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Tumor Suppressive Effects of the Beta-2 Adrenergic Receptor and the Small GTPase RhoBCarie, Adam E 24 March 2008 (has links)
Receptor tyrosine kinases such as ErbB2 contribute greatly to human malignant transformation, but the role that other receptors such as ß2 adrenergic receptor (B2 AR)play in cancer is ill defined. Furthermore, while some GTPases such as Ras and RhoA promote oncogenesis, RhoB has been suggested to have tumor suppressive activity. In this thesis the tumor suppressive activity of ß2 adrenergic receptors through blockade of the Ras/Raf/Mek/Erk pathway is demonstrated. Furthermore, this thesis provides strong evidence in support of a tumor suppressive activity of RhoB, but not RhoA, in delaying EbB2 mammary oncogenesis in a transgenic mouse model.
Chapter 1 describes a chemical biology approach that identifies a beta 2 adrenergic receptor agonist, ARA-211 (also known as pirbuterol) that suppresses the growth of cultured cells and of human tumors grown in nude mice by a mechanism involving stimulation of the ß2 AR, cAMP production and activation of PKA, which in turn leads to the inactivation of C-Raf, Mek1/2 and Erk1/2. Chapter 2 describes the translation of these findings by ex-vivo treatment of fresh human tumor biopsies, with the ultimate goal of validating this novel therapeutic approach. Chapter 3 describes the generation of transgenic mice that over express ErbB2 along with either RhoB or RhoA to determine the effects of these two small GTPases on ErbB2-mediated mammary tumorigenesis. The findings indicate that overexpression of RhoB, but not RhoA, results in decreased multiplicity and delay in the tumor onset mediated by ErbB2 overexpression.
In summary, this thesis work resulted in the discovery of how crosstalk between the ß2 AR/cAMP/PKA circuit with the Raf/Mek/Erk1/2 cascade leads to tumor suppression; and the discovery of the suppression of ErbB2-mediated breast cancer by the GTPase RhoB.
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Estimates of the nutritional cost of the development of immunity to gastrointestinal parasites in sheepGreer, Andrew Walter January 2005 (has links)
This thesis describes a series of three experiments designed to estimate the nutritional cost of the immune response to the gastrointestinal nematodes Trichostrongylus colubriformis and Teladorsagia circumcincta in sheep. For each experiment, animals were allocated hierarchically by liveweight into one of four groups that were either infected (group IF), similarly infected and concurrently immuno-suppressed with weekly intramuscular injections of 1.3mg kg liveweight (LW)-1 of methylprednisolone acetate (group ISIF), immuno-suppressed only (group IS) or remained as controls (group C). Body composition of all animals was estimated using x-ray computer tomography prior to infection and at the conclusion of each study with bodyweight and faecal nematode egg counts (FEC; eggs gram-1 of fresh faeces (epg)) measured along with blood samples taken for the determination of levels of serum proteins, phosphate and antibodies.
In the first trial (Chapter 3), the nutritional cost of both the acquisition and maintenance of immunity to gastro-intestinal nematodes was investigated using immunologically naive 5-month-old lambs and immunologically competent 17-month-old ewes during infection with 2,000 and 4,000 L3 infective T. colubriformis larvae d-1, respectively (80 L3 T. colubriformis larvae kgLW-1 d-1). Profiles of FEC and comparative worm burdens at slaughter indicated an effective immune response was maintained in IF ewes and developed in IF lambs while successfully suppressed in both ISIF lambs and ISIF ewes and was confirmed by serum antibody titres. The typical reduction in voluntary feed intake as a consequence of infection was observed in IF lambs (0.30, p<0.001) but not in IF ewes, ISIF lambs or ISIF ewes, and appeared to be associated with L3 IgA. Gross efficiency of use of metabolizable energy (ME) for net energy (NE) deposition was reduced by 0.20 in lambs during acquisition of immunity and by 0.16 in ewes maintaining an established immunity. Infection in immuno-suppressed animals reduced efficiency by 0.05 and 0.15 for lambs and ewes. These findings allowed the hypothesis that the reduction in feed intake and nutrient utilization in young parasitized sheep is caused by physiological signalling associated with the acquisition phase of the host immune response to infection, rather than simply the damage caused by the parasite per se.
