cell-Free Protein Synthesis (CFPS) systems has enabled flexible design, easy manipulation, and novel applications of protein engineering in therapeutic production, biocatalysis, and biosensors. This dissertation reports on three advances in the application of CFPS systems for 1) improving biocatalysis performance in industrial applications by site-specific covalent enzyme immobilization, 2) expressing and optimizing a difficult to express a mammalian protein in bacterial-based CFPS systems and its application for cost-effective, on-demand biosensors compatible with human body fluids, and 3) streamlining the procedure of an E. coli extract with built-in compatibility with human body fluid biosensors. Site-specific covalent immobilization stabilizes enzymes and facilitates recovery and reuse of enzymes which improves the net profit margin of industrial enzymes. Yet, the suitability of a given site on the enzyme for immobilization remains a trial-and-error procedure. This dissertation reports the reliability of several design heuristics and a coarse-grain molecular simulation in predicting the optimum sites for covalent immobilization of a target enzyme, TEM-1 ?-lactamase. This work demonstrates that the design heuristics can successfully identify a subset of favorable locations for experimental validation. This approach highlights the advantages of combining coarse-grain simulation and high-throughput experimentation using CFPS to efficiently identify optimal enzyme immobilization sites. Additionally, this dissertation reports high-yield soluble expression of a difficult-to-express protein (murine RNase Inhibitor or m-RI) in E. coli-lysate-based CFPS. Several factors including reaction temperature, reaction time, redox potential, and presence of folding chaperones in CFPS reactions were altered to find suitable conditions for m-RI expression. m-RI with the highest activity and stability was used to develop a lyophilized CFPS biosensor in human body fluids which reduced the cost of biosensor test by ~90%. Moreover, an E. coli extract with RNase inhibition activity was developed and tested which further streamlines the production of CFPS biosensors compatible with human body fluids. Mehran Soltani cell-free protein synthesis cell-free CFPS in vitro TEM-1 ?-lactamase antibiotics unnatural amino acid protein engineering site-specific covalent immobilization site-directed mutagenesis design heuristics coarse-grain simulation biosensor cell-free biosensor RNase inhibitor murine RNase inhibitor m-RI RNase inhibitor RNase A human body fluids saliva serum urine glutamine lyophilized point of care GroEL/ES folding chaperones E. coli extract Extract with RNase inhibition activity Engineering
77	Fauxtopia Kampf, Raymond William 01 January 2004 (has links) To all who come to this fictitious place:Welcome.Fauxtopia is your land. Here, age relives distorted memories of the past, and here, youth may savor the challenge of trying to understand the present. Fauxtopia is made up of the ideals, the dreams and the fuzzy facts which have re-created reality... with the hope that it will be a source of edutainment for all the world.Ray KampfFauxtopia DedicationApril 1st, 2004 swindle subterfuge story stooge stereotype stratagem spurious spiel soft-soap snow job simulated aspersion adventure artificial anecdote allegory advertising assumptions belie bluff bogus brainwash calumniation canard cheat chronicle cliche cliffhanger con man conceal counterfeit crooked deal deceit deception disguise dishonest distort double cross ersatz evangelism exaggerate fabrication facade fake falsehood fantasy fib fiction flim flam fool folktale forgery frame up fraudulence fudge gag grifter guile guise history hoax hokey hype hyperbole knavery legend likeness make believe malign mask memoir misrepresentation mockery myth parable parody performance perjury phony placebo player ploy potboiler prevarication propaganda publicity puppet put-on quasi record reproduction romance routine ruse saga scam shtick semblance sequel show serial synthetic tall story trick unnatural untruth veneer verisimilitude white lie whopper Disney politics hyper reality theme park entertainment Art and Design Arts and Humanities
78	A case for memory enhancement : ethical, social, legal, and policy implications for enhancing the memory Muriithi, Paul Mutuanyingi January 2014 (has links) The desire to enhance and make ourselves better is not a new one and it has continued to intrigue throughout the ages. Individuals have continued to seek ways to improve and enhance their well-being for example through nutrition, physical exercise, education and so on. Crucial to this improvement of their well-being is improving their ability to remember. Hence, people interested in improving their well-being, are often interested in memory as well. The rationale being that memory is crucial to our well-being. The desire to improve one’s memory then is almost certainly as old as the desire to improve one’s well-being. Traditionally, people have used different means in an attempt to enhance their memories: for example in learning through storytelling, studying, and apprenticeship. In remembering through practices like mnemonics, repetition, singing, and drumming. In retaining, storing and consolidating memories through nutrition and stimulants like coffee to help keep awake; and by external aids like notepads and computers. In forgetting through rituals and rites. Recent scientific advances in biotechnology, nanotechnology, molecular biology, neuroscience, and information technologies, present a wide variety of technologies to enhance many different aspects of human functioning. Thus, some commentators have identified human enhancement as central and one of the most fascinating subject in bioethics in the last two decades. Within, this period, most of the commentators have addressed the Ethical, Social, Legal and Policy (ESLP) issues in human enhancements as a whole as opposed to specific enhancements. However, this is problematic and recently various commentators have found this to be deficient and called for a contextualized case-by-case analysis to human enhancements for example genetic enhancement, moral enhancement, and in my case memory enhancement (ME). The rationale being that the reasons for accepting/rejecting a particular enhancement vary depending on the enhancement itself. Given this enormous variation, moral and legal generalizations about all enhancement processes and technologies are unwise and they should instead be evaluated individually. Taking this as a point of departure, this research will focus specifically on making a case for ME and in doing so assessing the ESLP implications arising from ME. My analysis will draw on the already existing literature for and against enhancement, especially in part two of this thesis; but it will be novel in providing a much more in-depth analysis of ME. From this perspective, I will contribute to the ME debate through two reviews that address the question how we enhance the memory, and through four original papers discussed in part three of this thesis, where I examine and evaluate critically specific ESLP issues that arise with the use of ME. In the conclusion, I will amalgamate all my contribution to the ME debate and suggest the future direction for the ME debate. 174

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