1978 pitch outline to McDonald's Restaurants

Outline of the 1978 pitch by Vickers & Benson to McDonald's Restaurants.

Isolation and characterization of genomic DNA sequences that enhance the stability of plasmid DNA in mammalian cells /

The ease of production and manipulation has made plasmid DNA a prime target for its use in gene transfer technologies such as gene therapy and DNA vaccines. The major drawback of plasmid however is its stability within mammalian cells. Plasmid DNA is usually lost by cellular mechanisms or as a result of mitosis by simple dilution. This study set out to search for mammalian genomic DNA sequences that would enhance the stability of plasmid DNA in mammalian cells.Creating a plasmid based genomic DNA library, we were able to screen the human genome by transfecting the library into Human Embryonic Kidney (HEK 293) Cells. Cells that contained plasmid DNA were selected, using G418 for 14 days. The resulting population was then screened for the presence of biologically active plasmid DNA using the process of transformation as a detector.A commercially available plasmid DNA isolation kit was modified to extract plasmid DNA from mammalian cells. The standardized protocol had a detection limit of -0.6 plasmids per cell in one million cells. This allowed for the detection of 45 plasmids that were maintained for 32 days in the HEK 293 cells. Sequencing of selected inserts revealed a significantly higher thymine content in comparison to the human genome. Sequences with high A/T content have been associated with Scaffold/Matrix Attachment Region (S/MAR) sequences in mammalian cells. Therefore, association with the nuclear matrix might be required for the stability of plasmids in mammalian cells.

The effects of postural threat on cognitive strategies used to maintain upright stance

It is well established that postural threat modifies postural control, although little is known regarding the underlying mechanism(s) responsible. It is possible that changes in postural control under conditions of elevated postural threat result from alterations in cognitive strategies. The purpose of this study was to determine the influence of elevated postural threat on cognitive strategies and to determine the relationship between postural control, psychological, and cognitive measures. It was hypothesized that elevated postural threat would cause a shift to more conscious control of posture. It was also expected that a relationship between fear of falling and postural control would exist that could be explained by changes in conscious control of posture. Forty-eight healthy young adults stood on a force plate at two different surface heights: ground level (LOW) and 3.2m above ground level (HIGH). Center of pressure (COP) summary measures calculated to quantify postural control were the mean position (AP-COP MP), root mean square (AP-COP RMS) and mean power frequency (AP-COP MPF) in the anteriorposterior direction. Trunk sway measures calculated in the pitch direction were trunk angle and trunk velocity. Psychological measures including perceived balance confidence, perceived fear of falling, perceived anxiety, and perceived stability were self reported. As a physiological indicator of anxiety, electrodermal activity was collected. The cognitive strategies assessed were movement reinvestment and attention focus. A modified state-sp-ecific version of the Movement Specific Reinvestment Scale was used to measure conscious motor processing (CMP) and movement self-consciousness (MSC). An attention focus questionnaire was developed to assess the amount of attention directed to internal and external sources. An effect of postural threat on cognitive strategies was observed as participants reported more conscious control and a greater concern or worry about their posture at the HIGH postural threat condition as well as an increased internal and external focus of attention. In addition changes in postural control, psychological, and physiological measures were found. The participants leaned away from the edge of the platform, the frequency of their postural adjustments increased, and the velocity of their trunk movements increased. Participants felt less confident, more fearful, more anxious, and less stable with an accompanying increase in physiological anxiety. Significant correlations between perceived anxiety, AP-COP MP, and cognitive measures revealed a possible relationship that could be mediated by cognitive measures. It was found that with greater conscious motor processing, more movement self-consciousness, and a greater amount of attention focused externally there was a larger shift of the mean position away from the edge of the platform. This thesis provides evidence that postural threat can influence cognitive strategies causing a shift to more conscious control of movement which is associated with leaning away from the edge of the platform. Shifting the position of the body away from the direction of the postural threat may reflect a cognitive strategy to ensure safety in this situation due to the inability to employ a stepping strategy when standing on an elevated platform.

Investigating the role of apoptosis regulator EndoG on exogenous DNA uptake, stability, replication and recombination

Endonuclease G (EndoG) is a well conserved mitochondrial nuclease with dual lethal and vital roles in the cell. It non-specifically cleaves endogenous DNA following apoptosis induction, but is also active in non-apoptotic cells for mitochondrial DNA (mtDNA) replication and may also be important for replication, repair and recombination of genomic DNA. The aim of our study was to examine whether EndoG exerts similar activities on exogenous DNA substrates such as plasmid DNA (pDNA) and viral DNA vectors, considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus (a cationic liposome transfection reagent), targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. To investigate possible effects of EndoG on viral DNA vectors, we constructed and evaluated AdsiEndoG, a first generation adenovirus (Ad5 ΔE1) vector encoding a shRNA directed against EndoG mRNA, along with appropriate Ad5 ΔE1 controls. Infection of HeLa cells with AdsiEndoG at a multiplicity of infection (MOI) of 10 p.f.u./cell resulted in an early cell proliferation defect, absent from cells infected at equivalent MOI with control Ad5 ΔE1 vectors. Replication of Ad5 ΔE1 DNA was detected for all vectors, but AdsiEndoG DNA accumulated to levels that were 50 fold higher than initially, four days after infection, compared to 14 fold for the next highest control Ad5 ΔE1 vector. Deregulation of the cell cycle by EndoG depletion, which is characterized by an accumulation of cells in the G2/M transition, is the most likely reason for the observed cell proliferation defect. The enhanced replication of AdsiEndoG is consistent with this conclusion, as Ad5 ΔE1 DNA replication is intimately related to cell cycling and prolongation or delay in G2/M greatly enhances this process. Furthermore, infection of HeLa with AdsiEndoG at MOI of 50 p.f.u./cell resulted in an almost complete disappearance of viable, adherent tumour cells from culture, whereas almost a third of the cells were still adherent after infection with control Ad5 ΔE1 vectors, relative to the non-infected control. Therefore, targeting of EndoG by RNAi is a viable strategy for improving the oncolytic properties of first generation adenovirus vectors. In addition, AdsiEndoG-mediated knockdown of EndoG reduced homologous recombination between pDNA substrates in HeLa cells. The effect was modest but, nevertheless demonstrated that the proposed role of EndoG in homologous recombination of cellular DNA also extends to exogenous DNA substrates.