The second trial (Chapter 4) investigated the influence of metabolizable protein (MP) supply on the metabolic disturbances associated with the acquisition phase of the immune response during infection with 2,000 L3 T. colubriformis d-1. Groups of lambs were offered either a low protein (L; 62g MP kgDM-1) or high protein diet (H; 95g MP kgDM-1). Patterns of total daily egg excretion indicated that an effective immune response was developed in HIF, but not LIF, HISIF nor LISF and was confirmed by comparative worm burdens. The proportionate reduction in feed intake in immunologically normal animals was reduced through the provision of additional protein, being 0.12 in HIF and 0.23 in LIF. Regardless of diet, infection did not cause a reduction in feed intake in immuno-suppressed animals (p>0.05). Infection proportionately reduced the gross efficiency of ME utilization in immunologically normal animals by 0.23 in HIF (p=0.09) and by 0.51 in LIF (p=0.01), but not in immuno-suppressed animals. Immuno-suppression did not suppress serum L3 IgA levels in seven of the eight HISIF and four of the eight LISIF animals. Furthermore, only four out of the eight immunologically normal animals from both the HIF and LIF groups displayed an L3 IgA response. Consequently, regardless of immuno-suppression treatment, animals were termed as IgA responders (HR or LR) or non-responders (HN or LN). Feed intake was proportionately reduced from day 22 by 0.15 in HR (p=0.03) and by 0.32 in LR (p=0.01), but was not significantly reduced in HN or LN. Gross efficiency of ME utilization was significantly reduced for LN animals only, being proportionately 0.59 (p<0.01). These findings allowed the conclusion that additional MP reduced the consequence of immunological signalling that was displayed in reduced feed intake and in nutrient utilization, both of which appeared to be associated with an IgA response. It is hypothesized that the lessening of nutritional disturbance observed in high protein and immuno-suppressed animals could be a consequence of altered physiological signalling during the immunological cascade.
The third trial (Chapter 5) utilized lambs infected with the abomasal parasite T. circumcincta to explore the possibility that the reduction in feed intake and nutrient utilization is a universal phenomenon of the acquisition phase of the immune response to nematode parasites inhabiting different organs along the gastrointestinal tract. In addition, immunological changes at the site of parasite infestation in the abomasal mucosa were measured from serial biopsy tissue samples taken from a further twelve animals that were surgically fitted with an abomasal cannula and either infected (CIF) or concurrently infected and immuno-suppressed as described previously (CISIF). The development of immunity in IF animals was accompanied by a 0.17 proportional decrease in feed intake between days 15 to 28 of infection (p<0.05) and a 0.20 proportional reduction in nutrient utilization (p=0.07), none of which were observed in ISIF animals. While FEC and worm burdens indicated successful immuno-suppression in ISIF animals, both serum IgA and total antibody production were not reduced. The development of immunity in CIF was reflected in an increase in both mast cells and globule leukocytes in serial abomasal tissue biopsies, both of which were reduced in CISIF (p<0.01 for both). In serial biopsy tissue, immuno-suppression did prevent a rise in tissue IgA that was apparent in CIF animals (p<0.01) although these changes were not reflected in serum IgA levels. It appears that the alleviation of the reduction in feed intake and nutrient utilization in young lambs through the use of corticosteroid induced immuno-suppression may be a universal phenomenon for both intestinal and abomasal parasites, but the association with and/or role of IgA during infection with T. circumcincta is unclear.
In summary, the reduction in feed intake and nutrient utilization in sheep during infection with both the abomasal nematode T. circumcincta and the small intestine nematode T. colubriformis appears to be associated with a component(s) of the acquisition phase of the host immune response, rather than, as conventionally assumed, the direct mechanical damage of the parasite per se. It is hypothesised that the nutritional disturbance as a consequence of infection in young lambs may be the result of pro-inflammatory cytokines involved in immunological signalling that may also be associated with the production of IgA, the effects of which can be reduced through the provision of adequate MP. These studies provide evidence that the immune response to gastrointestinal parasites is nutritionally costly to the animal and have implications for application of manipulations that are intended to promote the development of a strong immune reaction in high producing animals.
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Suppression et flot elliptique du J/psi dans les collisions Au+Au à 200~GeV dans la région à mi-rapidité de l'expérience PHENIX.Atomssa, Ermias Tujuba 02 December 2008 (has links) (PDF)
Le méson J/psi est considéré comme une sonde privilégiée de la formation du Plasma de Quarks et de Gluons (PQG) lors des collisions d'ions lourds. L'écrantage de la force de couleur a été proposé comme mécanisme de suppression anormale du J/psi dans un milieu déconfiné, au-delà de la suppression normale dans la matière nucléaire ordinaire. Le détecteur PHENIX est, des quatre expériences qui furent installées au démarrage de l'accélérateur RHIC, celle qui a une conception optimale pour la mesure du J/psi : dans les canaux de décroissance en dimuon à rapidité vers l'avant, et en dielectron à mi-rapidié. Le travail présenté ici couvre l'analyse de données prises par les spectromètres à mi-rapidité de PHENIX en collisions Au+Au à 200~GeV. Le facteur de modification nucléaire est mesuré en fonction de la centralité, et ensuite comparé à des estimations de la suppression due à la matière nucléaire froide. Malgré le fait que la suppression observée dans les collisions les plus centrales atteint un facteur plus grand que trois, une majeure partie peut être attribuée aux effets nucléaires froids. D'autre part, la comparaison avec les résultats des mesures effectuées au SPS du CERN et vers l'avant dans PHENIX a conduit à la spéculation que la régénération, un mécanisme de production des J/psi par association de quarks c et c-bar non corrélés, pourrait jouer un rôle important dans les collisions d'ion lourds les plus centraux. Le flot elliptique du J/psi en fonction de pT, une autre mesure présentée dans ce travail, est un outil potentiel pour tester le scénario de la régénération. Malgré l'obstacle statistique, la mesure est une démonstration de la faisabilité d'une mesure du flot elliptique dans un environnement à haute multiplicité.
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Investigation of Limiters For HPM and UWB Front-door Protection / Undersökning av limitrar för HPM och UWB framvägskopplingsskyddNilsson, Tony January 2006 (has links)
<p>An extensive investigation of front-door protection devices i.e. limiters has been made. The thesis work contains both HPM- and UWB-measurements done on various limiters, in order to characterize them. The measurements show that all limiters are not suitable as protection against HPM- and UWB-pulses. The limiters that were found to provide the best protection are limiters based on diode technologies. PIN- and Schottky-diodes generally shows very good performance and they fulfill many parameters that have been set by FOI. To obtain a full protection it is presumably necessary to use two or more limiters in combination, which complement each other.</p> / <p>En omfattande studie av framvägskopplingsskydd, dvs. limiters har gjorts. Examensarbetet innehåller resultat från både HPM- och UWB- mätningar som har gjorts på olika limitrar för att karaktärisera deras prestanda. Av resultaten från mätningarna kan man se att alla limitrar inte passar som skydd mot HPM- och UWB-pulser. De limitrar som tillhandahöll det bästa skyddet var baserade på olika diodtekniker. PIN- och Schottky-dioder visade sig överlag ha väldigt goda prestanda och de uppfyller många av de parametrar som bestämts av FOI. För att få ett heltäckande skydd är det förmodligen nödvändigt att man använder två eller flera limitrar i kombination, som kompletterar varandra.</p>
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Evaluation neurophysiologique des désordres temporo-mandibulaires (DTM) : la suppression extéroceptive du muscle temporal (EST)Marashi, Ramin 09 March 2005 (has links)
L'évaluation, le diagnostic et le traitement des douleurs et des dysfonctionnements de l'appareil stomatognathique, encore appelés « Syndrome algo-dysfonctionnel de l'appareil manducateur », ont évolué dans leur approche clinique. En fonction du choix des organes observés, de l'appréciation du rôle respectif des différents facteurs déclenchant ou entretenant la pathologie et en considérant l'étiopathogénie de ces désordres, de nombreuses propositions de dénomination se sont succédées au fil des années. Il s'agit maintenant de dysfonctions cranio mandibulaires ou de désordres temporo mandibulaires (DTM) qui prennent plus en considération les causes extérieures à la sphère oro faciale. Les désordres temporo-mandibulaires sont reconnus comme une cause majeure de douleurs oro-faciales non dentaire et représentent une classe de désordres musculo-squelettiques. La recherche conduit à considérer les DTM comme un ensemble corrélé de symptômes affectant l'appareil manducateur. De toute évidence, les signes et symptômes rencontrés lors de l'analyse des patients présentant des algies ou des dysfonctionnements de l'appareil manducateur, même s'ils intéressent un territoire bien délimité, ne peuvent pas être placés dans une urne commune. Ainsi, conformément à la conception moderne d'une étiologie multifactorielle, les désordres temporo-mandibulaires revêtent, par leurs manifestations cliniques, une sémiologie très polymorphe.
Les patients consultent pour des douleurs exquises ou diffuses de la mâchoire rebelles à toute thérapeutique médicale, pour des bruits au niveau de l'articulation temporo mandibulaire, pour une gêne lors des mouvements mandibulaires mais également pour des céphalées, des otalgies et des algies cranio-cervico-faciales. De plus, la multiplicité des signes objectifs et subjectifs peut constituer un véritable dédale dans lequel un diagnostic précis s'avère délicat.
Les neurophysiologistes reconnaissent que plus un appareil ou un système est important, plus son complexe sensitif apparaît hautement différencié. Ainsi, l'appareil manducateur, par le biais des nerfs trijumeaux, bénéficie d'une innervation sensitive particulièrement riche. En effet, l'arcade dentaire mandibulaire entre en contact avec son arcade dentaire antagoniste dans un très grand nombre de positions dont certaines sont très particulières. L'incision, la mastication, la déglutition, la respiration, la phonation et la mimique font largement appel à la mobilisation de la mandibule. Cette polyvalence fonctionnelle repose sur plusieurs mécanismes neurophysiologiques. Le sens de la proprioception, alimenté par les capteurs musculaires, articulaires, tendineux, parodontaux, muqueux, informe sur la position mandibulaire et sur les forces musculaires exercées. Il permet d'ajuster les mouvements, de les coordonner pour remplir au mieux ces fonctions. Enfin, la capsule de l'articulation temporo-mandibulaire (ATM) est particulièrement riche en récepteurs proprioceptifs pour améliorer la discrimination des situations et des postures.
Une régulation harmonieuse de cet appareil requiert des informations périphériques et centrales correctes ; en l'absence de celles-ci, des troubles et des douleurs peuvent apparaître. Vu la complexité des connexions du nerf trijumeau, il est tout à fait justifié de penser que des perturbations dans le système afférent, en provenance des muscles, des tendons, des articulations et des dents, peuvent entraîner des dysfonctions des circuits neuronaux. De plus, on sait que toute fonction physiologique requiert l'étude exhaustive de deux aspects, l'un sensitif, l'autre moteur. Les patients atteints de DTM présentent une altération du système neuromusculaire. Cette conception est admise par Nielsen dans une étude soulignant l'intérêt de l'Electromyographie (EMG) dans les troubles oro-faciaux. Certains auteurs se sont attachés à étudier d'autres techniques d'exploration neurophysiologique que l'EMG tels que les potentiels évoqués moteurs et somesthésiques, le réflexe de clignement ou encore la suppression extéroceptive du muscle temporal (EST).
Nous avons étudié une de ces explorations neurophysiologiques dans le cadre de quelques pathologies oro-faciales et principalement les désordres temporo-mandibulaires : l'examen de la suppression extéroceptive du muscle temporal (EST). Dans notre recherche, nous avons pour objectif d'effectuer dans un premier temps une synthèse et une évaluation de la littérature sur l'EST. Dans un second temps, nous avons évalué la technique de la suppression extéroceptive en mettant en place une méthodologie originale d'enregistrement. Nous avons examiné la reproductibilité de cette méthodologie au sein d'une population de sujets sains. Dans un troisième temps, nous avons évalué la technique de la suppression extéroceptive, dans une étude prospective randomisée en double aveugle, sur une population de sujets présentant des désordres temporo-mandibulaires. Enfin, nous avons évalué la technique de la suppression extéroceptive, dans une étude rétrospective sur des sujets présentant des désordres temporo-mandibulaires et différents troubles oro-faciaux. / The Aim of this study is the development of an exteroceptive suppression evaluation program (ESEP), wich uses the exteroceptive suppression of temporalis (EST) as complementary examination to diagnose temporomandibular disorders (TMD). The subjects, responding to different inclusion and exclusion criteria, undergo 6 tests EST where various procedures are carried out: reversible modification of the dental occlusion, ultrasounds application on the neck and structured cervical mobilizations. The measured parameters are the latencies and durations of ES1 and ES2 in various degrees of suppression. The EST of healthy group is different from pathological group. The parameters are reproducible in the two groups. The reversible modification of dental occlusion and the structured cervical mobilization change in a highly significant way the parameters of ES2 in marked degrees of suppression. EST remains perfectly reproducible. The installation of ESEP, makes it possible to qualify and quantify the results.
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Polymeric Loop Formation at Hard and Soft InterfacesAshcraft, Earl 01 August 2010 (has links)
Copolymers are used to increase the interfacial strength of immiscible components and suppress recombination of the minor phase by steric hindrance. The experiments conducted in these studies are designed to investigate in situ polymer loop formation at soft interfaces and functionalized nanotube surfaces. Block copolymers are the most effective type of copolymer for compatibilization because they extend perpendicular to the interface, allowing good entanglement with the homopolymer chains. Multiblock copolymers are more effective than diblock copolymers for strengthening the interface because they can cross the interface multiple times, forming “loops” in each phase that provide entanglement points for the homopolymer.
The first part of this dissertation focuses on understanding how telechelic variables influence their effectiveness to compatibilize an immiscible polystyrene (PS)/polyisoprene (PI) homopolymer blend. A fast reacting anhydride and amine telechelic pair (Anh-PS-Anh/NH2-PI-NH2) are compared with a slower reacting epoxy and carboxylic acid pair (Epoxy-PS-Epoxy/COOH-PI-COOH). Different molecular weight pairs are used to investigate the influence of end group concentrations and steric effects. We also investigate how the loading level affects the conversion of one telechelic pair. The PI telechelic has a fluorescent tag, which enables gel permeation chromatography (GPC) with fluorescence detection to be used for determining the amount of tagged PI converted and the molecular weight of the copolymer formed in situ as a function of mixing time. The effectiveness of these telechelic pairs as compatibilizers is quantified by annealing the samples and using scanning electron microscopy (SEM) to measure the domain size of the minor phase as a function of annealing time.
The second part of this study investigates the grafting of polymer loops to carboxylated multiwall nanotube (COOH-MWNT) surfaces and determining the reaction rate. These polymer loops will improve the nanotube dispersion by steric hindrance and improve energy transfer by creation of polymer chain entanglements. Fourier transform infrared spectroscopy (FT-IR) is used as a novel technique to measure the quantity of Epoxy-PS-Epoxy grafted to the nanotube surface. In addition, we determined the fraction of telechelics that form loops by further reacting the grafted nanotubes with monocarboxy terminated poly(4-methylstryrene) (COOH-P4MS), which only reacts with unbound Epoxy-PS-Epoxy chain ends.
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Royal Canadian Navy Evaluation of Handheld Aerosol ExtinguishersSheehan, Thomas David 16 April 2013 (has links)
Defence Research and Development Canada - Atlantic is currently under a project arrangement with Sweden and Holland to investigate new or emerging fire suppression technologies in naval applications. One possible outcome of this project arrangement could be the identification of a safe and effective Halon 1301 replacement suppression agent within the respective navies. The subject area Canada has agreed to investigate is aerosol fire extinguishing agent technologies. Although aerosols have been shown to be effective in suppressing demonstration fires, to date there has been little systematic scientific research into fire suppression using aerosol particulates. Therefore, there is a need for more in depth investigation of some of the commercial aerosol products available on the market to determine their fire suppression efficacy in naval applications, as well as any potential negative impacts that the aerosol may have on personnel, equipment and the environment. Aerosol suppression systems range from small handheld grenade extinguishers to large fitted and remotely activated aerosol dispersal units. The fire research and testing presented in this thesis looks specifically at the efficacy and safe use of two variants of the small handheld aerosol extinguishers, while also assessing aerosol agent suppression technologies overall.
The Royal Canadian Navy (RCN) currently uses a two tiered response to fire, consisting of first response by a Rapid Response Team (RRT), followed by full response by an Attack Team (AT). A Rapid Attack Team (RAT) has been introduced as an intermediate response team. To enhance efficiency of the RRTs or RATs, handheld aerosol units, in this evaluation the Dry Sprinkler Powder Aerosol (DSPA) and StatX fire knock down aerosol extinguishers, could potentially be stored throughout the ship or transported by the teams to a fire scene and used to control, suppress or even extinguish a fire prior to the AT arriving on the scene, particularly in the case of smaller enclosure fires. To fully evaluate their potential for use in this capacity, it is important to carefully study the suppression efficacy of these units under conditions similar to those in which they would be deployed, as well as to better understand their impact on a fire environment in terms of important parameters such as compartment temperature reduction, visibility, oxygen concentration, aerosol particulate dwell time, and toxicity. In terms of operational issues related to deployment of these pyrotechnic tools onboard RCN vessels, it is critical to assess the requirements for extinguisher safe storage and to gain an understanding of the incendiary potential of the units, as well as post suppression overhaul, smoke/agent clearing and compartment gas free certification.
The thesis includes a description of the experimental design, measurement techniques, and key results and conclusions for each of the 26 full-scale simulated marine enclosure live fire tests that were conducted. In general, handheld aerosol extinguishers have proven to be effective for fire control and even suppression under certain circumstances. They can improve the fire safety of RCN vessels when used correctly. Experimental data measured that relate to the consequences of accidental discharge and incendiary potential can also be used to ensure naval applications are safe and effective.
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Post Citizen United: The Lack of Political Accountability and Rise of Voter Suppression in a Time of Newly Defined CorruptionFullerton, Hannah S 01 April 2013 (has links)
In 2010, our definition of democracy in America was drastically changed by the Supreme Court case Citizens United v. FEC. The Court ruled that under the First Amendment, corporations have the right to free speech. The decision removed the final ban on corporations, which prohibited corporate money used for direct advocacy. The consequences of this have been tremendous. The decision has allowed for the creation and rise of Super PACs and political active nonprofits. As a result, Super PACs and nonprofits now act as “shadow campaigns”. Outside groups have the ability to engage in voter suppression tactics without politically hurting the candidate. Unlike political candidates, there are no direct ramifications for an outside organization to get caught engaging in voter suppression. They are not held accountable by anyone. The ability to take political action that is independent from the government or campaigns allows for a new form of corruption. Corruption is no longer a coordinated act between corporate money and a candidate, but rather political actions that take place outside the public sphere. Political actions that take place in the private sphere are outside the realm of political accountability. The people stand powerless against private outside organizations.
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Cognitive Mechanisms Underlying Second Language Listening ComprehensionHu, Guiling 30 March 2009 (has links)
This dissertation research investigates the cognitive mechanisms underlying second language (L2) listening comprehension. I use three types of sentential contexts, congruent, neutral and incongruent, to look at how L2 learners construct meaning in spoken sentence comprehension. The three types of contexts differ in their context predictability. The last word in a congruent context is highly predictable (e.g., Children are more affected by the disease than adults), the last word in a neutral context is likely but not highly predictable (e.g., Children are more affected by the disease than nurses), and the last word in an incongruent context is impossible (e.g., Children are more affected by the disease than chairs). The study shows that, for both native speakers and L2 learners, a consistent context facilitates word recognition. In contrast, an inconsistent context inhibits native speakers’ word recognition but not that of L2 learners. I refer to this new discovery as the facilitation-without-inhibition phenomenon in L2 listening comprehension. Results from follow-up experiments show that this facilitation-without-inhibition phenomenon is a result of insufficient suppression by L2 learners.
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Two Dimensional Genetic Approach to the Development of a Controllable Lytic Phage Display SystemSheldon, Katlyn 20 February 2013 (has links)
Bacteriophage Lambda (λ) has played a historical role as an essential model contributing to our current understanding of molecular genetics. Lambda’s major capsid protein “gpD” occurs on each capsid at 405 to 420 copies per phage in homotrimeric form and functions to stabilize the head and likely to compact the genomic DNA. The interesting conformation of this protein allows for its exploitation through the genetic fusion of peptides or proteins to either the amino or carboxy terminal end of gpD, while retaining phage assembly functionality and viability. The lytic nature of λ and the conformation of gpD in capsid assembly makes this display system superior to other display options.
Despite previous reports of λ as a phage display candidate, decorative control of the phage remains an elusive concept. The primary goal of this study was to design and construct a highly controllable head decoration system governed by two genetic conditional regulation systems; plasmid-mediated temperature sensitive repressor expression and bacterial conditional amber mutation suppression.
The historical λ Dam15 conditional allele results in a truncated gpD fragment when translated in nonsuppressor, wild-type E. coli cells, resulting in unassembled, nonviable progeny. I sequenced the Dam15 allele, identifying an amber (UAG) translational stop at the 68th codon. Employing this mutant in combination with a newly created isogenic cellular background utilizing the amber suppressors SupD (Serine), SupE (Glutamine), SupF (Tyrosine) and Sup— (wild type), we sought to control the level of incorporation of undecorated gpD products. As a second dimension, I constructed two separate temperature-inducile plasmids whereby expression of either D or D::eGFP was governed by the λ strong λ CI[Ts]857 temperature-sensitive repressor and expressed from the λ PL strong promoter.
Our aim was to measure the decoration of the λ capsid by a D::gfp fusion under varying conditions regulated by both temperature and presence of suppression. This was achieved utilizing this controllable system, enabling the measurement of a variable number of fusions per phage based on diverse genetic and physical environments without significantly compromising phage viability. Surprisingly, both SupE and SupF showed similar levels of Dam15 suppression, even though sequencing data indicated that only SupE could restore the native gpD sequence at amino acid 68 (Q). In contrast, SupD (S), conferred very weak levels of suppression, but imparted an environment for very high decoration of gpD::eGFP per capsid, even at lower (repressed) temperatures. The presence of albeit few wild-type gpD molecules allowed for an even greater display than that of the perceived “100%” decoration scenario provided by the nonsuppressor strain. It appears that the lack of wild-type gpD does not allow for the space required to display the maximum number of fusions and in turn creates an environment that affects both phage assembly and therefore phage viability. Finally, the use of Western blotting, confirmed the presence of gpD::eGFP fusion decoration by employing a polyclonal anti-eGFP antibody.
The significance of this work relates to the unique structure of λ’s capsid and its ability to exploit gpD in the design of controlled expression, which is guiding future research examining the fusion of different therapeutic peptides and proteins. Furthermore this approach has important implications specifically for the design of novel vaccines and delivery vehicles for targeted gene therapy in which steric hindrance and avidity are important concerns.
The execution of this project employed basic bacterial genetics, phage biology and molecular biology techniques in the construction of bacterial strains and plasmids and the characterization of the phage display system.
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